a figure given is for an average adult (male: 23 m³/day; female 21 m³/ day) with 8 hours of resting and 16 hours of light/non-occupational activity.

3.6 References

Eklund G and Oskarsson A (1999). Exposure of cadmium from infant formulas and weaning foods. Food Addit Contam 16, 509-519.

EPA (1997). Exposure Factors Handbook. Update to Exposure Factors Handbook. EPA/600/8-89/043 - May 1989. http://www.epa.gov/nceawww1/pdfs/efh/front.pdf

Ishøy T and Jensen K (1999). Forgiftninger hos børn: udviklingen gennem to årtier. Ugeskrift for Læger 161/20.

Larsen PB (1998). Afskæringskriterier for forurenet jord. Miljøprojekt nr. 425, Miljøstyrelsen, in Danish.

Larsen PB, Larsen JC, Fenger J and Jensen SS (1997). Evaluation of health impacts of air pollution from road traffic. Miljøprojekt nr. 352, Miljøstyrelsen, in Danish

Lawrie CA (1998). Different dietary patterns in relation to age and the consequences for intake of food chemicals. Food Addit Contam 15, Suppl., 75-81.

MAFF (1995). National diet and nutrition survey: children aged 1½ to 4½ years. 1: Report of the diet and nutrition survey.

MST (1990). Risikovurdering af forurenede grunde. Miljøprojekt nr. 123, 1990, in Danish.

National Food Agency (1996). Danskernes kostvaner 1995, in Danish.

Nordic Council of Ministers 1996. Nordiska näringsrekommendationer 1996:28.

Norén K and Meironyté D (1998). Contamints in Swedish human milk. decreasing levels of organochlorine and increasing levels of organobromine compounds. Organohalogen compounds 38, 1-4.

NRC (1993). Pesticides in the diet of infants and children. National Research Council, National Academy Press, Washington, D.C.

Osimitz T (1999). Handouts from "Exposure Assessment in Children: A Consumer Exposure Workshop" Swedish Chemical Inspectorate 9 - 10 September 1999.

SST/FDIR (1999). Indhold af dioxiner, PCB, visse chlorholdige pesticider, kviksølv og selen i modermælk hos danske kvinder 1993-94. Sundhedsstyrelsen, Fødevaredirektoratet.

WHO (1994). Assessing human health risks of chemicals: derivation of guidance values for health-based exposure limits. Environmental Health Criteria 170. International Programme on Chemical Safety, World Health Organization.

WHO (1989). Cadmium. In: Toxicological Evaluation of Certain Food Additives and Contaminants, WHO Food Additives Series No. 24. Prepared by the 33^rd meeting of the Joint FAO/WHO Expert Committee on Food Additives.

4. Testing methodology

Well-planned and documented epidemiological studies have a clear advantage over studies in experimental animals in providing the most relevant information on health effects in humans, thus avoiding extrapolation from experimental animals. However, owing to the lack of adequate epidemiological data for most chemical substances, toxicological studies in animal species play an important role in hazard identification and risk assessment.

In general there are two groups of basically different types of studies in epidemiology. One group includes different types of epidemiological experiments e.g. clinical trials (with patients as subjects), field trials (with healthy subjects) and community intervention trials (with the intervention assigned to groups of healthy subjects). For various reasons these types of experiments are hardly ever used in human toxicology (except for the registration of adverse effects in phase 2 and 3 of clinical trials). In the other group are the non-experimental trials of which there are three primary types: the cross-sectional study, the case-control study and the cohort study.

Cross-sectional study

In the cross-sectional study both the exposure and the effect are measured at the same time in a defined population (all persons in a limited area or a random sample). Because of the lack of longitudinal data this type of studies is often insufficient to elucidate a cause-effect relationship especially if there is a time delay before the effects occur. The advantage is that the data are collected within a limited time resulting in a relatively short time interval between generation of the hypothesis and the answer.

Case-control study

In the case-control study different exposures in two selected groups of people, the cases (the ill) and the controls (the reference group of well persons) are compared. This type of study is relatively simple and economical to carry out. The cases are defined by the investigator (case definition). A classic example of a case-control study was the discovery of the relation between thalidomide and unusual limb defects in babies born in the Federal Republic of Germany in 1959 and 1960; the study, undertaken in 1961, compared affected children with normal children. Of 46 mothers whose babies had typical malformations, 41 had taken thalidomide between the fourth and ninth weeks of pregnancy, whereas none of the 300 control mothers, whose children were normal, had taken the drug at these stages. In a case-control study the association of an exposure and an effect is measured by calculation of the odds ratio, which is the ratio of the odds of exposure among the cases to the odds of exposure among the controls.

Ccohort studies

In cohort studies (follow-up studies) the risk factor (exposure) in a defined group is defined and measured. Then the group is followed over time for development of the disease (or another end- point parameter). The ratio between the risk in the exposed group and the risk in the unexposed group is the Relative Risk (RR). In this kind of studies the entire population is known, which is not always the case in the case-control study. The disadvantage of this type of study is that they are usually very big and expensive. On the other hand they provide the best information about the causation of disease and the most direct measurement of the risk of development of disease.

The quality of the result of epidemiological investigations depends on the validity and accuracy of the data on the exposure as well as the effect. The validity depends on the number of systematic errors when data are collected (bias). The accuracy depends on the number of random errors. The two have a tendency to counterbalance each other. Information- and selection bias give rise to misclassification. Whether the study population is selected in a truly random manner or not is crucial for the results of epidemiological trials. Selection bias (e.g. healthy worker effect) might mask an association between an exposure and a health effect. A small vulnerable group might appear insignificant in a big less vulnerable group (maybe they are already dead due to the exposure) and a small heavily exposed group might appear insignificant in a big less exposed group. A low rate of participation increases the risk for selection bias.

In a study of the association between exposure to a cause (or risk factor) and the occurrence of disease, confounding can occur when another exposure exists in the study population and is associated both with the disease and the exposure being studied. A problem arises if this extraneous factor – itself a determinant or risk factor for the health outcome – is unequally distributed between the exposure subgroups. It could be age, gender, smoking habits, socio-economic status etc. One way of reducing the effect of confounding is by matching cases and controls with regard to the potential confounders.

In case-control studies the controls act as representatives for the background exposure. In order to fulfil this they have to be chosen independently of their exposure and they should have been submitted to the same selection mechanisms as the cases (i.e. as potential cases). When selecting the control group there is a potential for selection bias. Random controls selected from the community often introduce recall bias. They are less prone to recall a certain exposure in the past than cases, resulting in a drift towards a false positive association. Problems of similar kinds arise when using other not randomly selected controls as family, friends or cases with another end-point.

In general, case descriptions are suitable to show a possible effect of an exposure but they are insufficient to evaluate a risk. Case-control studies may be limited by the small study groups with few cases and rare specific exposures. Cohort studies may be limited by the rare occurrence of effects associated with the exposure in question. Hence, the strength of the statistical calculations are often limited in case-control and cohort studies.Traditional epidemiology is often challenged by incorrect information about exposure and effect, resulting in a low sensitivity. This, and a small gap between the exposure in the exposed and the unexposed group, makes it difficult to study the effects of low dose exposures. To get statistically significant results either large study populations, a strong cause-effect relationship or a very rare effect parameter are required. A small study population may in itself be the reason for overlooking a relevant association. This makes it risky to conclude that there is no cause-effect relationship when the study indicates that the exposure-effect relationship is not existing. In reviewing the literature there is an additional risk of publication bias, as both the scientists and the editors are more prone to publish positive correlations than negative results.

In general epidemiological studies has a number of advantages as compared to animal experiments: they study effects in humans, they are often cheaper, they can be based on already existing data, and they can use intellectual and psychological end-points.

Many different experimental methods for investigating toxic effects of chemicals on reproduction and development are in use. Several tests are standardised and guidelines have been issued by various governmental agencies and international organisations, others are still undergoing scientific evaluation. In the following sections, standardised and regulatory accepted methods are discussed concerning procedures and endpoints with emphasis on effects induced during the prenatal and postnatal period. Table 4.2 summarises the tests discussed. In the last section the effects assessed during the prenatal and postnatal period is compared to the possibility for detection of effects in toxicity testing in adults, e.g. in repeated dose toxicity studies.

Other test than those included can reveal effects which indicate a potential of a chemical to interfere with normal reproduction, e.g. the dominant lethal test, fertility assessment by continuous breeding, and repeated dose toxicity testing where the gonads are subjected to pathological examination. These tests, however, provide only information on effects of dosing adult animals.
During recent years many in vitro test systems have been proposed as alternatives to animal testing for developmental toxicity. These tests may be useful for screening of closely related chemicals and for pinpointing mechanisms underlying developmental effects, but they cannot replace animal testing. Consequently, they are not considered in the following sections.

Table 4.2
Overview of in vivo tests for reproductive toxicity testing.

Modified from Hass et al. (1994).

Design considerations in reproductive toxicology studies

When designing experimental in vivo studies in reproductive toxicology some important factors must be considered such as selection of animal species, dose, time of dosing, route of exposure, and housing and handling of the animals.

The animal species usually used for screening studies are rats, mice and rabbits. There are important species differences between humans and rodents especially concerning the timing of brain development in relation to the birth of the offspring. These differences must be taken into account when extrapolating from animal data to the human situation.

The dose levels used in animal studies are often higher than the expected exposure levels in humans. There are several reasons for this. Firstly, the limited number of animals per group limits the sensitivity of the experimental studies; secondly, the human exposure may continue for a long time, even throughout life, while the exposure period in experimental studies is shorter; and thirdly, comparisons of human and animal data seem to indicate that humans are more sensitive than the commonly used laboratory animals when the dose is expressed as mg/kg (Jakobsen & Meyer 1989). The highest dose level should normally be chosen with the aim to induce (slight) general toxicity in adult animals but not death (OECD 1983, OECD 1994).

The time and duration of treatment in reproductive toxicity tests influences the types of effects found. Tests for developmental toxicity are, for example, often restricted to the period of organogenesis, i.e. days 6-15 for mouse and rats, and days 6-18 for rabbits. This period is sensitive to induction of structural malformations while functional effects may be induced at all stages of development. Ideally, treatment in the mouse, rat and rabbit should continue from day 6 of pregnancy through to weaning, though there may be advantages in separating prenatal and postnatal phases in different groups of animals (Barlow & Sullivan 1975).

The route of exposure in studies designed for hazard assessment should ideally be the same as the expected exposure of humans. In the workplaces the main routes of exposure are via inhalation or uptake through the skin. However, oral exposure is often used and recommended in guidelines (OECD 1983, OECD 1994). An important difference between inhalation exposure and e.g. oral exposure is that there is no first-pass effect in the liver for inhaled chemicals, i.e. the chemical in the bloodstream reach the placenta before the liver. Therefore, extrapolation from experimental inhalation studies to inhalation exposure of humans is easier than extrapolation from oral studies. Inhalation studies are, however, not easily performed during the birth period and the early neonatal period. Therefore, inhalation exposure is often postponed a few days before expected birth and only in some cases continued after birth at day 2 or 3.

Standardisation of litter size to 8 pups (culling) was previously commonly used in generation studies, and the procedure is described in the guidelines for generation studies (OECD 1983). An argument in favour of culling is that since pup weight is related to litter size, culling might lead to a more uniform pup weight at weaning. However, in a study of data from approximately 500 litters culling seemed only to increase the mean pup weight in the litters, while the variation remained unaffected (Palmer 1986). Actually culling results in elimination of 25-40% of the offspring and may introduce bias for example by random elimination of runts (Palmer 1986).

Postnatal effects, especially on body weight and behaviour of the pups, may be induced via effect on the mother. For example, lactation or maternal care may be affected and potentially any alteration in maternal physiology and behaviour may in turn influence the offspring. To control for this, cross-fostering techniques may be used where prenatally exposed litters are reared by non-exposed mothers and vice versa. This obviously requires more animals and demands more resources, and is therefore not normally used in screening studies.

Indications of gender differences in response level were among other examples seen in the Collaborative Behavioral Teratology Study (CBTS) in the USA (Kimmel & Buelke-Sam 1985). There were no indications that female behaviour were more variable than male behaviour in the CBTS study. Therefore, there is no reason to exclude females because of possible response variability due to oestrus cycling. Especially in a screening situation, behaviour of both sexes should be monitored (Kimmel & Buelke-Sam 1985).

Housing and handling of animals may influence the results. For example, frequent handling of rats during infancy may alter their physical response to stress and their behaviour in tests for emotionality and learning. To control for environmental influences, the conditions under which the animals are kept must be standardised within experiments with respect to variables such as temperature, humidity, noise level, lighting, cages, handling, and cage cleaning (Barlow & Sullivan 1975).

One-two-, and multigeneration studies

Guidelines for carrying out one-, two-, and multigeneration studies have been published by the US-FDA, OECD (1983), and the EEC, amongst others. In the EEC, the test is requested for new industrial chemicals (i.e. introduced after 1980) with a production volume reaching 100 tons per year.

The purpose of generation studies is to examine successive generations to identify possible increased sensitivity to a chemical, effects on the fertility of male and female animals, pre-, peri-, and postnatal effects on the ovum, foetus and progeny, including teratogenic and mutagenic effects, as well as peri- and postnatal effects on the mother.

In a one-generation study, the test substance is administered in graduated doses to groups of males and females. Males should be dosed during growth and for at least one complete spermatogenic cycle (approx. 56 days in the mouse and 70 days in the rat) in order to elicit any adverse effect on spermatogenesis. Females should be dosed for at least two complete oestrus cycles in order to elicit any adverse effect on oestrus. The test substance should be given continuously during mating and for the females also during pregnancy and the nursing period.

