Toxicological Evaluation and Limit Values for Nonylphenol, Nonylphenol Ethoxylates, Tricresyl, Phosphates and Benzoic Acid

4. Toxicity, animal data

4. Toxicity, animal data
4.1 Short term toxicity
4.1.1 Studies on all possible TCP isomers
4.1.2 Studies on o-TCP
4.1.3 Studies on other TCP´s
4.1.4 Study on o-TCP
4.2 Long term toxicity
4.2.1 o-TCP
4.2.2 Other TCP´s
4.2.3 o-TCP
4.3 Reproductive and developmental effects
4.3.1 o-TCP
4.3.2 Other TCP´s
4.4 Mutagenic and genotoxic effects
4.5 Carcinogenic effects

Animal testing is of relevance for detecting, if a substance can induce OPIDN. There is a great difference in the sensitivity of the different species in the induction of OPIDN. The chicken and cat have been widely utilised to investigate the syndrome because their responses are similar to those of man. Rabbit, guinea pig, monkey and dog react inconsistently. Rats and mice have been reported to be relatively resistant to the severe paralysis in spite of nervous tissue damage. The preferred test organism is the domestic hen. This is used in both the acute and subchronic testing for delayed neurotoxicity of organophosphorous substances in OECD Test Guidelines 418 and 419 (OECD 1993), as experience has shown that this organism is the most relevant for extrapolating the results to human effects after organophosphorous exposure.

Inhalation

No data have been found.

Oral administration

For mixed isomers of TCP, the oral LD₅₀-values range from >4640 to >15.800 mg/kg b.w. in the rat. In the mouse a value of 3900 mg/kg b.w. has been reported.

For o-TCP, a range of LD₅₀-values from 1160 to 8400 mg/kg b.w. has been reported for the rat, and in the rabbit a value of 3700 mg/kg b.w. has been found. In chicken, o-TCP is more acutely toxic than in the other species tested with LD₅₀-values of 100-500 mg/kg b.w. being reported.

For m-TCP, the LD₅₀-value is reported as >3000 mg/kg b.w. in the rabbit, and >2000 mg/kg b.w. in chicken.

For p-TCP, the LD₅₀-value is reported as >3000 mg/kg b.w. in the rabbit, and >2000 mg/kg b.w. in chicken.

(EHC 1990).

Henschler (1959) studied the relative neurotoxicity of the 10 different isomers of tricresyl phosphates after single oral doses in hens. The most potent isomers for inducing OPIDN were the three isomers with only one ortho-cresyl group, these were equally potent. When the toxicity of these is set to 100%, then the two isomers with two ortho-cresyl groups had a relative toxicity of 50%, and the tri-ortho-cresyl phosphate had a relative toxicity of 10%. The four tricresyl phosphates without the ortho-isomer did not induce OPIDN. Information on dose ranges are not given in detail.

White leghorn adult hens were given a single oral dose of 0, 300, 400, 500, 600, or 700 mg o-TCP/kg b.w.. Hens were sacrificed 14 and 21 days after dosing for neuropathological study, and other hens were sacrificed day 1 and 2 after dosing for determination of neurotoxic esterase (NTE) inhibition in brain and peripheral nerve. Symptoms of OPIDN appeared in the 400, 500, 600, and 700 mg/kg b.w. group on day 3 and in the 300 mg/kg b.w. group on day 4 in the form of weakness of the legs. By day 16 all o-TCP dosed groups had complete prostration.

The inhibition of NTE in brain and peripheral nerve increased dose-dependently compared to controls from 70% in the 300 mg/kg b.w. group to 90% in the 700 mg/kg b.w. group on day 1 and 65% to 80% on day 2 for the two groups, respectively.

In the neuropathological study, o-TCP-treated hens showed progressive neuropathological degenerative changes in brain tissue and in peripheral nerves. In the 300 mg/kg b.w. group the mean damage score was 20% and 38% for brain and peripheral nerve, respectively. This score increased dose-dependently to 35% and 60% in the 700 mg/kg b.w. group for brain and peripheral nerve, respectively. (Nanda & Tapswi 1995).

It has been shown that a single oral dose of o-TCP in the range of 58 to 580 mg/kg b.w. induces mild to severe paralysis in the hen (Cavanagh 1954, Hine et al. 1956 - quoted from EHC 1990).

Certain animal species (e.g. cats, dogs, cows, sheep and chicken) are susceptible to OPIDN-related paralysis after single oral doses of down to 100 mg o-TCP/kg b.w., whereas others (rats and mice) are less susceptible to the ataxia but susceptible to the pathological changes. (EHC 1990, Wilson et al. 1982).

In the studies given below, the isomer composition is given, when stated in the study.

Groups of 10 male and 10 female F344/N rats received TCP in corn oil by gavage at doses of 0, 360, 730, 1450, 2900, or 5800 mg/kg b.w. 5 days per week for a total of 13 or 14 doses during a 16-day period (NTP 1994). The tricresyl phosphate used in this and the subsequent NTP studies contained 21% m-TCP, 4% p-TCP and less than 0.1% o-TCP. Gas chromatographic analysis revealed two major peaks (24 and 30% of the total area) being isomers for which the chemical composition was not revealed. Dicresyl phosphate esters comprised 17% of the mixture.

One female receiving 1450 mg/kg b.w./day and five males and eight females receiving 2900 mg/kg b.w./day died before the end of the study. No deaths occurred in the other dose groups including the 5800 mg/kg b.w./ day group. The only clinical finding related to TCP was diarrhoea, which occurred from 730 mg/kg b.w./day. Final mean body weights of male and female rats that received 1450, 2900, or 5800 mg/kg b.w./day were significantly lower than those of the controls. Necrosis of the mandibular lymph node, spleen, and thymus occurred primarily in rats receiving 2900 and 5800 mg/kg b.w./day. Diffuse aspermatogenesis occurred in the testes of male rats that received 2900 and 5800 mg/kg b.w./day.

Changes in neurobehavioural parameters (spontaneous motor activity, forelimb and hindlimb grip strength, startle response, and paw-lick latency) were seen in groups that received 1450, 2900, or 5800 mg/kg b.w./day. The neurobehavioural results were confounded by mortality and reduced body weights and were not attributed to a direct neurotoxic response, so no detailed results were given.

Groups of 10 male and 10 female B6C3F1 mice received TCP in corn oil by gavage at doses of 0, 360, 730, 1450, 2900, or 5800 mg/kg b.w. 5 days per week for a total of 13 or 14 doses during a 16-day period (NTP 1994).

Five males and all females that received 1450 mg/kg b.w./day, all mice that received 2900 mg/kg b.w./day, and four males and one female that received 5800 mg/kg b.w./day died before the end of the study. Final mean body weights of males in the 1450 and 5800 mg/kg b.w./day groups were significantly lower than those of the controls. Final mean body weight of females of the 360, 730, and 5800 mg/kg b.w./day groups were significantly greater than those of the controls.

Necrosis of the mandibular lymph node, thymus, and spleen occurred primarily in mice receiving 2900 and 5800 mg/kg b.w./day.

Hindlimb grip strength of male mice that received 360 and 1450 mg/kg b.w./day and male and female mice that received 730 and 5800 mg/kg b.w./day were significantly lower than those of the controls at the end of the study.

Male and female CD-1 mice were given a diet containing 0, 0.437, 0.875, 1.75, 3.5, or 7% TCP for 14 days. No signs of toxicity developed at doses up to 0.875%. All animals in the groups given 1.75, 3.5, or 7% TCP (with the same chemical composition as the NTP study above) in the diet exhibited piloerection, tremors, and diarrhoea, and were lethargic before death during the 14 day exposure. (Chapin et al. 1988).

For mixed TCP isomers, the LD₅₀-value has been reported to be > 7900 mg/kg in rabbits and 1500 mg/kg in cats.