The main idea in two- and multigeneration studies is to incorporate dosing during the time of the organogenesis of the ovaries and testis and to investigate whether a chemical causes increasing toxicity and reproductive problems during subsequent generations. The dose levels and dosing period are similar to the one-generation study but continues until the third generation is weaned. The endpoints assessed in the offspring are viability, growth and histopathology of sex organs, brain and identified target organs. In a revised proposal for the OECD TG 416 Two-generation reproductive toxicity study, assessment of effects on sperm quality and oestrous cyclicity in offspring have been added (OECD 1999).

There are some limitations of generation studies concerning endpoints such as neonatal death and malformations, since the commonly used laboratory animals may eat dead or seriously malformed pups immediately after birth. An effect may, therefore, only be indicated indirectly by a smaller litter size. If only a few pups were malformed or dead, the reduction in litter size will be small compared to the normal variation in litter size and may therefore go undetected or not reach statistical significance. Preimplantation losses and resorptions are indicated in an indirect way as a decreased litter size and the sensitivity for these effects may be rather low.

Birth weight and postnatal growth can be assessed, but it is important to include variations due to different litter sizes or different sex distribution in the litters in the analysis. A change in offspring body weight is a sensitive indicator of developmental toxicity, in part because it is a continuous variable. In some cases, weight reduction in offspring may be the only indicator of developmental toxicity in a generation study. While there is always a question remaining as to whether weight reduction is a permanent or transitory effect, little is known about the long-term consequences of short-term foetal or neonatal weight changes. Therefore, weight reduction should be used to establish the NOAEL (OECD 1989).

Teratology study, prenatal developmental toxicity

This is the most used in vivo method for studying developmental toxicity. Current guidelines include those issued by USFDA, OECD and EEC (Meyer et al. 1989). In the EEC, the test is requested for new industrial chemicals (i.e. introduced after 1980) with a production volume reaching 100 tons per year.

In this protocol, pregnant animals are dosed during the period of organogenesis, since this period is most sensitive to the induction of structural, anatomical malformations. The day before expected birth the foetuses are removed and examined. The main endpoints recorded include resorptions, retarded growth, and visceral and skeletal anomalies.

The teratology test was designed to detect malformations. In the past, there was a tendency to consider only malformations or malformations and death as relevant endpoints in teratology studies. Today it is assumed that all of the four manifestations of developmental toxicity (death, structural abnormalities, growth alterations, and functional deficits) are of concern (OECD 1989). As a consequence, the name of the test is changed to "prenatal developmental toxicity test" and the exposure period is extended to the day before birth in a revised proposal for the OECD guideline 414 (OECD 1999).

The sensitivity of the test for detection of rare events such as malformations is limited, due to the use of a relatively small number of animals. With the normal group sizes of 20 pregnant rats, it is not possible to identify any increase in major malformations unless high dose levels are administered or the substance studied is highly embryo/foetotoxic (Palmer 1981). To assess the developmental toxicity of a chemical, it is therefore important to include information on other developmental effects such as minor anomalies, variations, foetal death and growth. In addition, malformations of organs developing after the period of major organogenesis, e.g. the sex organs and the brain, may at present not be detected in the teratology study. An example is the suspected endocrine disrupter dibutyl phthalate where exposure during the period of male sexual differentiation resulted in major disturbances in the morphological and functional development of the male reproductive system (Mylchreest et al. 1999).

Teratology studies are very suitable for the demonstration of intra-uterine death after implantation (resorptions). In studies where dosing is started before implantation, preimplantation loss may also be assessed.

Foetal weight can be assessed rather exact in a teratology study, but it is important to include variations due to different litter sizes or sex distribution in control vs. exposed groups in the analysis.

Developmental neurotoxicity studies, postnatal studies

A number of chemicals are known to produce developmental neurotoxic effects in humans and other species. A guideline for developmental neurotoxicity study was issued by USEPA in 1991 and a revised US guideline was proposed in 1995 (USEPA 1991). During recent years a proposal for OECD TG 426 Developmental Neurotoxicity Study has been developed based on the US guideline (OECD 1999).

Developmental neurotoxicity studies are designed to develop data on the potential functional and morphological hazards to the nervous system arising in the offspring from exposure of the mother during pregnancy and lactation.

The protocol is designed to be performed as a separate study, however, the observations and measurements can also be incorporated into e.g. a two-generation study.

The evaluation of the offspring consists of observations to detect gross neurological and behavioural abnormalities, assessment of physical development, reflex ontogeny, motor activity, motor and sensory function, and learning and memory; and evaluation of brain weights and neuropathology during postnatal development and adulthood.

The behavioural functions assessed cover many important aspects of the nervous system, however, some functions of relevance for e.g. endocrine disruption such as social interaction and mating behaviour are not included in guidelines at present.

The limitations mentioned in the section on generation studies concerning endpoints such as neonatal death, malformations, preimplantation loss and resorptions apply also to developmental neurotoxicity studies.

Other potentially relevant postnatal endpoints as e.g. kidney function, liver function and immunotoxicity, are not included in guidelines.

Reproduction/developmental toxicity screeing tests

Recently, the OECD introduced guidelines for screening tests for reproductive toxic effects, i.e. the Reproduction/Developmental Toxicity screening tests, as part of the Screening Information Data Sets (SIDS) for high production volume (HPV) chemicals (OECD 1994). The "Combined repeat dose and reproduction/developmental toxicity screening test" is a combination of a 28 days toxicity study and a reduced generation study whereas the "Preliminary reproduction toxicity screening test" is a reduced generation study.

The purpose of the test is to generate limited information concerning the effects of a test substance on male and female reproductive performance such as gonadal function, mating behaviour, conception, development of conceptus and parturition. It is not suggested as an alternative to or as a replacement for the existing test guidelines for generation and teratology studies.

The dosing of the animals is initiated 2 weeks prior to mating and continued until the end of the study on postnatal day 4. The number of animals per group is at least 10 animals of each sex and is expected to provide at least 8 pregnant females per group. Effects on fertility and birth are registered. Live pups are counted and sexed and litters weighed on days 1 and 4 postpartum.

The test does not provide complete information on all aspects of reproduction and development. In particular, it offers only limited means of detecting postnatal manifestations of prenatal exposure or effect induced during postnatal exposure.

The value of a negative study is more limited than data from generation and teratology studies due to the lower number of animals per group, the shorter period of exposure as well as the limited number of endpoints measured.

Repeated dose toxicity testing in adult animals provide information on the potential for systemic toxicity by investigations of growth, clinical symptoms, haematology, biochemistry, organ weights, pathology and histopathology of organs.

Reproductive toxicity testing can provide information on a number of developmental effects, such as malformations, growth retardation, foetal and postnatal death, fertility, and functional effects on the CNS. However, the investigations of systemic effects are not similar to the repeated dose toxicity studies in adults, since haematology and biochemistry is not investigated. In addition, the investigations of organ weight, pathology and histopathology are limited to the brain, sexual organs and identified target organs. Consequently, systemic effects induced during pre- or postnatal development on e.g., liver and kidneys may not be investigated.

In most cases, the effects of chemicals have not been assessed in the two-generation study, the prenatal developmental toxicity study as well as the developmental neurotoxicity study. In these cases, some of the developmental effects mentioned above will not be covered. For example, information on developmental effects on fertility and sex organs are only provided in the two-generation study, while effects on brain development is investigated only in the developmental neurotoxicity study.

In order to have a sufficient background to determine the sensitivity of the developmental period compared to adulthood there is a need for studies where end-points are investigated similarly for both age groups. Ideally, this would require a two-generation study incorporating developmental neurotoxicity end points and supplemented with similar investigations of systemic effects in offspring as in repeated dose toxicity studies.

Guidelines on Clinical Investigation of Medicinal Products in Children have been issued by the European Committee for Proprietary Medicinal Products (CPMP 1997a). According to the guidelines, children should not be given medicines which have not been adequately evaluated for use in that age group. The rationale for this is that adequate evaluation of medicinal products for use in children cannot be achieved in adult studies because there are physiological differences between children and adults, and because children suffer from different diseases from adults, or show a different natural history for the same disease.
Furthermore, the guidelines state the scientific data required before medicinal product testing in children:
When paediatric patients are included in clinical trials, safety data from previous adult human exposure should generally be available before paediatric clinical trials are started.
In all cases, in addition to appropriate repeated dose toxicity studies, all reproductive toxicity studies and the standard battery of genotoxicity tests should be completed prior to the initiation of trials in paediatric populations. Juvenile animal safety studies should be considered on an individual basis.
The need for carcinogenicity testing should be addressed prior to long term exposure in paediatric clinical trials, taking into account the duration of treatment and/or cause for concern.

The ICH Guideline on Non-Clinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals (CPMP 1997b) also points at juvenile animal safety studies when previous animal data and human safety data are insufficient.

The ICH draft Guideline on Clinical Investigation of Medicinal Products in the Paediatric Population (CPMP 1999) provides an outline of critical issues in paediatric drug development and approaches to the safe, efficient, and ethical study of medicinal products in the paediatric population. The specific clinical study issues addressed include considerations when initiating a paediatric program for a medicinal product, timing of initiation of paediatric studies during medicinal product development, types of studies (pharmacokinetic/pharmacodynamic, efficacy, safety), age categories for studies, and ethics of paediatric clinical investigation.

4.4 References

Barlow SM and Sullivan FM (1975). 6. Behavioural teratology. Teratology - trends and applications, Berry CL, Poswillo DE (eds): Springer Verlag, 103-120.

CPMP (1999). Note for Guidance on Clinical Investigation of Medicinal Products in the Paediatric Population. ICH Topic E 11. The European Agency for the Evaluation of Medicinal Products, Human Medicines Evaluation Unit, CPMP/ICH/2711/99.

CPMP (1997a). Note for Guidance on Clinical Investigation of Medicinal Products in Children. The European Agency for the Evaluation of Medicinal Products, Human Medicines Evaluation Unit, CPMP/EWP/462/95.

CPMP (1997b). Note for guidance on Non-Clinical Safety Studies for the Conduct of Human Clinical Trials for Pharmaceuticals. ICH Topic M 3. The European Agency for the Evaluation of Medicinal Products, Human Medicines Evaluation Unit, CPMP/ICH/286/95.

Hass U, Hansen EV and Østergaard G (1994). Experimental studies in laboratory animals. In Hass U et al. (eds) Occupational reproductive toxicity - Methods and testing strategy for hazard assessment of workplace chemicals. Nordic Council of Ministers and National Institute of Occupational Health, Denmark 1994.

Jakobsen BM and Meyer O. Extrapolation from in vivo/in vitro experiments to human beings. In: Andreasen PB, Brandt NJ, Cohr K-H, Hansen EV, Hass U, Hauge M, Jakobsen BM, Knudsen I, Lauritsen JG, Melchior JC, Meldgaard L, Meyer O, Olsen JH, Palludan B, Poulsen E. Embryo- foetal damage and chemical substances. Working party report, Copenhagen: Levnedsmiddelstyrelsen, 1989. 1-127. (Translated version of Danish report from 1986)

Kimmel CA and Buelke-Sam J (1985). Collaborative behavioral teratology study: Background and overview. Neurobehav Toxicol Teratol 7, 541-546.

Meyer O, Jakobsen BM and Hansen EV (1989). Identification of embryo-foetal toxicity by means of animal studies. In: Andreasen PB, Brandt NJ, Cohr K-H, Hansen EV, Hass U, Hauge M, Jakobsen BM, Knudsen I, Lauritsen JG, Melchior JC, Meldgaard L, Meyer O, Olsen JH, Palludan B, Poulsen E. Embryo-foetal damage and chemical substances. Working party report, Copenhagen: Levnedsmiddelstyrelsen, 1989. 1-127. (Translated version of Danish report from 1986).

Mylchreest E, Sar M, Catley RC and Foster PMD (1999). Disruption of androgen-regulated reproductive development by di(n-butyl) phthalate during late gestation in rats is different from flutamide. Toxicol Appl Pharmacol 156, 81-95.

OECD (1999). Guideline for the Testing of Chemicals. TG 414 Prenatal developmental toxicity study (draft proposal); TG 416 Two-generation reproductive toxicity study (draft proposal); Proposal for a new guideline 426. Developmental neurotoxicity study.

OECD (1994). Guideline for the Testing of Chemicals. TG 421. Reproduction/Developmental Toxicity Screening Test; TG 422. Combined Repeat Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test.

OECD (1989). Report on approaches to teratogenicity assessment (draft). Room document no 31.

OECD (1983). Guideline for the Testing of Chemicals. TG 417. One generation reproduction toxicity study. TG 418. Two generation reproduction toxicity study.

Palmer AK (1986). A simpler multigeneration study. International Congress of Pesticide Chemistry 1986 (Abstract).

Palmer AK (1981). Regulatory requirements for reproductive toxicology: theory and practice. In: Developmental toxicology, Kimmel CA and Buelke-Sam J (eds), New York, Raven Press, 259-288.

US-EPA (1991). Guideline 83-6 Developmental neurotoxicity. Washington DC:US Environmental Protection Agency.

5. Specific substances, examples of exposure and effects

This section will illustrate some differences in biological susceptibility and/or exposure between children and adults by giving a brief overview of selected chemical substances for which such differences have been reported in the literature. It should be stressed that the section is only intended for presenting some illustrative cases and therefore by no means should be considered as an exhaustive overview covering all cases and aspects.

5.1 Alcohol

Consumption of alcohol during pregnancy is associated with a great potential for developmental defects in the unborn child.
Alcohol is distributed with body water, and adverse effects in most organs have been reported. The critical target organs are the central nervous system and the liver. (Andrews & Snyder 1991, ILSI 1999).