4.1.4 Study on o-TCP

Groups of three male cats received a single dose of 0, 100, 250, 500, 1000, 1500, or 2000 mg o-TCP/kg b.w. on a 15 cm² clipped area of the neck. There was no wash off after the dosing. In order to reduce the acute effects of o-TCP dosing, cats treated with 1000 mg/kg and higher doses received 2 ml subcutaneous injections of atropine sulphate and PAM (pyridine-2-aldoxime-methylchloride) immediately after o-TCP administration and then one to three times daily until the disappearance of acute cholinergic effects of o-TCP. The cats were observed for up to 120 days with respect to body weight, development of OPIDN, and electromyographic changes.

In the 2000 mg/kg b.w. group, two cats died after 6 and 7 days while the third one died on day 25 without recovering from the acute poisoning from o-TCP. Cats treated with 250 to 1000 mg o-TCP/kg b.w. lost weight after administration, and the weight loss was generally dose dependent. As time passed, these animals regained all of the lost weight and continued to gain weight until the end of the experiment. Cats given 500 or 1000 mg o-TCP/kg b.w. had significantly higher gains (157 and 141% of initial weight, respectively) than those of the untreated control group, which at termination was 124% of the cats initial weight.

The functional disturbances of delayed neurotoxicity were always more severe in the hind legs than the fore limbs. The onset of these showed a dose dependent latency period being 17 days in the 1500 mg/kg b.w. group and 27 days in the 250 mg/kg b.w. group for leg weakness, and 31 to 36 days for mild ataxia of these two groups. In the 100 mg/kg b.w. group no signs of delayed neurotoxicity developed. Histopathologic examination of spinal cord showed changes in virtually all cats of the 250 to 2000 mg/kg b.w. groups. Histopathological examination of the peripheral nerves showed changes in cats of the 1000 to 2000 mg/kg b.w. groups. In the 100 mg/kg b.w. group no histopathologic signs of delayed neurotoxicity were observed. In electromyography, pathologic changes were observed in the 250 to 1500 mg/kg b.w. dose groups starting on day 37 to 50 with no apparent dose relationship. In the 100 mg/kg b.w. group no electromyographic change was observed. (Abou-Donia et al. 1986).

No data have been found.

As o-TCP and other isomers with at least one o-cresyl group are causing OPIDN, studies with o-TCP will be described before studies, where the content of o-TCP is low or not specified. Further as there is an OECD guideline for testing for OPDIN in 90 day studies using hens, the hen study will be described first.

In 90-day studies in hens, functional (ataxia) and morphological neuropathological changes were found at daily oral dose levels of 5 to 20 mg o-TCP/kg b.w., but not at 2.5 mg/kg b.w./day and lower dosages. The degree of ataxia correlated well with the degree of neuropathological changes (types not specified). The functional changes were graded on a scale from 0 to 8, with the score 0 representing no ataxia and score 8 representing a situation, where the bird is unable to stand, have weak limb movements, and tail and leg reflexes are virtually non-existent. (Prentice et al. 1983 and Roberts et al. 1983).

In order to test whether the absolute dose or the dosing regime (low daily (group A) versus high biweekly (group B) doses) were important for the neurotoxic action of o-TCP, a group of 40 male Long Evans rats received 116 mg o-TCP/kg b.w. by gavage 5 days/week for 24 weeks, another group of 70 rats received every two weeks a dose of 1160 mg o-TCP/kg b.w. for a period of 24 weeks, and finally a control group of 30 animals received 1 ml corn oil/kg. Group B animals received a prophylactic dose of 5 mg atropine sulphate 4 h after o-TCP dosing. Six to 10 animals from each group were killed for morphological study at 2, 6, 12, 18 and 24 weeks of exposure. Every six weeks, the rats were tested for plantar extension (placing reflex), when placed on a flat surface, general hindlimb co-ordination, when walking, and hindlimb splay.

Over 60% group B animals survived the acute cholinergic (acetyl choline esterase inhibition) effects. Body weights increased by 54, 67, and 86% in group A, B and the control group, respectively.