A distinct dysmorphic condition, the foetal alcohol syndrome (FAS), has been associated with alcoholism in the pregnant mother. The abnormalities most typically associated with FAS include central nervous system dysfunction characterised by mental deficiency and microcephaly, growth deficiencies (both length and weight), a characteristic cluster of facial abnormalities (short palpebral fissures, hypoplastic upper lip with thinned vermilion, diminished or absent philtrum, deficient eye growth, short nose), and variable major and minor malformations (cardio-vascular and skeletal defects).
The critical gestational stage at which the foetus is most vulnerable to the effects of alcohol is not yet known; however, it may be about the time of conception or early pregnancy, when heavy maternal drinking is particularly risky.
The severity appears to be related to the extent of alcohol consumption by the mother during pregnancy. Data are insufficient to state a threshold and no level of maternal drinking can be established as absolutely safe for the foetus. The National Board of Health in Denmark (personal communication), recommends that pregnant women generally should avoid drinking alcohol and maximum 1 glass a day corresponding to around 12 g of alcohol per day.
The pathogenesis of FAS still remains undefined. However, three main, nonexclusive mechanism that may explain the genesis of FAS include impaired placental/foetal blood flow, deranged prostaglandin balance, and direct effects of alcohol (or acetaldehyde, the primary metabolite) on cellular processes.
(Schardein 2000a, Andrews & Snyder 1991, ILSI 1999).

Many studies in laboratory animals clearly demonstrate that alcohol is a potent developmental toxicant inducing growth deficiency, mortality, and malformations similar to those seen in humans. Acetaldehyde, the primary metabolite of ethanol, is teratogenic in mice and induces growth retardation and malformations in rat foetuses similar to those of ethanol when injected into the maternal animal during organogenesis. (Schardein 2000a).
Animal models have provided evidence that foetal alcohol effects are dose-dependent and have clearly demonstrated that the peak maternal blood alcohol concentration and the pattern of drinking are the most important determinants of the magnitude of alcohol-related birth defects. Different aspects of development may be sensitive to different levels of alcohol. (ILSI 1999).

5.2 Tobacco smoke

Suspicions that exposure to tobacco smoke could be hazardous to reproductive function and foetal development date back to early in the present century. It is now generally considered that smoking during pregnancy increases the risk of perinatal mortality, lowers mean birth weight, increases the risk of spontaneous abortion, and has a significant influence of risks of premature delivery, placenta previa, and abruptio placentae. However, it appears that congenital malformations are not associated with smoking. (Schardein 2000b).

Exposure to environmental tobacco smoke (passive smoking) has been reported to be associated with a variety of respiratory disorders in children, including asthma, wheezing, dyspnoea, cough, bronchitis, pneumonia, otitis media, and laryngospasm with anaesthesia induction. Children exposed to passive smoke in the home have more days of school absence, bed confinement, and restricted activity than children in a smoke-free environment. (McCance & Huether 1998).

Tobacco smoke contains more than 3800 different compounds. About 10% of these constitute the particulate phase, which contains nicotine and tar. The remaining 90% contains volatile substances such as carbon monoxide, carbon dioxide, cyanides, various hydrocarbons, aldehydes, and organic acids. Although all of these substances affect the smoker to some degree, nicotine is generally considered to be the primary substance responsible for the pharmacological responses to smoking.
The nicotine content is approximately 3 mg/cigarette smoked in 5 to 10 minutes. Pharmacologically, it is a classic cholinergic agonist initiating nervous stimulation and then blocking cholinergic receptors. By releasing catecholamines, nicotine increases heart rate, oxygen consumption, utilisation of free fatty acids, hypoglycaemia, all of which can be expected to affect foetal development. Nicotine freely crosses the placenta but levels in foetal tissues remain relatively low compared with those of maternal tissues, except in the early stages of development.
(Schardein 1993b, IARC 1986).

Smoking reduces birth weight of offspring and the consensus of over 200 published studies is that smokers’ babies weigh, on the average, 170 to 200 g less at birth than non-smokers’ babies, with about twice as many babies weighing less than 2500 g at birth. Reduced birth weight is primarily due to intrauterine growth retardation, rather than prematurity. The cause of the growth retardation in utero remains highly controversial, but the available published information supports the direct effects of nicotine and carbon monoxide as factors causing intrauterine hypoxia as the most likely mechanism for the effect of smoking on birth weight. There seems to be a dose-response relation as it has been calculated that the decrease in birth weight is about 8 to 9 g for each cigarette smoked daily. Smoking fewer than seven to ten cigarettes per day or early cessation of smoking in pregnancy results in foetal body weights that do not differ significantly from those of nonsmokers.
Increased perinatal or neonatal mortality is also associated with smoking during pregnancy. Perinatal mortality increases 20% for less than one-pack-per-day and 35% for more than one-pack-per-day and in some studies, the risk is more than doubled. The incidence is proportional to the birth weight. Prematurity, anoxia, and placental complications (abruptio placentae and placenta previa) are generally regarded as being responsible for most of this increase.
Spontaneous abortion rates may also be higher in mothers who smoke. The frequency of abortion appears to be directly related to the number of cigarettes smoked with the risk perhaps twofold greater among heavy (more than one pack per day) smokers than in nonsmokers. Heavy smokers also appear to abort earlier in pregnancy.
Data are conflicting concerning a possible causal association between congenital malformations and cigarette smoking. Of 21 studies published since 1970, 11 studies reported positive associations for congenital malformations; cardiac defects, cleft lip and palate, and anencephaly were most often identified as specific malformations associated with smoking. The remaining 10 studies failed to associate smoking in pregnancy and malformations.
The effects of maternal smoking on functional development have not been defined precisely but several reports suggest some causal association between smoking and impaired function. New-borns of smoking mothers have been reported to perform poorer in two operant tasks (head turning and sucking), to be less visually alert, and to have atypical sleep patterns. Childhood hyperkinesis is reportedly more common among children of smoking mothers. Maternal smoking may also have an adverse effect on learning. Recent studies have shown that these types of effects are not limited to offspring of active smokers alone.
(Schardein 2000b).

Some of the developmentally toxic effects observed in humans have been observed in laboratory animals as well. Foetal growth retardation is also a characteristic in rats and rabbits, in the absence of malformations, at human exposure levels. Increased stillbirths have also been observed in rabbits exposed to the equivalent of 20 cigarettes per day during pregnancy. (Schardein 2000b).

5.3 Ambient air pollution

In the past decades the incidence of respiratory allergic diseases in children has increased significantly in the Western world. Current data do not indicate that ambient air pollution attribute significantly to this rise, however, air pollution may cause significant increases in respiratory symptoms and their intensity in persons suffering from asthma and other respiratory diseases.

Children in this respect may be considered as a special risk group as they breathe more air relative to their body weight and lung surface, and thus receive proportionally higher doses of air pollutants systemically and locally. Furthermore children spend more time outdoor, and are more active, resulting in mouth-breathing and increased respiratory rate (see section 2).

Recently Jedrychowski et al. (1999) in a Polish study followed groups of children in two different areas, and over time compared their growth in lung function. They found a significantly greater growth in lung function among the boys from the area with low level air pollution compared to boys from the area with the higher air pollution level. For girls a similar difference did not reach the level of significance. The authors suggested that air pollution may lead to retardation in pulmonary function growth during the pre-adolescent years.

Several epidemiological studies indicate that children are susceptible to ambient air pollution. Increasing levels of ambient ozone levels have been shown to be associated with a decrease in lung function in children (Krzyzanowski et al. 1992, Hoek et al. 1993a, Hoek et al. 1993b, Braun-Fahrländer et al. 1994, Stern et al. 1994, Kinney et al. 1996).

In the study by Krzyzanowski et al. (1992), children together with asthmatics and adults spending long time outdoor were identified as having the most significant decline in lung function in relation to ozone exposure.

Burnett et al. (1994) studied the association between ambient air pollution levels and the number of daily hospital admissions at 168 hospitals in the Ontario area, representing 8.1 million people. Summer respiratory admissions were found to be closely related to ozone levels, and children was found to be especially affected as 15% of the admissions for children were associated with ambient air pollution compared with 4% for the elderly population.

Recently Loomis et al. (1999) and Woodruff et al. (1997) found increased infant mortality associated with ambient levels of fine particles. Loomis et al. (1999) found that a 10 m g/m³ increase in mean level of fine particles during a period of three days was associated with an 6.9% increase in infant mortality. The authors noted that this particle-related excess mortality observed among children was greater in relative risk terms than excess mortality observed among elderly people in several other studies.

In relation to average particle level (long term exposure) Woodruff et al. (1999) compared post-neonatal mortality in areas with different levels of air pollution. They found a significantly increased odds ratio of 1.10 for postneonatal mortality (adjusted for other covariates) in highly polluted areas compared to low pollution areas (mean particle levels of 44.5 µg/m³ and 23.6 µg/m³, respectively). The increased mortality was primarily due to respiratory-related mortality and sudden infant death syndrome. The authors suggested that children may be considered as a susceptible group in relation to particulate air pollution.

5.4 Pesticides

5.4.1 Organophosphates

Organophosphorus insecticides are normally esters, amides, or thiol derivatives of phosphoric, phosphonic, phosphorothioic, or phosphonothioic acids. More than 100 different organophosphorus insecticides are known. Organophosphorus insecticides exert their acute effects in both insects and mammals by inhibiting acetylcholinesterase (AChE) in the nervous system with subsequent accumulation of toxic levels of acetylcholine (ACh), which is a neurotransmitter. Delayed neuropathy is initiated by attack on a nervous tissue esterase distinct from AChE. (WHO 1986a).

Many thousands of cases of acute poisoning by organophosphorus insecticides have been recorded, the majority being due to parathion and methyl parathion (WHO 1986a).
Great differences in response to parathion exposure have been observed among individuals but children are generally more sensitive than adults.
In a number of cases, the oral dose of parathion was known to be exactly 900 mg (around 15 mg/kg b.w.), and it was uniformly fatal. However, in one case the ingestion of 120 mg parathion (around 2 mg/kg b.w.) led rapidly to the death of a man. On the other hand, children (5-6 years old) died after eating 2 mg of parathion (around 0.1 mg/kg b.w.) In instances in which parathion-contaminated food was eaten by people of different ages, death occurred mainly or exclusively among children. Also the epidemiological evidence indicates that parathion is generally more toxic to children than to adults. (Gallo & Lawryk 1991).

The acute toxicity of different organophosphates ranges from highly toxic to only slightly toxic (oral LD₅₀-values for the rat range from less than 1 mg/kg b.w. to over 3000 mg/kg b.w.) (WHO 1986a). Several studies have reported higher sensitivity based on lethality in young animals compared to adults following acute exposure to organophosphorus insecticides. The age-related differences in sensitivity may differ qualitatively and quantitatively with different organophosphates and varying exposure conditions (e.g., high vs. low dose, acute vs. repeated). A study by Liu et al. (1999) showed that repeated exposures to chlorpyrifos were associated with relatively similar degrees of cholinesterase inhibition among the age groups (neonatal, 7 days of age vs. adult, 90 days of age). In contrast, cholinesterase activity and muscarinic receptor binding were generally more reduced in neonatal relative to adult brain regions following repeated exposures to methyl parathion.

Many organophosphorus insecticides are embryotoxic at doses that are toxic for the mother, but only few teratogenic effects have been reported (WHO 1986a).
Parathion is toxic to the foetus causing prenatal and postnatal death of the young and reduced weight gain of the surviving young; no developmental abnormalities have been observed. The greater susceptibility of the foetus is in spite of the fact that inhibition of cholinesterase activity in foetal blood is less than that in maternal blood following dosing of the mother. Weanlings are also more susceptible than adults to parathion due to their poorly developed microsomal enzymes and also greater inherent susceptibility of the young brain. (Gallo & Lawryk 1991).

In reproduction studies of methyl parathion at maternally toxic dose levels (ChE inhibition), no consistent effects on litter size, number of litters, pup survival rates, and lactation performance were observed; no primary teratogenic or embryotoxic effects were noted. (WHO 1993b).
In a toxicokinetic study in rats orally exposed to chlorpyrifos during late gestation (gestational days 14 to 18), the concentration of 3,5,6-trichloro-2-pyridinol (TCP - the only metabolite detected) in the foetal brain was two- to fourfold higher than the concentration in the maternal brain. The concentration of TCP in the maternal liver was approximately fivefold higher than in the foetal liver. Thus, the foetal nervous system may be exposed to a higher concentration of chlorpyrifos than the maternal nervous system when the dam is exposed during late gestation.
Cholinesterase activity levels were determined in liver and brain from maternal and foetal rats. Maternal liver cholinesterase was two-fold more inhibited than foetal liver activity. Similarly, the maternal brain exhibited more cholinesterase inhibition than foetal brain at 5 hours after the last dose of chlorpyrifos. Foetal brain cholinesterase activity recovered to control levels by 24 hours after the last dose, whereas the maternal brain activity was still inhibited (44%) 5 days after the last dose.
(Hunter et al. 1999).

In USA, chlorpyrifos is one of the most commonly used pesticides in the indoor environment today. A recent study (Gurunathan et al. 1998) showed that after a single broadcast spraying of chlorpyrifos in the indoor environment, chlorpyrifos continued to accumulate on children’s toys and hard surfaces 2 weeks after spraying. Based on this study and other research studies, the estimated chlorpyrifos exposure levels for children from indoor spraying are approximately 20 to 120 times above the current recommended reference dose (US-EPA) of 3 g/kg b.w./day for chlorpyrifos exposure to children from all sources. (Davis & Ahmed 1998). In Denmark, pesticides are not used very much in the indoor environment (MST 2000 - personal communication).