Functional abnormalities of group B animals were first noted at week 6 in the form of curled hind limb claws. After 12 weeks exposure, the same animals walked on their heels in stead of their toes and failed to maintain lateral extension of their hindlimbs, when lifted by the tail. After 18 weeks, these animals developed a peculiar rasping sound to their squeak, when excited by handling. After 24 weeks, over 50% of group B animals showed hindlimb crossing, when lifted by the tail.

Functional abnormalities in group A animals rarely went beyond abnormalities in walking e.g. heel walking even after 24 weeks of exposure.

Animals from both dosed groups showed visible loss of the thigh muscles after 24 weeks exposure. In morphological and histopathological studies, the neuronal damage was most pronounced in group B, with the ascending and descending tracts being damaged concurrently.

At week 12, a study was carried out to see, which axons of the PNS were most profoundly affected. In both dosed groups the large diameter fibres, particularly tibial nerve branches, were preferentially damaged. (Veronesi 1984).

This study shows that few dosings employing larger amounts of o-TCP given weeks apart produce more severe neurological toxicity, both behavioural and pathological, than smaller daily dosings, even though the same total dose was given to both groups.

Groups of 10 male Fischer 344 rats received 0, 10, 50, or 100 mg o-TCP/ kg b.w./day orally in corn oil for 63 days. A positive control group of three chickens were given oral doses of 100 mg o-TCP/kg b.w./day for 18 days. The rats were tested in four sensorimotor tests after the last dose and after sacrifice, half of the rats were used for neuropathological studies and the other half for biochemical assays.

In the 50 and 100 mg/kg b.w./day groups body weight gain was slightly reduced and signs of acute cholinergic toxicity (diarrhoea, salivation, and ocular discharge) were observed in these two dose groups only. At no time during the dosing period did rats show signs of delayed neurotoxicity. Chickens treated with o-TCP developed ataxia on day 7 and paralysis on day 15 of treatment.

In rats, the peak intensity of movement, horizontal motor activity, and vertical motor activity were non-significantly dose-related different from control values. In all dosed rats, forelimb grip strength was significantly decreased (p<0.05).

Brain acetylcholinesterase activity was significantly and dose-dependently decreased in the 50 and 100 mg/kg b.w./day groups with 20.1% of the control value being found in the 100 mg/kg b.w./day group. Brain NTE activity was also dose-dependently decreased in the 50 and 100 mg/kg b.w./day groups with 34.2% of the control value being found in the 100 mg/kg b.w./day group.

Neuropathological changes revealed swollen axons without fragmentation or loss of myelin staining in the spinal cord of only one rat receiving 100 mg/kg b.w./day.

(Somkuti et al. 1988).

Groups of 10 male and 10 female F344/N rats received tricresyl phosphate in corn oil by gavage at doses of 0, 50, 100, 200, 400, or 800 mg/kg b.w./day for 13 weeks (NTP 1994) The tricresyl phosphate used in this and the subsequent NTP studies contained 21% m-TCP, 4% p-TCP and less than 0.1% o-TCP. Gas chromatographic analysis revealed two major peaks (24 and 30% of the total area) being isomers, for which the chemical composition was not revealed. Dicresyl phosphate esters comprised 17% of the mixture.

All rats survived to the end of the study. Final mean body weights of male rats receiving 200, 400, and 800 mg/kg b.w./day were significantly lower than that of the controls. Cytoplasmic vacuolisation of the adrenal cortex occurred in all dosed groups and the severity increased with dose.

Ovarian interstitial cell hypertrophy occurred in all dosed groups of females, and atrophy of the seminiferous tubules occurred in male rats that received 400 and 800 mg/kg b.w./day.

There was a significant change in hindlimb grip strength in female rats that received 400 and 800 mg/kg b.w./day accompanied by a statistically lower body weight in 200, 400, and 800 mg/kg b.w./day females.

Groups of 10 male and 10 female F344/N rats received a diet containing 0, 900, 1700, 3300, 6600, or 13000 ppm tricresyl phosphate for 13 weeks, equivalent to average daily doses of 55, 120, 220, 430, and 750 mg/kg b.w./day for males and 65, 120, 230, 430, and 770 mg/kg b.w./day for females (NTP 1994).