5.4.2 Carbamates

A considerable number of reproduction and teratogenicity studies have been carried out with different carbamates and various animal species. Generally, the foetal effects included an increase in mortality, decreased weight gain in the first weeks after birth, and induction of early embryonic death. Certain carbamates also induce teratogenic effects, mainly at high dose levels applied by stomach tube. When the same dose levels was administered with the diet, no effects were seen. (WHO 1986b).

5.4.3 Lindane

Lindane is the g -isomer of 1,2,3,4,5,6-hexachlorocyclohexane (HCH). Lindane interacts with cellular membranes and may produce several generalised cytotoxic effects associated with impaired membrane function. (ATSDR 1997).
Several cases of fatal poisoning and of non-fatal illness caused by lindane have been reported. The toxic or lethal dose appears to vary considerably; under certain conditions, 10 to 20 mg/kg b.w. can represent a lethal hazard to humans, but higher doses can be tolerated when followed by appropriate medication. Some information indicates that children are more sensitive to lindane than adults. (WHO 1991b).
The acute toxicity in experimental animals is moderate (oral LD₅₀-values for rats and mice range from 60 to 250 mg/kg b.w. depending on the vehicle used). Young animals are generally more sensitive than adults. (WHO 1991b).
Lindane had no teratogenic effect in experimental animals after oral or parenteral administration. Foetotoxic and/or maternal toxic effects were observed with doses of 10 mg/kg b.w. and above when given by oral gavage. (WHO 1991b).

5.4.4 Paraquat

Paraquat is the dichloride salt of 1,1’-dimethyl-4,4’-bipyridinium ion.
Following exposure to paraquat, a characteristic lung injury is observed.
A large number of cases of suicidal or accidental poisoning from paraquat has been reported. Among individuals who experience lung damage the mortality rate is high. The minimum lethal dose is stated to be about 35 mg/kg b.w.; however, some individuals have survived after ingesting 10-20 g of paraquat (about 140-280 mg/kg b.w.).
The acute toxicity in experimental animals is moderate (oral LD₅₀-values for rats range from 100 to 200 mg/kg b.w.). Young rats were more resistant to the toxicity of paraquat than older rats; and some authors have paralleled this resistance with that of young rats to oxygen toxicity. One study (Smith & Rose 1977b) found a more than 40% increase in cumulative mortality in 180 g rats compared with 50 g rats, after oral dosing with paraquat at 175 mg/kg b.w.; according to the authors, the difference in renal function between young and mature rats accounted for the difference in paraquat toxicity.
Oral administration of high doses of paraquat to pregnant rats, mice, or rabbits on various days of gestation produced no evidence of teratogenicity, but did produce slight embryotoxicity; however, the doses used induced significant maternal toxicity.
(WHO 1984).

5.5 Drugs

Several drugs are known to induce various adverse effects in children including the unborn child. The classical example is the known human teratogen thalidomide which caused unusual limb defects in babies born of mothers who had taken thalidomide between the fourth and ninth weeks of pregnancy. A few other examples are given here.

5.5.1 Chloramphenicol

Chloramphenicol is a broad-spectrum antibiotic having an antibacterial spectrum and potency very similar to those of the tetracyclines. It is well absorbed from the gastrointestinal tract, metabolised in the liver, and excreted rapidly in the urine predominantly as metabolites. Chloramphenicol crosses the placenta and the concentration in the foetus varies from 30 to 80% of the concentration in maternal blood. Chloramphenicol is also secreted into the milk.
Premature babies and infants (up to one month of age) do not have the detoxifying liver enzyme (glucuronyltransferase) and are therefore extremely sensitive to the serious toxic effects associated with exposure to chloramphenicol. The symptom complex observed in this age group, often referred to as the "grey baby syndrome", consist of gastrointestinal effects (vomiting and abdominal distension), dyspnoea, cyanosis, and vascular collapse.
Because of the serious toxicity such as aplastic anaemia (complete suppression of bone-marrow activity) observed in adults and in foetuses and the "grey baby syndrome" in infants following systemic administration, chloramphenicol is only indicated for the treatment of very serious infections; it is contraindicated for pregnant and lactating women. No data on teratogenic effects in humans are available.
(Lægemiddelkataloget 1996a, Goth 1978).

Chloramphenicol caused closure defects, among other abnormalities, in rats and non-specific malformations in rabbits. The teratogenic activity in the rat was attributed to interference with activity of the electron transport systems and oxidative energy formation in the embryo during embryogenesis. In mice and rhesus monkeys, no teratogenic effects have been noted under the regimens employed. Some postnatal behavioural effects, including reduction in learning ability, have been described in mice following prenatal treatment. (Schardein 2000c).

5.5.2 Sulfonamides

Sulfonamides are derivatives of sulfanilic acid and used for treatment of infections predominantly of the urinary tract. Only one sulfonamide, sulfamethizole, is registered in Denmark.
Sulfonamides are rapidly and extensively absorbed following oral administration, metabolised (acetylation or glucuronidation) in the liver and excreted in the urine predominantly as metabolites. Sulfonamides cross the placenta and are secreted into milk.
The most common adverse effects (dermatitis and drug fever) of sulfonamides are related to acquired hypersensitivity. Serious toxic effects (icterus because of damaged liver cells, aplastic anaemia, neutrocytopenia, acute haemolytic anaemia) are infrequently reported.
Because of the risk of development of icterus in new-borns and young infants (age up to one month), sulfonamides are contraindicated to pregnant women during the last 4 weeks before expected delivery, to lactating women, and to infants (age up to one month).
(Lægemiddelkataloget 1996b).

One study of 458 women taking sulfonamides over the entire pregnancy reported that there were more congenital malformations among their offspring than in the young of untreated controls. Four other studies found no relation to sulfonamide therapy in early pregnancy and malformations. A large collaborative study found no significant malformations associated with the use of specific sulfonamides, including sulfamethizole.
Sulfonamides have shown a mixed teratogenic potential in experimental animals with about one-third indicating activity.
(Schardein 2000d).

5.5.3 Diethylstilboestrol (DES)

DES is an artificial non-steroid oestrogen used as an antineoplastic agent for treatment of prostate cancer. DES is absorbed rapidly following oral administration, metabolised in the liver and excreted in the urine and faeces. (Lægemiddelkataloget 1996c, Schardein 2000e).

DES is a transplacental carcinogen in humans and development of adenocarcinomas in the cervix and vagina have been observed in young females of mothers treated with DES during pregnancy.
DES was apparently thought to be efficacious in the definitive and preventive treatment of abortion and premature delivery. For this reason, it was given to a large number of pregnant women, being approved for use in pregnancy by the US-FDA in 1947. Early in 1970, seven cases of vaginal adnocarcinoma were reported in young women aged 15 to 22 and it was found that the patient’s mothers had ingested oestrogen, DES specifically, in the first trimester of their respective pregnancies many years earlier. In 1971, a registry of clear cell adenocarcinoma of the genital tract in young females was established. By June 1997, 695 cases of clear cell adenocarcinoma were listed in the registry, two-thirds of which
were associated with prenatal DES exposure. In all of the patients who have vaginal and cervical carcinoma, maternal ingestion of DES occurred before the 18^th week of pregnancy and thus, early first-trimester exposure appears to be mandatory in its subsequent toxicity. A peak in the age incidence curve of DES-related cases has been observed at about 19 years, with the age range (latency) being 7 to 30 years.
By far, most of the cases reported have been in the USA, but case stories have also been reported from other countries; however, countries where DES was never used (e.g., Denmark and West Germany) did not have cancer cases.
A mechanism for the vaginal lesions has been theorised. DES may act to sensitise the proliferating stroma of the lower mullerian duct so that it is incapable of fostering upgrowth of urogenital sinus epithelium to spread over and replace the epithelium covering the vagina and cervical protico by 18 weeks when this event should occur. DES may also preferentially affcet the stroma of the developing cervix.
(Schardein 2000e).

5.6 Polychlorinated biphenyls (PCBs)

Because of their high persistence, and their other physical and chemical properties, PCBs are present in the environment all over the world. The general population is exposed to PCBs mainly through contaminated food (aquatic organisms, meat, and dairy products). Infants are exposed through the mother’s milk and it has been estimated that the nursing period contributes about 1.3% of the life-time intake. (WHO 1993a).
In Denmark, an investigation has been performed to determine the content of dioxins and PCBs in the breast milk of Danish mothers. The average concentration of total PCB was 469 ng/g fat corresponding to an average daily intake in the sucking child of 2.4 µg/kg b.w./day. In the report, a NOAEL of 0.33 µg/kg b.w./day was considered for behavioural effects. However, these two figures should not be compared as the TDI relates to a life long exposure. (SST/FDIR 1999).

In general, PCBs appear to be rapidly absorbed, particularly via the gastrointestinal tract after oral exposure; information on the rates of human absorption is limited. PCBs are rapidly cleared from the blood and accumulate in the liver and adipose tissue. There is evidence of placental transport, foetal accumulation, and distribution to milk. (WHO 1993a).

There are great difficulties in assessing human health effects separately for PCBs and polychlorinated dibenzofurans (PCDFs) since, quite frequently, PCB mixtures contain PCDFs.
Therefore, in many cases, it is not clear which effects are attributable to the PCBs themselves and which to the much more toxic PCDFs. Much of the data of the human toxicity come from large episodes of intoxication, e.g., the Yusho and Yu-Cheng episodes. The most striking signs of intoxication in Yusho and Yu-Cheng patients include hypersecretion in the eyes, pigmentation of the nails and mucous membranes, and acneiform eruptions of the skin.
Furthermore, oedema of the extremities, liver enlargement and disorders, central nervous disturbances, respiratory problems, and changes in the immune status were also observed. In children of Yusho and Yu-Cheng patients, diminished growth, dark pigmentation of the skin and mucous membranes, gingival hyperplasia, xenophthalmic oedematous eyes, dentition at birth, abnormal calcification of the skull, rocker bottom heel, and a high incidence of low birth weight were observed. (WHO 1993a).

The Michigan Maternal and Infant Study has reported adverse developmental health outcomes in new-born infants of mothers who consumed more than 12 kg of contaminated Great Lakes fish. Statistically significant decreases in infant’s birth weight, gestational age, and head circumference compared to controls were observed. The infants also exhibited neurodevelopmental and behavioural deficits based on tests of visual recognition and memory at 7 months and 4 years of age. At 11 years of age, many of the neurobehavioural deficits had persisted, e.g., poorer short- and long-term memory and lower IQ scores. (Johnson et al. 1999).

Lesions induced in experimental animals exposed to PCB mixtures or individual congeners concern the liver, skin, immune system, reproductive system, oedema, and disturbances of the gastrointestinal tract and thyroid gland.
The acute toxicity of PCBs is generally low in rats; young animals appear to be more sensitive than adults.
The Rhesus monkey is the most sensitive species with regard to general toxicity and particularly with regard to reproductive toxicity. PCBs adversely affected the reproductive performance of female Rhesus monkeys, mated with control males after 6 months of dietary exposure to a toxic dose (0.09 mg/kg b.w./day) and continuation of the exposure for an average of up to 10 months. Neonates of nursing mothers exposed to PCBs (during gestation and lactation of the first generation) showed adverse effects similar to those seen in their mothers and, in addition, persistent behavioural disturbances as well as several other adverse effects. PCBs may also bind to the cytoplasmic oestrogen receptor. Effects have also been observed on the oestrus cycle of female rats and monkeys, on the sex organs of male rats, and on the implantation rate of fertilised ova following exposure of female mice or male rats.
Available studies in rats and monkeys did not indicate any teratogenic effects (malformations), when animals were dosed orally during organogenesis at doses that produced foetotoxicity and/or maternal toxicity.
(WHO 1993a).

5.7 Polychlorinated dibenzo-p-dioxins (PCDDs)

Consumption of food (including human milk) is the most important pathway for exposure to PCDDs for the general population (adults and children) representing over 90% of the total daily intake. Other pathways include inhalation and direct contact with PCDDs. Exposure of infants and young children may be very high because of their relatively high consumption of milk, including breast milk. (ATSDR 1998).
In Denmark, an investigation has been performed to determine the content of dioxins and PCB in the breast milk of Danish mothers. The average concentration of dioxins + PCB was 30 pg TEQ/g fat (TEQ = 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity equivalents) corresponding to an average daily intake of the sucking child of 150 pg TEQ/kg b.w./ day. A TDI of 1-4 pg TEQ/kg b.w./day (dioxins and dioxin-like PCBs) has been set (WHO 1999) as an average daily intake fo the whole lifetime (SST/FDIR 1999).

Humans can absorb PCDDs by the inhalation, oral, and dermal routes of exposure; absorption is vehicle-dependent and congener-specific. For most mammalian species, the liver and adipose tissue are the major storage sites of PCDDs. Tissue deposition is congener-specific and depends on the dose, the route of administration, and age; 2,3,7,8-substituted PCDDs are the predominant congeners retained in tissues. PCDDs are very slowly metabolised. The major routes of excretion are the bile and the faeces; smaller amounts are excreted via the urine. In mammalian species, lactation is an effective way of elimination PCDDs from the liver and other tissues. Human studies show that infants may absorb up to 95% of the amount ingested via breast milk. (ATSDR 1998).