All rats survived to the end of the study. Final mean body weights of males and females given 6600 and 13000 ppm and females given 3300 ppm TCP were significantly lower than those of controls. Feed consumption by male and female rats given 13000 ppm were significantly lower than that by controls during the first week of the study. In male rats at 13000 ppm a significant reduction in hindlimb grip strength was observed, possibly caused by the reduced body weight (120 g less than control). Cytoplasmic vacuolisation of the adrenal cortex occurred in all exposed groups of rats. Hypertrophy of ovarian interstitial cells and inflammation of the ovarian interstitium occurred in all dosed groups of females. Renal papillary oedema and renal papillary necrosis occurred in 13000 ppm males and females and in 6600 ppm females. Basophilic hypertrophy of the pituitary gland pars distalis and atrophy of the seminiferous tubules occurred in 6600 and 13000 ppm males.

Groups of 95 male and 95 female F344/N rats received diets containing 0, 75, 150, or 300 ppm TCP for two years (NTP 1994). The average daily doses were 3, 6, and 13 mg/kg b.w. to males and 4, 7, 15 mg/kg b.w. to females. An additional group of 95 male and 95 female rats were given diets containing 600 ppm TCP for 22 weeks and then received only control feed. After 3, 9, and 15 months of exposure, up to 15 males and 15 females per group were evaluated for forelimb and hindlimb grip strength, then necropsied and evaluated for histopathologic lesions.

Survival, feed consumption, and final mean body weights of all groups of rats given TCP were similar to those of the controls. Cytoplasmic vacuolisation of the adrenal cortex, which might be a general indication of stress, occurred in 300 ppm males and in 75, 150, and 300 ppm females at the 3 months interim evaluation. At 9 and 15 months, cytoplasmic vacuolisation occurred only in female rats, primarily in the 300 ppm group. Cytoplasmic vacuolisation of the adrenal cortex and ovarian interstitial hyperplasia occurred in female rats given 300 ppm TCP throughout the 2-year study and the incidence and severity increased at the end of the study. No dose-related neurotoxicity was observed.

Groups of 10 male and 10 female B6C3F1 mice received TCP in corn oil by gavage at doses of 0, 50, 100, 200, 400, or 800 mg/kg b.w./day for 13 weeks (NTP 1994). All mice survived to the end of the study. Final mean body weight of male mice receiving 200 mg/kg b.w./day and of male and female mice receiving 400 and 800 mg/kg b.w./day were significantly lower than those of the controls. Cytoplasmic vacuolisation of the adrenal cortex occurred in all dosed groups and the severity increased with dose. Ovarian interstitial cell hypertrophy was present in all dosed female mice. Multifocal degeneration of the spinal cord occurred in males and females that received 100, 200, 400, and 800 mg/kg b.w./day, and multifocal degeneration of the sciatic nerve occurred in males that received 200, 400, and 800 mg/kg b.w./day and females that received 100, 200, 400, and 800 mg/kg b.w./day. Hind limb grip strengths of male mice that received 200, 400, and 800 mg/kg b.w./day were significantly lower than that of controls at the end of study. (NTP 1994).

Groups of 10 male and 10 female B6C3F1 mice were fed diets containing 0, 250, 500, 1000, 2100, or 4200 ppm tricresyl phosphate for 13 weeks (NTP 1994). Dietary levels of 250, 500, 1000, 2100, and 4200 ppm TCP were equivalent to average daily doses of 45, 110, 180, 380, and 900 mg/kg b.w./day to males and 65, 130, 230, 530, and 1050 mg/kg b.w./day to females. All mice survived to the end of the study. Mean body weights of 4200 ppm males and of 2100 and 4200 ppm females were lower than those of the controls throughout the study. Feed consumption in females given 1000, 2100, or 4200 ppm TCP was lower than that by controls during week 12. Cytoplasmic vacuolisation of the adrenal cortex occurred in all dosed groups of males and females with the exception of 250 ppm males. Papillary hyperplasia of the gallbladder mucosa occurred in male mice given 500 ppm or more and in female mice given 1000 ppm or more. Axonal degeneration occurred in males and females given to 2100 and 4200 ppm and in females of the 1000 ppm group. Renal tubule degeneration occurred in all 4200 ppm male mice, and not in any other groups. The interpretation of the grip strength changes observed in the groups receiving 2100 or 4200 ppm TCP were confounded by the reduced body weight of these groups. There was a significant loss of both fore- and hindlimb grip strength in both sexes of the high dose group. (NTP 1994).

Groups of 95 male and 95 female mice were fed diets containing 0, 60, 125, or 250 ppm TCP for two years (NTP 1994). The dietary levels were estimated to correspond to average daily doses of 7, 13, and 27 mg/kg b.w. to males and 8, 18, and 37 mg/kg b.w. to females. At 3, 9, and 15 months of chemical exposure, up to 15 males and 15 females per group were evaluated for forelimb and hindlimb grip strength, then necropsied and evaluated for histopathologic lesions.

Survival, feed consumption, and final mean body weights of males and females given TCP were similar to those of the controls. Incidences of clear cell foci, fatty change, and ceroid pigmentation of the liver were significantly increased in male mice that received 125 or 250 ppm TCP. There were no statistically significant differences in fore- and hindlimb grip strength at the 3, 9, and 15 months examinations.

Dermal contact

Groups of at least three male cats received daily dermal doses of 0, 0.5, 1, 5, 10, or 100 mg o-TCP/kg b.w. for 90 days. There were no washing off of the substance. The surviving cats were further observed for a 30-day period. Cats that developed acute toxicity signs received daily 1-ml subcutaneous injections of atropine and PAM.

In the 100 mg/kg/day group body weight decrease was observed steadily throughout the survival period of the cats which lasted to day 40, when the cats of this group were killed for humane reasons. In the 10 mg/kg/ day group body weight initially fell, and during the recovery phase there was an increase in body weight. In the 5 mg/kg b.w./day group body weight at the end of dosing was 135% of the initial body weight compared to 122% for the control group. The 1 mg/kg b.w./day and the 0.5 mg/kg b.w./day groups had a body weight of 116 and 110% of the initial body weight at the end of dosing.

Signs of toxicity (cholinergic effects) developed in the 100, 10, and 5 mg/kg b.w./day groups 9, 25, and 35 days after onset of dosing, respectively. In the two last groups recovery from toxicity began 35 and 45 days after dosing, respectively. In the 1 and 0.5 mg/kg b.w./day groups no sign of acute toxicity was seen.

Delayed neurotoxicity developed in the 100 mg/kg b.w./day group on day 23 with mild ataxia and developed further to paresis on day 33. In the 10 mg/kg b.w./day group leg weakness started on day 21 and developed further to paresis on day 40 in one cat. In the 5 mg/kg b.w./day group leg weakness started on day 52 and developed into mild ataxia on day 65. In the 1 mg/kg b.w./day group leg weakness started on day 74 and had recovered on day 92. No sign of delayed neurotoxicity was seen in the 0.5 mg/kg b.w./day group.

In four cats of the 10 mg/kg b.w./day group electromyography was carried out at intervals. Electromyographic changes were seen in the cats from day 49-51 and recovery had taken place on day 68-72. Comparing the onset of clinical signs and electromyographic signs of delayed neurotoxicity, it appears that the clinical signs appear first. (Abou-Donia et al. 1986).