A wide variety of effects have been observed in adults exposed to 2,3,7,8-TCDD. The primary targets appear to be the skin, liver, thyroid, and cardiovascular, endocrine, and immune systems; an increased cancer risk has also been observed. It is likely that these organs/systems will also be sensitive targets in children. Children exposed to 2,3,7,8-TCDD appear to be more sensitive than adults to the dermal effects (chloracne). A number of human studies have investigated the potential of 2,3,7,8-TCDD to induce developmental effects. In one study, no significant increases in the incidence of birth defects have been observed in the children of parents living in Seveso at the time of the accident or during the next 6-year period. In contrast, other studies have found increases in specific types of defects, although the total number of defects was not significantly altered. (ATSDR 1998).
In animal oral toxicity studies, toxic effects of PCDDs have been observed in most organs/systems. The studies clearly demonstrate that the developing organism is very susceptible to the toxicity of PCDDs, in particular 2,3,7,8-TCDD. Prenatal or perinatal exposure has resulted in structural malformations (e.g., cleft palate, hydronephrosis), functional alterations (e.g., damage to the immune system, impaired development of the reproductive system), decreased growth, and foetal/new-born mortality in several animal species. The organ system most sensitive during development is the reproductive system (alterations in androgen levels, secondary sex organs, spermatogenesis, fertility, and sexual behaviours). Also neurobehavioral effects are observed. Additionally, several animal studies provide evidence that exposure to 2,3,7,8-TCDD via mother’s milk alone can adversely affect the developing animal. (ATSDR 1998).

5.8 Polybrominated diphenyl ethers (PBDEs)

PBDEs are widely used in a variety of materials and products including textiles, many types of electronic devices, cabins for and circuit boards in personal computers and TV sets, electrical cables, switches and capacitors, and building materials. Furthermore, PBDEs are found in several foods of animal origin (fish, meat, and cow’s milk) (Darnerud et al. 1998, WHO 1994, WHO 1997b).
In Swedish human milk samples, the concentration of PBDEs has increased continuously with levels showing an exponential increase from 1972 and a doubling time of 5 years (Norén & Meironyté 1998).

Studies on the reproductive toxicity of PBDEs are limited. Only one study on fertility (decaBDE in rats) is available; no treatment-related effects in reproductive performance or maturation of pups were reported.
The developmental toxicity studies available on decaBDE, octaBDE, and pentaBDE are equivocal. Foetotoxicity (resorptions, delayed ossification) but no malformations have been observed in rats exposed to decaBDE (one study) and octaBDE (two studies) and in rabbits exposed to octaBDE (one study) at dose levels which did not induce maternal toxicity. In contrast to these findings, foetotoxicity was observed in rats exposed to pentaBDE (one study) only in the presence of maternal toxicity. (WHO 1994, Darnerud et al. 1998).

A very recent study investigating possible neurobehavioural effects in neonatal mice (10 days old) following a single exposure to pentaBDE suggests differences in behavioural patterns between treated and control mice (Eriksson et al. 1998).

5.9 Phthalates

Phthalates are high production volume chemicals widely used as additives in PVC plastics. Due to the ubiquitous use, phthalates are found everywhere in the environment.

In Denmark, the intake of the phthalates DEHP, DBP, and BBP has been analysed in a number of samples (29) from a double portion study where adults participated; estimates of the mean and maximum intake were 0.13-0.29, 0.02-0.03, and 0.19-0.3 mg/person/day for DBP, BBP, and DEHP, respectively. For children, the mean and maximum intake of phthalates from infant formulae has been estimated to <0.042, 0.0006-0.0009, and 0.009-0.021 mg/child/day (assumed child weight of 3 kg) for DBP, BBP, and DEHP, respectively. (Petersen 1999).

Some phthalates affect fertility and reproduction in rodents of both sexes and also produce developmental effects in the offspring.

Generally, phthalates with side chains of 4 to 6 carbons atoms in length, e.g., di-2-ethylhexyl phthalate (DEHP), dibutyl phthalate (DBP), and butyl benzyl phthalate (BBP), affect the reproductive system of male rodents whilst phthalates with side chains shorter than 4 carbon atoms and longer than 6 carbon atoms appear to be without effect. Observed effects include marked reductions in the weights of the testes and accessory sex glands, decreased numbers of spermatocytes, degeneration of the seminiferous tubules, a reduction in testicular zinc and iron levels and serum testosterone levels, an increase in testosterone levels in the testes, sloughing of germ cells, and vacuolisation of Sertoli cells (DEHP). Spe cies differences have been observed as the reproductive system of the male rat appears to be more sensitive than that of the mouse, which appears to be more sensitive than that of the hamster, guinea pig, and non-human primates.
The effects on the male reproductive system are influenced by the age at which the animal is exposed. Studies of DEHP have shown that developing and sexually immature male rats are more sensitive to DEHP-induced testicular toxicity than sexually mature rats and that the onset of the effects in young animals is more rapid. Furthermore, exposure (DEHP, DBP) of rats prenatally and during suckling has produced irreversible testicular damage at dose levels inducing only minimal effects in adult animals. (CSTEE 1998, WHO 1997, Nielsen & Larsen 1996, Mylchreest & Foster 1998).

Numerous studies have shown that some phthalates induce embryotoxic and teratogenic effects in the offspring. DEHP is embryotoxic and teratogenic in mice and embryotoxic in rats at maternally non-toxic dose levels (CSTEE 1998, Nielsen & Larsen 1996). DBP generally induce foetotoxic effects in rats and mice in the absence of maternal toxicity, and teratogenic effects only at high maternally toxic doses (WHO 1997a).
In very recent studies in rats exposed to DBP during the prenatal and early neonatal periods, a number of effects were seen in the male offspring, including decreased anogenital distance, absent or underdeveloped epididymis and seminal vesicles, cleft phallus (hypospadias), decreased reproductive organ weights, and widespread germ cell loss in the testis. In contrast, vaginal opening, age at first oestrus, and oestrous cyclicity were not affected in the female offspring indicating that DBP is not oestrogenic but rather antiandrogenic. (Mylchreest & Foster 1998).

5.10 Lead

In the non-smoking adult general population, the major exposure pathway for lead is from food and water. The level of dietary exposure to lead depends upon many factors, including foodstuffs consumed, processing technology, use of lead solder, lead levels in water, and use of lead-glazed ceramics.
For infants and young children, food, air, water and dust/soil are the major potential exposure pathways. For infants up to 4 or 5 months of age, air, breast milk, formulae and water are the significant sources of lead exposure. For infants and young children, lead in dust and soil often constitutes a major exposure pathway. Lead levels in dust depend upon such factors as the age and condition of housing, the use of lead-based paints, lead in petrol and urban density. The intake of lead will be influenced by the age and behavioural characteristics of the child and the bioavailability of lead in the source material. (WHO 1995).
A recent Swedish study (Berglund et al. in press - quoted from CSTEE 2000) has shown that food is now the main source of lead exposure even in young children living in areas with high soil lead concentrations, i.e. downtown Stockholm (<10-330 mg/kg in soil) and mining areas (20-5000 mg/kg). It was concluded that lead in soil and dust contributed little to the total intake of lead.

In humans, lead adversely affects several organ systems and organs, including the nervous, haematopoietic, reproductive, and cardiovascular systems, the liver, the kidney, and the gastrointestinal tract. Neurodevelopmental effects and subcellular changes, particularly the effects on haem synthesis, appear to be the most sensitive endpoints. (WHO 1995).
Cognitive and sensory motor deficits have been shown in children to be associated with blood lead levels as low as 100 to 150 µg/l. An average IQ decrement between 1 to 3 points with increasing blood lead levels from 100 to 200 µg/l correspons to 20% or less of the standard deviation of a typical IQ distribution. These and more recent data indicate, that even below 100 µg/l effects might occur and that no clear threshold for effects has been identified. (CSTEE 2000).

Children, in comparison with adults, are more susceptible to lead in several respects. Children have a greater absorption of ingested lead than adults, resulting in a higher body burden from a given external exposure. About 40 to 50% of dietary lead is absorbed from the gastro-intestinal tract in infants and young children compared to around 5 to 10% in adults. Absorption of lead from ingested dust and soil is somewhat lower than from food, approximately 30% in infants and young children. It also appears that children are generally more sensitive to the toxicological effects of lead at a given internal exposure level (measured as the blood lead level) as the lowest observed effect levels (LOAELs) for various end-points (e.g. slowed nerve conduction velocity, impaired neurobehavioural function, encephalopathy, anaemia, reduced haemoglobin) are lower in children than in adults. (WHO 1995, WHO 1996).

5.11 Mercury

The general population is primarily exposed to inorganic mercury and methyl mercury through the diet. In most foodstuffs, mercury is largely in the inorganic form. Fish and fish products are the dominant source of methyl mercury in the diet. Air and water can also contribute significantly to the total daily intake of total mercury. Furthermore, dental amalgam may also be a source of exposure to inorganic mercury due to release from amalgam restorations. (WHO 1990, WHO 1991a).
No specific information was found concerning exposure of infants and children.

Methyl mercury is a well-established neurotoxicant that can cause serious adverse effects on the development and function of the human central nervous system, especially when exposure occurs prenatally (Harada 1995). The neurotoxic potential was first described from industrial exposures as the Hunter-Russell syndrome, and then reappeared in the fishing town of Minamata, Japan, in the early 1950s (Igata 1993). Most surprisingly, while unaffected themselves by mercury toxicity, many pregnant women exposed to mercury-contaminated fish bore infants that suffered from severe congenital poisoning (Harada 1995, Igata 1993). The characteristics of this form of developmental neurotoxicity are now relatively well known at high exposure levels, where a cerebral palsy syndrome occurs. In less severe poisoning, blindness, deafness, and mental retardation may be apparent. In a poisoning incident in Iraq, a dose-response relationship was established between maternal hair-mercury concentrations during pregnancy and the prevalence of severe psychomotor retardation in the children (Marsh et al. 1990). This evidence from poisoning outbreaks clearly documents the hypersusceptibility of the developing nervous system with regard to this neurotoxicant.

Current concerns relate to the neurotoxic risks at lower exposure levels prevalent in fishing communities (Grandjean et al. 1997). A birth cohort of 1000 Faroese children was examined at age 7 years, where clinical examination did not reveal any clear-cut abnormalities associated with the cord-blood mercury concentrations. However, mercury-related neuropsychological deficits at this age occurred in the domains of language, attention, and memory, and to a lesser extent in visuospatial and motor functions. The associations could not to be explained by various possible confounders such as polychlorinated biphenyls (PCBs) from seafood, and they remained after exclusion of highly-exposed children with a maternal hair-mercury concentrations above 10 µg/g. This limit was thought to represent an upper safe level as based on the data from Iraq (WHO 1990).

Supporting evidence has now emerged from a fishing community in Madeira, where 149 children from the first grade in school showed mercury-related delays in the electrical signals of the brain, as recorded by the evoked potentials technique (Murata et al. 1999). A similar pattern was seen in the Faeroes (Grandjean et al. 1997). Other cross-sectional studies in Brazil (Grandjean et al. 1999) and French Guyana (Cordier et al. 1999), have shown mercury-associated developmental effects in agreement with the Faroese findings. However, a prospective study in the Seychelles has not revealed any clear adverse effects related to maternal hair-mercury concentrations, but results beyond 5 years of age are not yet available (Davidson et al. 1998).

Although the question as to the safety of fish consumption during pregnancy has not been settled, the preponderance of evidence indicates that the unborn child is much more susceptible to methyl mercury neurotoxicity than adults are. This substantial age-dependency may be a more general phenomenon for neurotoxicants, as similar evidence is available for other substances, especially lead and PCBs (Steuerwald et al. 2000).

5.12 Copper

In the general population, the major route of exposure to copper is oral. Variations in dietary copper intake reflects different dietary habits as well as different agricultural and food processing practices used world-wide. In some cases, drinking water may make a substantial additional contribution to the total daily intake of copper, particularly in households where corrosive waters have stood in copper pipes. All other intake for copper (inhalation and dermal) are insignificant in comparison to the oral route. Women using copper-containing intra uterine devices (IUDs) are exposed to only minor amounts from this source. (WHO 1998a).

Copper is an essential element and adverse health effects are related to deficiency as well as to excess intake. The relationship between intake and risk has a U-shaped curved, with risk for deficiency associated with low intakes and risk for toxicity associated with high intakes. The range of acceptable intakes which meet the biological requirement without causing toxicity may be rather narrow.
New-borns appear to absorb copper more readily than adults whereas the aged appear to absorb copper less efficiently. Copper can cross the placental barrier and is taken up by the foetus. The copper concentration in the liver of new-borns is 6-10 times higher than in adults but decreases during the first 3 months of life.
The concentration of copper in the body is kept relatively constant by homeostatic mechanisms, and toxic effects of long-term ingestion of excess copper are not frequently observed in the general population. However, children below one year of age are probably more susceptible than adults to copper toxicity because the homeostatic mechanisms may not have fully developed. A number of cases (some of them fatal) in which development of liver damage (early childhood cirrhosis) has been related to excess intake of copper from drinking water has been described.
(Nielsen 1997).

5.13 Boric acid

The most frequent and appreciable general population exposures to boron are likely to be from ingestion of food and, to a lesser extent, from ingestion of drinking water. Other potential sources include absorption of boron from cosmetic and medical preparations through mucous membranes or damaged skin; and the inhalation, dermal absorption, or accidental ingestion of boron-containing household cleaning products, pesticides, or fertilisers. (WHO 1998b).

Boric acid is readily absorbed from the gastrointestinal and respiratory tracts. The absorption is essentially complete (approximately 95% in humans) following ingestion. Dermal absorption across intact skin is negligible in all species evaluated, including humans (infants and adults). Boric acid is rapidly excreted mainly (95%) by the kidneys. (WHO 1998b).