Groups of three roosters received 0 or 100 mg o-TCP/kg b.w./day for 18 days, whereafter reproductive parameters were assessed. Two groups of roosters received 750 mg o-TCP/kg b.w. once and were sacrificed day 1 and day 18 after treatment, respectively. In the roosters sacrificed day 1 after dosing no clinical signs were observed. In the animals sacrificed day 18 developed typical signs of OPIDN at approximately day 12. In the animals given daily doses of 100 mg/kg b.w., testis weight and percent motile sperm were significantly decreased, whereas those animals that received a single dose of 750 mg/kg b.w. and were sacrificed on day 18 no adverse effects on these parameters were seen. In 5 of 10 roosters treated with 100 mg/kg b.w./day for 18 days, there was a significant disorganisation of the seminiferous epithelium, which affected 20-80% of the tubules per animal. The remaining 5 roosters of this group showed no consistent pattern of pathology. The two single dose o-TCP (1 and 18-day sacrifice) treatment groups showed no histopathological damage in the testis. (Somkuti et al. 1987).

Groups of 10 to 18 pregnant Long-Evans rats received 0, 87.5, 175, 350 mg o-TCP/kg b.w. from day 6 to 18 of gestation. Standard OECD guideline teratology laboratory procedures were used in the assessment of the teratogenic potential of o-TCP. In the 350 mg/kg b.w. group 28% (5) of the animals died and one had totally resorbed foetuses. No maternal deaths or toxicity were observed in the 87.5 and 175 mg/kg b.w. groups. There were no significant differences noted among the dosed groups and the control group for preimplantation loss or resorption. Foetal weights for both sexes in the o-TCP dosed groups were significantly greater than in the control group. The results of this study indicate that o-TCP is not teratogenic in the Long-Evans rat. (Tocco et al. 1987).

Groups of 12 male Long-Evans rats received 0, 100, or 200 mg TCP (less than 9% o-TCP)/kg b.w./day by gavage in 10.0 ml corn oil for 56 days prior to breeding and throughout a 10 day breeding period and groups of 24 female Long-Evans rats received 0, 200, or 400 mg TCP/kg/day for 14 days prior to breeding, and throughout breeding, gestation, and lactation. The males and females were mated - one male to two females - as follows: control to control, low dose to low dose, and high dose to high dose. During the mating period females were checked for sperm every morning until sperm cells were detected, whereafter the mated females were housed individually.

No clinical signs or body weight depression were observed in any of the TCP dosed groups relative to controls. The observed rate of sperm positive females was similar in all female groups i.e. mating capacity was not affected. However, the percent of sperm positive females giving birth dropped from 95% in the control group to 45% in the low dose group and further to 5 % in the high dose group females i.e. fertility was affected by TCP dosing. Pups of the only litter in the high dose group died on lactation day 5 due to dehydration, and no milk was found in their stomachs. An evaluation of reproductive organ weights and sperm parameters was carried out on male rats at the end of dosing. Epididymis weight and sperm concentration in the high dose group were significantly decreased and in both the low and high dose groups motility, progressive movement and sperm morphology were adversely affected. Histopathologic changes were observed in the testes and epididymides of male rats and in the ovaries of female rats exposed to TCP.

(Carlton et al. 1987).

Male F344 rats and male B6C3F1 mice were treated with 0, 50, 100, or 200 mg/kg b.w. (mice), 0, 50, 100, 200, 400, or 800 mg/kg b.w. (rats) TCP containing <0.1% o-TCP by gavage in corn oil, or 0, 1700, 3300, or 6600 ppm (rats) or 0, 500, 1000, or 2100 ppm (mice) TCP in feed for 13 weeks. Sperm concentration, motility, and morphology were evaluated. The reproductive tract was examined for histopathologic lesions.

Mice exposed by gavage exhibited a decrease in sperm concentration and an increase in abnormal sperm morphology at 200 mg/kg b.w.; a decrease in sperm motility was found in all TCP dosed groups. Tricresyl phosphate treatment significantly affected (p<0.01) all male reproductive parameters in a dose-dependent manner in rats given TCP by gavage. In animals given TCP in the feed, sperm parameters were adversely affected in the 2100 ppm group mice and 6600 ppm group rats. Preliminary histopathologic findings indicate multifocal testicular degeneration in rats but not mice by both dosing routes. At comparable dose levels (mg/kg b.w.) corn oil gavage administration of TCP results in greater male reproductive toxicity in rats and mice than dosing in the diet.
(Carlton et al. 1986).