Only a few human studies have been conducted to assess health effects associated with exposure to boron compounds, including boric acid.
Fatalities among young children have resulted from skin absorption of boric acid used as dusting powder on diapers; in 120 reported cases of poisoning, the mortality was 52.5% (Deichmann & Gerade 1969).
Accidental use of boric acid solution in the preparation of baby formula has resulted in poisoning in infants and a lethal dose of 2 to 3 g has been reported. Based on the reported lethal doses, which are not well documented in the literature, infants appear to be more sensitive than adults to boron compounds. (WHO 1998b).

After repeated oral administration to experimental animals, growth inhibition, organ weight changes, and testicular damage are the most striking effects observed.
The testis is the critical target organ with adverse effects being observed ranging from inhibited spermiation to degeneration of the seminiferous tubules with variable loss of germ cells, to complete absence of germ cells resulting in atrophy and transient or irreversible loss of fertility but not of mating behaviour.
Developmental toxicity has also been demonstrated in experimental animals with lower foetal bodyweight being the critical effect
(WHO 1998b).

5.14 Nitrate and nitrite

Nitrate and nitrite are naturally occurring ions that are part of the nitrogen cycle. The nitrate ion is the stable form for oxygenated systems although it can be reduced to nitrite by microbial action.
In general, vegetables will be the main source of nitrate intake in the general population. When the nitrate concentration in drinking water exceeds 50 mg/l (the limit value in Denmark), drinking water will be the major source of total nitrate intake, especially for bottle-fed infants. For the bottle-fed infant, daily intake from formula made with water containing 50 mg/l of nitrate would average about 8.5 mg/kg b.w. per day.
(WHO 1996).

Ingested nitrate is readily and completely absorbed from the gastrointestinal tract and rapidly distributed throughout the tissues. Approximately 25% of ingested nitrate is actively secreted into saliva, where it is reduced to nitrite by the oral microflora. Bacterial reduction of nitrate may also take place in other parts of the human gastrointestinal tract. (WHO 1996).

The toxicity of nitrate to humans is thought to be solely the consequence of its reduction to nitrite. The major biological effect of nitrite in humans is its involvement in the oxidation of normal haemoglobin to methaemoglobin, which is unable to transport oxygen to the tissues. The reduced oxygen transport becomes clinically manifest when methaemoglobin concentrations reach 10% of that of haemoglobin and above; the condition, called methaemoglobinaemia and also known as the "blue baby syndrome", causes cyanosis and, at higher concentrations, asphyxia. The haemoglobin of new-borns and young infants is more susceptible to methaemoglobin formation than that of older children and adults. (WHO 1996).

5.15 References

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ATSDR (1997). Toxicological Profile for alpha-, beta-, gamma- and delta-hexachlorocyclohexane. Draft for Public Comment. U.S. Department of Health & Human Services, Public Health Service, Agency for Toxic Substances and Disease Registry.

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CSTEE (1998). CSTEE opinion on phthalate migration from soft PVC toys and childcare articles. The Scientific Committee on Toxicity, Ecotoxicity and the Environment (CSTEE) of DG XXIV, Consumer Policy and Consumer Health Protection. November 1998.

Davidson PW, Myers GJ, Cox C, Axtell C, Shamlaye C, Sloane- Reeves J, Cernichiari E, Needham L, Choi A, Wang Y, Berlin M and Clarkson TW (1998). Effects of prenatal and postnatal methylmercury exposure from fish consumption on neurodevelopment. JAMA 280, 701-707.

Davis DL and Ahmed AK (1998). Exposures from indoor spraying of chlorpyrifos pose greater health risks to children than currently estimated. Environ Health Perspect 106, 299-301.

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Hoek G, Brunekreef B, Kosterink P, van den Berg R and Hofschreuder P (1993a). Effect of ambient ozone on peak expiratory flow of exercising children in the Netherlands. Archiv Environ Health 48, 27-32.

Hoek G, Fischer P, Brunekreef B, Lebret E, Hofschreuder and Mennen MG (1993b). Acute effects of ambient ozone on pulmonary function of children in the Netherlands. Amer Rev Resp Diseases 147, 111-117.

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IARC (1986). Tobacco smoking. IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans. Volume 38. IARC, Lyon, France.

Igata A (1993). Epidemiological and clinical features of Minamata disease. Environ Res 63, 157-169.

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Jedrychowski W, Flak E and Mróz E (1999). The adverse effect of low levels of ambient air pollutants on lung function growth in preadolescent children. Environ Health Perspect 107, 669-674.

Johnson BL, Hicks HE and De Rosa CT (1999). Key environmental human health issues in the Great lakes and St. Lawrence River Basins. Environ Res Section A 80, S2-S12.

Kinney PL, Thurston GD and Raizenne M (1996). The effects of ambient ozone on lung function in children: A reanalysis of six summer camp studies. Environ Health Perspect 104, 170-174.

Krzyzanowski M, Quackenboss JJ and Lebowitz MD (1992). Relation of peak expiratory flow rates and symptoms to ambient ozone. Arch Environ Health 47, 107-115.

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Lægemiddelkataloget (1996a). Chloramphenicol, 264. In Danish.

Lægemiddelkataloget (1996b). Sulfonamides, 276-277. In Danish.

Lægemiddelkataloget (1996c). Diethylstilboestrol, 569; oestrogens, 216-217. In Danish.

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Nielsen E and Larsen PB (1996). Toxicological evaluation and limit values for DEHP and phthalates other than DEHP. Environmental Review No. 6 1996, Danish Environmental Protection Agency.

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6. Regulations

Chemical substances are present around us and consequently we are all exposed to a number of such substances e.g., via the environment, food and beverages, cosmetics, and through a number of different products which many of us use daily. Additionally, adults and young people may be exposed to chemical substances via the working environment and children by contact with toys and childcare products (see section 3).

In order to assure the best possible protection against adverse effects resulting from exposure to chemical substances, several regulations within the different areas have been issued. This part of the report briefly summarises the most relevant Danish regulations on chemical substances with focus on the protection of children and pregnant women, including their unborn child, exposed to chemical substances.

It should be noticed that the regulations of chemical substances are based on the existing knowledge with regard to adverse health effects and risks related to the use of the substances. It must be emphasised, that for the great majority of substances used, very little - if any - knowledge exists regarding the relevant exposure scenarios and/or vulnerability of children, including the unborn child.

6.1 Environmental contamination

In Denmark, health based limit values (quality criteria) are set for chemical substances in soil, drinking water and ambient air according to the principles laid down in the Guideline for health based evaluation of chemical substances in drinking water (MST 1992a) and in Appendix A of the Industrial Air Pollution Control Guidelines (MST 1992b). The principles as they are used today will briefly be outlined here:

When the available toxicological data have been evaluated, the hazard considered most important, "the critical effect", for setting the quality criteria for a given substance is identified. Generally, the effects are considered to be of more concern the lower the concentration or dose at which they occur, and the effect observed at the lowest concentration or dose level often forms the basis for the calculation of a tolerable concentration or tolerable daily intake (TDI).
In doing this, a "no observed adverse effect level" (NOAEL) or when this is not possible a "lowest observed adverse effect level" (LOAEL) is identified. Having identified a NOAEL (or a LOAEL), three safety factors (uncertainty factors) SF_I, SF_II, and SF_III are used for the calculation of the tolerable concentration or TDI. SF_I accounts for interspecies variation in susceptibility and SF_III accounts for the quality and relevance of the data. SF_II accounts for differences in intraspecies susceptibility, i.e. this safety factor is used for the protection of groups in the population that may be more susceptible than the general population such as children, pregnant women and their unborn child, elderly or sick people; in general a default value of 10 is used for SF_II.

Following calculation of the tolerable concentration or TDI, the health based limit values are derived for the relevant media by division of the value for the tolerable concentration or TDI by a given standard exposure level of the relevant media. For derivation of the limit value for drinking water and ambient air, a daily standard exposure for an adult person is used. Thus, no specific attention is normally given to exposure of children. For soil exposure, however, the standard exposure used for calculation of the limit value is a standard exposure for soil exposure of children (values for daily soil ingestion or soil in contact with skin), as exposure to soil is considered especially important (and critical) for children. To ensure that the total daily intake for various media does not exceed the tolerable concentration or TDI, a certain percentage (often 10%) of the tolerable daily exposure level can be assigned to the relevant media and in this way account for other important exposure routes such as exposure through the food.

6.2 Chemical substances and products

Classification and labelling

Within the EU, regulations on classification and labelling of chemical substances are based on total harmonised directives (EEC 1967, with amendments). In Denmark, the Statutory Order on classification, packaging, labelling, sale and storage of chemical substances and preparations (MEM 1997) implements the relevant EU Council Directives into national legislation. The Statutory Order provides the regulations for evaluating the inherent properties of a chemical substance or preparation in relation to hazard identification and assessment.
The hazard identification and assessment cover a number of important toxicological endpoints such as acute toxicity, irritative and corrosive properties, sensibilisation, repeated dose toxicity, reproductive and developmental effects, and genotoxic and carcinogenic properties. Chemical substances considered dangerous according to the regulations are classified and labelled with symbols and indications of danger, nature of special risks (R-phrases) and safety advises (S-phrases).

Classification is normally based on results from specific animal experiments according to EU guidelines (Annex V to EEC 1967, with amendments). In most of these studies, no specific attention is paid to whether specific age groups could be identified as sensitive subgroups.

A number of R- and S-phrases are, however, specifically related to protection of children and/or pregnant women:

Chemical substances which are classified as reproductive toxicants in category 1 (substances known to impair fertility or to cause developmental toxicity in humans) and category 2 (substances which should be regarded as if they impair fertility or cause developmental toxicity in humans) are labelled with R60 "may impair fertility" or R61 "may cause harm to the unborn child", respectively.

Chemical substances which are classified as reproductive toxicants in category 3 (substances which cause concern for human fertility or for humans owing to possible developmental toxic effects) are labelled with R62 "possible risk of impaired fertility" or R63 "possible risk of harm to the unborn child", respectively.

Chemical substances which are known or suspected to be secreted into the breast milk at toxic levels are labelled R64 "may cause harm to breastfed babies".

The S-phrase S2 "keep out of the reach of children" is assigned to every classified chemical substance or preparation.

Packaging, sale and storage

The Statutory Order on classification, packaging, labelling, sale and storage of chemical substances and preparations (MEM 1997) also include regulations which ensure that children will not come into contact with most of the dangerous chemicals.
Chemical substances and preparations which are classified as carcinogenic, mutagenic, or reproductive or developmental toxicants in category 1 and 2 must not be sold to the general public.
Chemical substances and preparations which are classified as "very toxic" (Tx) and "toxic" (T) can only be sold to individuals of the general public who have a special permission from the police.
Chemical substances and preparations must not be marketed in containers which are considered to attract children’s attention. For certain classified chemical substances and preparations, special closures (child-resistant fastenings) should be used for the containers and such products should be stored out of the reach of children.

Risk assessment in EU and OECD

Within the EU and OECD, specific programs on risk assessment for chemical substances are on-going. In the EU, a directive (EEC 1993a) provides the regulation on risk assessment of new notified chemical substances and two Council regulations (EEC 1993b, EEC 1994a) on risk assessment of existing substances. Denmark takes part in the risk assessment programs according to the EU regulation, but no specific regulation have been issued in Denmark to implement this regulation.
The risk assessment process, in relation to both human health and the environment, entails a sequence of actions: effect assessment (hazard identification and dose (concentration) - response (effects) assessment), exposure assessment (estimation of the concentrations/doses to which human populations or environmenal compartments are or may be exposed) and risk characterisation (estimation of the incidence and severity of the adverse effects likely to occur in a human population or environmental compartment due to actual or predicted exposure to a substance). The risk characterisation is carried out separately for three subgroups of the population: workers, consumers, and man exposed indirectly via the environment; only the last two subgroups include children. In the risk characterisation for consumers and man exposed indirectly via the environment, the interspecies variation (see 6.1) is taken into account when considering the margin of safety (the ratio between the NOAEL or LOAEL and the estimated exposure).
For some specific substances (e.g., phthalates), children have been considered as a specific subgroup of consumers as children may be exposed to these substances in a way which is not considered relevant for adults.

No specific EU or OECD guidelines for risk assessment for children and pregnant women and their unborn child have been issued.

Assessment based on available data

Classification and risk assessment are based on available data. In general, the available data on a specific chemical substance seldom allow for an evaluation with respect to whether children or the unborn child should be considered more susceptible than adults. Concerning fertility and teratogenicity, data exist only for 20-30 % of the 2500 high production volume chemicals, and for thousands of chemicals produced in smaller quantities this percentage is expected to be considerably lower (Danish Board of Technology 1996).

Testing of chemical substances new chemical substances

For new notified chemical substances (substances which were not on the market within the EU at any time in the 10 years prior to 18 September 1981), step-wise, tonnage related data requirements have been issued. The number of tests required is dependant on the supply level. The results of one study should generally be evaluated before another study is initiated.

base-set testing

Base-set testing is demanded for a new substance produced or imported in amounts of 1-10 tonnes/year per producer or importer in an EU member state. No specific tests to detect adverse effects on reproduction are required. However, it is indicated that there is a requirement at this level to "screen" new substances for their potential to cause reproductive toxicity.

level 1 testing

Level 1 testing is additional to the base-set testing and includes a fertility study (one species, one generation, males and females, most appropriate route of administration) and a teratogenicity study (one species, most appropriate route of administration). Level 1 testing may be required when supply levels reach 10 tonnes/year per producer or importer (or 50 tonnes/year cumulative per producer or importer in the EU) and shall be required at supply levels of 100 tonnes/year (500 tonnes cumulative) unless the notifier can give good reason why a particular test is not appropriate or an alternative scientific study would be preferable.

level 2 testing

Level 2 testing is required for high production volume chemicals, i.e. when supply levels reach 1000 tonnes/year (5000 tonnes cumulative) and includes fertility study (three-generation study; only if an effect on fertility has been established at Level 1), developmental toxicity study on peri- and post-natal effects, and teratogenicity study (species not employed in the respective Level 1 study).

less than 1 tonne/year

For new substances produced in amounts less than 1 tonne/year, no tests on reproductive toxicity are required.

existing chemical substances

For existing high production volume chemical substances (substances which were on the market within the EU at any time in the 10 years prior to 18 September 1981 and produced in amounts above 1000 tonnes/year), the data to be made available shall at least include the base-set required for new substances. Any gaps in the base-set data should be filled, unless the producers or importers can justify not providing it.