In a continuous breeding protocol pairs of 20 F344 rats received 0.4 g TCP/kg b.w./day for 7 days prior to mating, through a 63 day breeding period, a 28-day post breeding interval and a crossover mating period. The TCP used is stated to be free of o-TCP and consisted of p- and m- isomers (62 wt%), cresyl-xylyl (18 wt%) and cresyl-ethylphenyl (18 wt%) phosphates. A group of 40 pairs served as control group. During the breeding period only 9 litters were delivered by the TCP dosed animals compared to 107 litters being delivered by the control group i.e. fertility was affected by TCP dosing. In the crossover mating, where control group males were mated to TCP dosed females and vice versa, it was shown that TCP caused infertility in TCP dosed males but not in the TCP dosed females. (Latendresse et al. 1994).

In a continuous breeding protocol, groups of at least 20 pairs of CD1 mice received 0, 0.05, 0.1, or 0.2% TCP in the diet for 98 days, equivalent to 62.5, 124, and 250 mg/kg b.w./day. Fertility parameters and maternal body weight were used to assess the reproductive toxicity of TCP, containing 74.9% TCP with virtually no o-TCP (<0.1%). The remaining 25% were dicresyl phenyl and di- and tricresylxylyl phosphates. In the 0.1 and 0.2% dose groups reproductive toxicity was seen in the form of reduced numbers of pairs giving litters (for the 0.1% dose at the last litter and for the 0.2% dose group from the second litter and onwards). In the 0.2% dose group the numbers of live pups were decreased, and the numbers of dead pups were increased significantly. In the 0.1 and 0.2% dose groups the combined mean live pup weight was significantly decreased.

In order to determine which sex was affected, control males were mated to 0.2% females, and 0.2% males were mated to control females. In a control group control males were mated to control females. No difference was observed in mating index, but the fertility index was reduced in both of the tested groups compared to the control pairs. Additionally the combined number of live pups per litter was significantly reduced in both test groups most in the combination control male mated to 0.2% female. This indicates that fertility of both sexes was affected by the 0.2% TCP dosing.

In sperm parameters the 0.2% TCP dosed males had reduced percentage of motile sperm, reduced sperm concentration and increased percentage of abnormal sperm.
(Chapin et al. 1988).

Technical TCP (no further specifications given) was tested in the Ames test by two independent laboratories using Salmonella typhimurium strains TA100, TA1535, TA 1537, and TA98 with and without metabolic activation. TCP did not show mutagenic activity in the tests. (Haworth et al. 1983).

TCP was tested for its ability to induce Unscheduled DNA Synthesis (UDS) in hepatocytes from male Fischer-344 rats after in vivo dosing by gavage and after in vitro exposure. All of the tests were negative. No information on doses in the in vivo study and concentration of TCP in the in vitro culture is provided in the abstract. (Mirsalis et al. 1983).

NTP (1994) tested TCP for genotoxicity in three different in vitro genotoxicity tests with and without metabolic activation. In the Ames test Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 was used and TCP was tested in concentrations up to 10,000 mg/plate.

In hamster ovary cell cultures sister chromatid exchange and chromosomal aberrations was studied in at least four different doses of TCP with 5,000 mg/ml as the highest concentration. None of the tests gave positive evidence for in vitro genotoxicity of TCP.

4.5 Carcinogenic effects

Groups of 95 male and 95 female F344/N rats received diets containing 0, 75, 150, or 300 ppm TCP for two years (NTP 1994). The dietary levels were estimated to correspond to average daily doses of 3, 6, and 13 mg/kg b.w. to males and 4, 7, 15 mg/kg b.w. to females. No carcinogenic effects were observed in the study.

Groups of 95 male and 95 female B6C3F1 mice were fed diets containing 0, 60, 125, or 250 ppm TCP for two years (NTP 1994). The dietary levels were estimated to correspond to average daily doses of 7, 13, and 27 mg/kg b.w. to males and 8, 18, and 37 mg/kg b.w. to females. No carcinogenic effects were observed.