6.3 Cosmetics

The use of cosmetics by different age groups, including infants and children, has increased during the last years. Consequently, exposure to chemical substances via cosmetics is increasing and it has been suggested that this is a contributing factor for the increased incidence of allergy observed among the general population. Illustrative examples of the increasing use for infants and children include bath and shower preparations, suncare products, wet tissues, hair care products (including colours and bleaches), and special cosmetics intended for children.

Within the EU, regulations on cosmetics are based on totally harmonised directives (EEC 1976a, with amendments). In Denmark, the Statutory Order on cosmetic products (MEM 1998a) implements the relevant EU Council Directives into national legislation. The regulation on exposure to chemical substances in cosmetic products is built up of different lists:

Annex 1 lists the different cosmetic products which are regulated.

Annex 2 lists the chemical substances which are not allowed intentionally as ingredients in cosmetic products.

Annex 3 lists the chemical substances to be used in cosmetic products according to specific limitations.

Annex 4-6 are positive lists for pigments, preservatives and UV-filters, respectively.

In order to protect children against exposure to specific chemical substances in cosmetics, a few specific substances have been banned from cosmetic products intended for children below 3 or 12 years of age.

The evaluations of chemical substances to be used in cosmetic products are discussed in the EU Scientific Committee on Cosmetics. In the Committee’s evaluation of a specific substance, the safety margin for expected daily use is estimated; however, no specific considerations are taken regarding the use of the substance in products which will be used for children.

6.4 Toys and childcare products

Within the EU, regulations on toys are based on total harmonised directives. The Statutory Order on safety regarding toys and products which can be mistaken for foodstuffs (FST 1995) implements the EU Council Directives (EEC 1987, EEC 1988a, with amendments) into national legislation. According to these regulations, toys may generally not contain dangerous chemical substances (those substances which are classified according to the EU Council Directive (EEC 1967) on classification, packaging and labelling of chemical substances and preparations) in such amounts, which will give rise to development of adverse effects in children. Furthermore, specific migration limits have been set for 8 metals (antimony, arsenic, barium, cadmium, chromium, lead, mercury, selenium). It should be noted, however, that a safe migration limit for a substance in a specific toy only can be set on the basis of a realistic worst-case exposure scenario with respect to toy exposure. The scenarios may differ considerably for different kind of toys and the possibility for exposure during use (and misuse).
Denmark has prohibited the use of phthalates in toys and childcare products for children of the age of 0 to 3 years (MEM 1999).
EU has adopted measures prohibiting the placing on the market of toys and childcare articles intended to be placed in the mouth by children under three years of age made of soft PVC containing one or more of 6 specific phthalates (DINP, DEHP, DBP, DIDP, DNOP, BBP) (EU 1999).

Several other national regulations have been issued in order to protect children from exposure to chemicals through their use of toys.

6.5 Food additives, pesticides and contaminants in food

6.5.1 Food additives

In Denmark, food additives are regulated by the Statutory Order on food additives (FM 1997) which implements the relevant EU Council Directives (EEC 1978, EEC 1988b, with amendments) into national legislation. The use and use levels of permitted food additives are given in "Positivlisten" (VFD 1997). The establishment of the use levels is based on the ADI (acceptable daily intake) which is estimated from the "no observed adverse effect level" (NOAEL) considered from evaluations of studies on experimental animals or, in cases where a NOAEL cannot be identified, the "lowest observed adverse effect level" (LOAEL), by applying a safety factor of normally 100. This safety factor is considered to take into account the intraspecies variation, i.e. to take into account that the observed effects may vary in severity within the population, thereby protecting children and pregnant women against possible adverse health effects from exposure to the substance.

The ADI is not considered to cover the use of food additives in infant formula, since the usual toxicity test regimen does not cover this situation; therefore, specific evaluations are carried out for the use of food additives in infant formulas.

Budget method

Having obtained the ADI, the use levels are derived by applying the so-called ‘Budget method’. The Budget method, which has been developed by the National Food Agency, now the Danish Veterinary and Food Administration, is based on considerations of the total daily intake of energy (calories) and liquid. The intake of liquid per kg body weight varies with age, being highest in small children, thereafter declining rapidly during childhood and more slowly in adult age. The Budget method uses the recommended liquid intake at the age of two, 100 ml/kg b.w., as the basis for the calculations of maximum intakes. Intake of food (energy) per kg body weight is also dependent on age. The Budget method uses the energy requirement of 100 kcal for a one-two year old child as a basis for the limit of food intake which will sufficiently protect adults also. (Larsen 1992).

6.5.2 Pesticides

Sale and use

Before sale and use, the active substance in the formulated pesticide product has to be authorised in the EU, whereas the formulation itself has to be authorised in the actual Member State for each crop. The active substances are evaluated according to the EU regulations (EEC 1967, with amendments) on classification, packaging and labelling of chemical substances and preparations, see 6.2. In Denmark, the formulations are evaluated according to the Statutory Order on pesticides (MEM 1998b) which implements the relevant EU Council Directives (EEC 1991, EEC 1976, with amendments) into national legislation.
The evaluation of the formulations are based on risk assessment for specific uses and therefore generally only apply to adult workers. Although children and pregnant women may also be exposed to formulated pesticides, e.g. following their use in gardens and indoor such uses are generally not included in the evaluation of the formulations.

To protect the general population, the most toxic formulations which are labelled as "very toxic" or "toxic" according to the EU regulations on classification, packaging and labelling of chemical substances and preparations (see 6.2), are not allowed for use in gardens, public areas, kindergartens etc. This includes substances which are classified as reproductive or developmental toxicants in category 1 and 2. Formulations are banned in Denmark when the substance is carcinogenic or mutagenic in category 1 and 2. Also when exposure is greater than the AOEL (Acceptable Operator Exposure Level), use of the product cannot be approved. The AOEL is based on the no effect level (NOAEL) from animals studies with a safety factor of at least 100. Formulated products classified as "irritant" with the R-phrase R41 "risk of serious damage to eyes" are not allowed in Denmark.

A separate risk evaluation is not done for re-entry in private homes after professional application of products not allowed for private use. However, in green-houses, where relevant, this evaluation is done for re-entry exposure.

Specific considerations related to protection of children and/or pregnant women are taken into account in the evaluation of a given formulation. Data from studies on reproductive and developmental effects as well as effects in sucking pups of the active substance(s) are requested for the authorisation.
Furthermore, when estimating the ADI for the active substance one of the safety factors, often set to 10, takes into account the intraspecies variation, i.e. that the observed effects may vary in severity within the population.

MRLs in foodstuffs

EU establishes harmonised maximum residue levels (MRLs) for pesticides in foodstuffs, which are in force in all Member States. In Denmark, the Statutory Order on maximum residue levels for pesticides in foodstuffs (FM 1999a) implements the relevant EU Council Directives (EEC 1976b, EEC 1986a,b, with amendments) into national legislation; the MRLs for a number of substances are given in the Annexes of the Statutory Order. The establishment of MRLs is based on the principles of Good Agricultural Practice (GAP¹) and the estimated food consumption per person. From the MRL the TMDI (theoretical maximum daily intake) is calculated and compared with the ADI estimated according to the same practice as already mentioned for food additives, see 6.5.1. If the TMDI exceeds the ADI, more refined methods are used to calculate a more realistic intake using e.g. the actual median residue levels determined after GAP supervised trials, and reduction factors from the processing of food. If the ADI is still exceeded, then the proposed MRLs cannot be endorsed.
For pesticides with a highly acute toxicity, acute reference doses are also estimated and compared with the MRL to protect against such effects.

The Danish Veterinary and Food Administration has analysed the residue levels of pesticides in many different foodstuffs. The estimated intake of the most frequently detected pesticides indicates that the average intake for adults is maximally a few percent of the ADI; therefore, it may be considered that most children also are protected against possible adverse health effects from exposure to the actual pesticides in foodstuffs.

In the EU, a general limit value of 0.01 mg/kg of residues of individual pesticides in processed baby foods and infant formulae has been issued (EEC 1999a,b). This limit value is considered to provide a very high degree of protection of children against adverse health effects from exposure to pesticides. Furthermore, specific pesticides (listed in Annexes to the Directives) must not be used in agricultural products intended for the production of baby foods and infant formulae. These regulations will be implemented in a revision of the Statutory Order on pesticides.

6.5.3 Veterinary medicinal products

EU establishes harmonised maximum residue levels (MRLs) for veterinary medicinal products in milk and foods of animal origin, which are in force in all Member States (EEC 1990, with amendments); the MRLs for a number of substances are given in the Annexes to the Council Regulation (EEC 1990, with amendments).

MRLs for veterinary medicinal products are established on the basis of the ADI assuming that a person weighing 60 kg consumes a certain amount of meat and fish and 1.5 l of milk (cheese and butter included) ("standard food package"). By using this "standard food package" in relation to children, the milk intake is underestimated as children often drink more than 1.5 l of milk (see section 3); however, the intake of meat and/or fish will often be overestimated as children generally consume less of these foods than adults. Thus, the ADI is generally not exceeded for children under normal circumstances. However, if an MRL is only set for milk (e.g. products for intramammary administration) and the ADI is solely allocated to this commodity; the underestimation of the milk intake for children may consequently result in exceeding the ADI even if the level of the given residue in milk is within the MRL.

6.5.4 Contaminants

Chemical substances arising from different sources may occur in foodstuffs. In Denmark, the Statutory Order on certain contaminants in foodstuffs (FM 1999b) implements the relevant EU Council Directives (EEC 1993c, EEC 1997, with amendments) into national legislation; limit values for some metals (lead, cadmium, mercury and tin), nitrate, and mycotoxins (aflatoxin and ochratoxin A) are given in the Annexes. The evaluation of contaminants in foodstuffs are carried out according to the same practice as already mentioned for food additives, see 6.5.1. However, a TDI (tolerable daily intake) instead of an ADI is estimated to signal that the contaminants are not added to foodstuffs deliberately. Furthermore, the database for evaluation of contaminants is less comprehensive than for food additives and pesticides and therefore, higher safety factors are often applied when the TDI is estimated.

Since 1983, the Danish Veterinary and Food Administration has analysed the levels of a number of contaminants in relevant foodstuffs. The levels of the analysed contaminants have significantly decreased during recent years and no specific dietary advice to pregnant women are considered necessary.

6.6 Working environment

In Denmark, the general regulations for employers and workers are given in the Danish Working Environment Act (1998) which include specific regulations regarding young people; these regulations are further specified in the Statutory Order on young people’s employment (AM 1996). Specific regulations are also given for pregnant women in the Statutory Order on the performance of the work (AM 1994). These regulations have been issued to protect young people and pregnant women against exposure to chemical substances and preparations via their working environment.

Young people

The specific regulations for young people below the age of 18 is given by the Statutory Order on young people’s employment (AM 1996); the Statutory Order goes beyond the relevant EU Council Directive (EEC 1994b).
Generally young people below the age of 18 are not allowed to work with or in other ways be exposed to specific chemical substances and materials listed in Annex 2a to the Statutory Order. Most of the listed chemical substances are covered by the EU regulations (EEC 1967, with amendments) on classification, packaging and labelling of chemical substances and preparations (see 6.2) and the following are included:

	substances classified as "very toxic" (Tx), "toxic" (T), "corrosive" (C), "explosive" (E)
	substances with the R-phrases R12, R39, R45, R46, R60, R61
	substances classified as "harmful" (Xn) and at least one of the following R-phrases R40, R42, R48, R62, R63
	substances labelled as "irritant" (Xi) and R43
	substances and materials included on the Danish Working Environment Service’s list of chemicals and processes considered as carcinogenic as well as materials containing more than 0.5% of the chemicals
	organic solvents included on the Danish Working Environment Service’s list of organic solvents as well as materials containing more than 0.5% solvents.

Pregnant women

The specific regulations for pregnant women are given by the Statutory Order on the performance of the work (AM 1994); the Statutory Order implements certain regulations in the relevant EU Council Directives (EEC 1989, EEC 1992, with amendments). Other regulations are implemented by collective agreements or other legislation.

Generally, a specific evaluation of the pregnant woman’s working environment, including the influence of exposure to chemical substances, is requested to identify possible influence on the pregnancy. If this evaluation identify any influences, a more detailed evaluation is requested regarding the nature, extent and duration of the influence(s) to identify any risks for the pregnant woman and the unborn child. If any risk is identified, the working environment of the pregnant woman should be improved or, in cases where such an improvement is not possible, the pregnant woman should be removed from the actual working environment.

Annex 4 to the Statutory Order lists the chemical substances which must at least be considered when evaluating the risk during pregnancy and lactation. Included are:

	chemical substances and materials classified and labelled with R40, R45, R46, R60, R61, R62, R63, and R64
	chemical substances and materials included in the Danish Working Environment Service’s Statutory Order no 300, 1993 regarding carcinogens
	chemical substances and materials included in the Danish Working Environment Service’s Statutory Order no 52, 1988 regarding materials containing volatile chemicals, including solvents
	pesticides
	volatile anaesthetics
	mercury
	lead
	cytostatics
	carbon monoxide
	chemical substances, which pose a proven risk by dermal uptake.

6.7 Drugs

6.7.1 Registration of drugs

In EU, there is at present no mandatory requirement for pharmaceutical companies to investigate new medicinal products in children.
The pharmaceutical industry is however encouraged to investigate the safety and efficacy of a product in children, if it is likely to be of therapeutic benefit to this age group (CPMP 1997). In case sufficient documentation is provided, the license may cover treatment of children.

6.7.2 Principles for dosing children, and pregnant and lactating women

The unborn child as well as infants are considered potentially more susceptible to adverse effects of drugs than adults. The principles for medical treatment of children and pregnant and lactating women are outlined below. The principles and recommendations are described in the catalogue of registered pharmaceuticals in Denmark, Lægemiddelkataloget (1997).

Children
According to Lægemiddelkataloget (1997), schematic dosing of infants and children is difficult and is generally practised as mg drug per kg body weight.

Because of the differences between children and adults in response to drugs, simple proportionate correction in the adult dose for body weight may not be adequate to determine a safe and effective dose for children, especially for the very young. Dosage requirements, cardiac output, hepatic and renal blood flow, and glomerular filtration rate in children and in adolescents of widely differing sizes have been found to correlate better with body surface area than body weight. According to this concept, a child’s dose can be calculated from the formula:

	surface area child (m2)
child’s dose = 1.4 x		x adult dose
	1.8 m2

where 1.8 m² is the surface area of an average 70-kg adult. The surface area of a child can be determined from its body weight using the observation that surface area is proportional to body weight to the 0.7 power. (Rowland & Tozer 1995).

Another approach for determining the dose for children is to test the drug in this age group and the Committee for Proprietary Medicinal Products (CPMP) has issued a Note for Guidance on Clinical Investigation of Medicinal Products in Children (CPMP 1997).

Drug therapy in new-borns and particularly premature babies requires special attention because of the very rapid changes in renal and hepatic function in these early stages of life. Therefore, dosing of this age group is generally referred to paediatric specialists.

Pregnant women

Most chemical substances, including drugs, taken by pregnant women can cross the placenta and enter the tissues of the developing embryo and foetus with consequent risk of teratogenic effects in the form of malformations or other developmental effects (see section 2 and 3 for further details).
Relatively few chemical substances and drugs are known human teratogens; however, a potential for inducing teratogenic effects by the remainder cannot be fully excluded as the mechanisms by which teratogenic effects can be induced are poorly understood and are probably multifactorial. Therefore, every drug should be considered as a potential human teratogen and medical treatment of pregnant women and women who plan pregnancy should be avoided if possible. However, some women with chronic diseases are dependent on medical treatment. Regarding medical treatment of pregnant women with chronic diseases, benefits and risks of the treatment should be carefully evaluated for the mother and the unborn child and in case of medical treatment, these women have to be very carefully monitored during their pregnancy.
(Lægemiddelkataloget 1997).

As a general practise, medical treatment during pregnancy should be based on the following principles (Lægemiddelkataloget 1997):

	Avoid new drugs because preclinical studies do not fully exclude a risk to the human foetus.
	Avoid medical treatment during the first trimester where the risk for malformations are greatest.
	Manage medical treatment by monitoring the plasma concentration.
	Avoid combination of drugs.

Lactating women

Most drugs administered to lactating women can be detected in the breast milk (see section 2 and 3 for further details). The concentration of drugs in the breast milk is highly variable and is dependent on many different factors; however, the knowledge within this area is still very limited. Therefore, as a general practise, medical treatment of lactating women should be avoided. If medical treatment cannot be avoided, the sucking child should be very carefully followed. (Lægemiddelkataloget 1997).

6.8 The Precautionary principle

The precautionary principle is a principle that opens up for regulatory action on the basis of suspicions for harmful consequences rather than waiting for cases or a full scientific evidence. In the context of this report, the use of the precautionary principle may be relevant for the aim of protecting defined vulnerable groups in the population against potential hazardous exposures for which the harmful consequences are not fully understood.

The first actual mentioning of the precautionary principle in connection with legislation was in German legislation in 1976, where it is called the Vorsorgeprinzip: "Environmental policy is not fully accomplished by warning off imminent hazards and the elimination of damage which occurred. Precautionary environmental policy requires furthermore that natural resources are protected and demands on them are made with care." Since then, and in various wording, the precautionary principle has been incorporated in a series of international treaties (e.g., the EU Amsterdam Treaty from 1998) and declarations. In many of these the principle has been connected mainly to hazardous chemical substances, but there are also examples of broader approaches to environmental issues as such. (MST 1998).

In Danish legislation the precautionary principle is not explicitly mentioned, but it is often reflected in the introductory comments to various environmental acts, the central ones being the Environmental Protection Act, the Chemical Substance and Product Act, the Marine Environment Act, and the Gene Technology Act. (MST 1998).

Both in Denmark and abroad, there has been increasing focus on the precautionary principle in recent years. In line with the increasing complexity of environmental issues, politicians, enterprises, and authorities are expected to allow this doubt to benefit the environment and through this public health. However, it is not clear either in Denmark nor in other countries, what the principle specifically involves and how it should be applied in the day-to-day work. To have a debate on the issue and to give a better understanding of the principle, the Danish EPA held a conference on 29 May 1998 which was attended by industry people, researchers, green organisations, politicians, authorities, and other interested parties. The conference provided no clear answers and no easy solutions. However, in general there was broad agreement among the presenters and during the debates that the precautionary principle must be regarded as a political norm for both legislation and administration. It was expressed in the discussion that there was a great need for more guidance on how to apply the precautionary principle in practice. (Danish EPA 1998).

Recently, the Commission of the European Communities has published a communication on the precautionary principle (EU 2000).

6.9 Overall

As mentioned in section 1, the Danish Government initiated a cross-ministerial overview concerning the administrative measures for protecting children and pregnant women. From this overview, which also covered the chemical areas described in this section, it was concluded that in specific areas increased effort should be made to increase the protection of children and pregnant women and several future initiatives in different ministerial sectors were proposed.

With respect to environmental factors and chemical regulations, it was concluded that more specific focus on protection of children and/ or pregnant women should be put in regulatory procedures in relation to: the use of chemicals in cosmetics and toys; risk assessment procedures in the EU; risk assessment procedures in relation to the national derivation of limit values for air, soil and water; and the procedures for approval of pesticides (especially pesticides for domestic use) and for risk assessment of food additives and food contaminants.

6.10 References

AM (1998). The Danish Working Environment Act, Statutory Order from the Ministry of Labour no 497 of 29 June, 1998. In Danish.

AM (1996). The Statutory Order from the Ministry of Labour no 516 of June 14, 1996, on young people’s employment. In Danish.

AM (1994). The Statutory Order from the Ministry of Labour no 867 of 13 October, 1994, on the performance of the work. In Danish.

CPMP (1997). Note for Guidance on Clinical Investigation of Medicinal Products in Children. The European Agency for the Evaluation of Medicinal Products, Human Medicines Evaluation Unit, CPMP/EWP/462/95.

Danish EPA (1998). The precautionary principle. Extracts and summary from the Danish Environmental Protection Agency’s conference on the precautionary principle, Eigtveds Pakhus, Copenhagen, 29 May 1998.

EEC (1999a). Commission directive 1999/39/EC of 6 May 1999 amending Directive 96/5/EC on processed cereal-based foods and baby foods for infants and young children.

EEC (1999b). Commission directive 1999/50/EC of 25 May 1999 amending Directive 91/321/EC on infant formulae and follow-on-formulae.

EEC (1997). Commission Regulation (EC) No 194/97 of 31 January 1997 setting maximum levels for certain contaminants in foodstuffs.

EEC (1994a). Commission Regulation (EC) No 1488/94 of 28 June 1994 on risk assessment for existing substances.

EEC 1994b). Council Directive 94/33/EC of 22 June 1994 on the protection of young people at work.

EEC (1993a). Commission Directive 93/67/EEC of 20 July 1993, laying down the principles for the assessment of risks to man and the environment of substances notified in accordance with Council Directive 67/548/67.

EEC (1993b). Council Regulation 793/93/EEC of 23 March 1993 on the evaluation and control of the risks of existing substances.

EEC (1993c). Council Regulation (EEC) No 315/93 of 8 February 1993 laying down Community procedures for contaminants in food.

EEC (1992). Council Directive 92/85/EEC of 19 October 1992 on the introduction of measures to encourage improvements in the safety and health at work of pregnant workers and workers who have recently given birth or are breastfeeding.

EEC (1991). Council Directive 91/414/EEC of 15 July 1991 concerning the placing of plant protection products on the market.

EEC (1990). Council Regulation (EEC) No 2377/90 of 26 June 1990 laying down a Community procedure for the establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin.

EEC (1989). Council Directive 89/391/EEC of 12 June 1989 on the introduction of measures to encourage improvements in the safety and health of workers at work.

EEC (1988a). Council Directive 88/378/EEC of 3 May 1988 on the approximation of the laws of the Member States concerning the safety of toys.

EEC (1988b). Council Directive 89/107/EEC of 21 December 1988 on the approximation of the laws of the Member States concerning food additives authorised for use in foodstuffs intended for human consumption.

EEC (1987). Council Directive 87/357/EEC of 25 June 1987 on the approximation of the laws of the Member States concerning products which, appearing to be other than they are, endanger the health or safety of consumers.

EEC (1986a). Council Directive 86/362/EEC of 24 July 1986 on the fixing of maximum levels for pesticide residues in and on cereals.

EEC (1986b). Council Directive 86/363/EEC of 24 July 1986 on the fixing of maximum levels for pesticide residues in and on foodstuffs of animal origin.

EEC (1978). Council Directive 79/112/EEC of 18 December 1978 on the approximation of the laws of the Member States relating to the labelling, presentation and advertising of foodstuffs for sale to the ultimate consumer.

EEC (1976a). Council Directive 76/768/EEC of 27 July 1976 on the approximation of the laws of the Member States relating to cosmetic products.

EEC (1976b). Council Directive 76/895/EEC of 23 November 1976 relating to the fixing of maximum levels for pesticide residues in and on fruit and vegetables.

EEC (1967). Council Directive 67/548/EEC of 27 June 1967 on the approximation of the laws, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances.

EU (2000). Communication from the Commission on the precautionary principle. Commission of the European Communities, Brussels, 2.2.2000 COM(2000) 1 final.

EU (1999). Commission Decision of 7 December 1999 adopting measures prohibiting the placing on the market of toys and childcare articles intended to be placed in the mouth by children under three years of age made of soft PVC containing one or more of the substances di-iso-nonyl phthalate (DINP), di(2-ethylhexyl phthalate (DEHP), dibutyl phthalate (DBP), di-iso-decyl phthalate (DIDP), di-n-octyl phthalate (DNOP), and butylbenzyl phthalate (BBP). 1999/815/EC.

FM (1999a). The Statutory Order from the Ministry of Food, Agriculture and Fisheries no 465 of June 15, 1999, on maximum residue levels for pesticides in foodstuffs. In Danish.

FM (1999b). The Statutory Order from the Ministry of Food, Agriculture and Fisheries no 57 of January 22, 1999, on certain contaminants in foodstuffs. In Danish.

FM (1997). The Statutory Order from the Ministry of Food, Agriculture and Fisheries no 942 of December 11, 1997, on food additives. In Danish.

FST (1995). The Statutory Order from the National Consumer Agency of Denmark no 329 of May 23, 1995, on safety regarding toys and products which can be mistaken for foodstuffs. In Danish.

Larsen JC (1992). Food additives, Positive list. Philosophy, regulation, special conditions. In: Risk Management and Risk Assessment in Different Sectors in Denmark, Proceedings from the ATV Conference "Risk Management, Hazard and Risk Assessment in Connection with the Setting of Limit Values for Chemicals" June 1992, ATV Danish Academy of Technical Sciences, 109-124.

Lægemiddelkataloget (1997). Dosing of drugs to children, 38-40; drugs and pregnancy, 45-46; secretion of drugs into breast milk, 46. In Danish.

MEM (1999). The Statutory Order from the Ministry of Environment and Energy no 151 of March 15, 1999, on prohibiting the use of phthalates in toys to children at the age of 0 to 3 years and in certain childcare products etc. In Danish.

MEM (1998a). The Statutory Order from the Ministry of Environment and Energy no 303 of May 18, 1998, on cosmetic preparations. In Danish.

MEM (1998b). The Statutory Order from the Ministry of Environment and Energy no 241 of April 27, 1998, on pesticides. In Danish.

MEM (1997). The Statutory Order from the Ministry of Environment and Energy no 801 of October 23, 1997, on classification, packaging, labelling, sale and storage of chemical substances and preparations. In Danish.

MST (1998). The precautionary principle. Extracts and summary from the Danish Environmental Protection Agency’s conference on the precautionary principle, Eigtveds Pakhus, Copenhagen, 29 May 1998.

MST (1992a). Health based evaluation of chemical substances in drinking water. Guideline Nr. 1 1992 from the Danish Environmental Protection Agency. In Danish.

MST (1992b). Industrial Air Pollution Control Guidelines. Guideline Nr. 9 1992 from the Danish Environmental Protection Agency.

Rowland M and Tozer TN (1995). Age and weight. In: Clinical pharmacokinetics. Concepts and applications. Third edition. Williams & Wilkins, 241-243.

VFD (1997). List of food additives to be used in foodstuffs. "Positivlisten". Danish Veterinary and Food Administration