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
4.2 Long term toxicity
4.3 Reproductive / developmental effects
4.4 Genotoxic effects
4.5 Carcinogenic effects

4.1 Short term toxicity

nonylphenol

The LC₅₀-value of NP is unknown. Four hours was the maximum survival time for rats inhaling a "concentrated vapour" of NP, the concentration was not stated (Smyth et al. 1962, 1969 - quoted from Talmage 1994).

The sensory irritation potential of nonylphenol has been investigated (EU-RAR 1998). Atmospheres of saturated vapour concentration and one tenth saturated vapour concentration, nominally 3636 mg/m³ (400 ppm) and 267 mg/m³ (30 ppm), respectively, were tested. Groups of five female CD-1 mice were exposed, nose only, to each concentration and the respiration rate was monitored using pressure plethysmography. The duration of exposure to the nonylphenol vapour was not reported. The proportion of liquid particulate material in the test atmospheres was determined, and found to be approximately 1% of the nominal concentration, an amount considered unlikely to have a significant influence on the results. At 3636 mg/m³ a mean respiratory rate depression of about 25% was found during exposure. However, at 267 mg/m³ there were no changes in the respiratory rate. These results suggest that nonylphenol can cause mild sensory irritation to the respiratory tract at high exposure levels.

nonylphenol ethoxylate

The LC₅₀-value for NPE is unknown, although several studies of acute inhalatory toxicity have been performed (Table 1). No further details about the concentrations of the test atmospheres were given in the reports.

Oral administration

nonylphenol

Three unpublished studies performed according to OECD test guideline 401 have yielded oral LD₅₀ values of 1200 to 2400 mg/kg for males, and 1600 to 1900 mg/kg for females; presumably the test species was the rat (EU-RAR 1998).
LD₅₀ values for NP of 580 (Texaco Chemical Company 1985 - quoted from Talmage 1994), 1300 (Monsanto Chemical Company 1985 - quoted from Talmage 1994), and 1620 mg/kg in rats (Smyth et al. 1969 - quoted from Talmage 1994) have been reported.

nonylphenol ethoxylate

A number of oral LD₅₀ values for NPE with various ethylene oxide chain lengths have been reported. These values are presented in Table 2.

Dermal contact

For NP, a dermal LD₅₀-value of 2031 mg/kg in rabbits has been reported (EU-RAR 1998)
For NPE, the acute dermal toxicity for a number of NPEs with a varying number of ethoxylene oxide units has been determined in rabbits (Table 3).

NP is corrosive on contact with skin and is a severe eye irritant. Exposure to the saturated vapour may lead to mild sensory irritation of the respiratory tract (EU-RAR 1998).

Inhalation

No data have been found.

NP 28-day study

In a 28-day study (quoted in IUCLID 1996) performed according to OECD guideline 407 (in 1981), doses of 0, 25, 100 or 400 mg/kg/day of NP were administered to Sprague-Dawley rats in the diet. At the highest dose level, body weight, food consumption, and food utilisation was statistically significantly reduced in both sexes. Also at the highest dose level, for male animals only, relative kidney, liver and testes weights were statistically significantly increased (by 20%), blood urea and cholesterol levels were statistically significantly increased, and glucose was statistically significantly reduced. Histopathological examination revealed hyaline droplet accumulation in the renal proximal tubules, and a minor vacuolation in the periportal hepatocytes. Females did not show these effects. In the EU-RAR (1998), the NOAEL is considered to be 100 mg/kg/day.

NP 90-day study

In a 90-day study performed according to EPA guidelines and of GLP quality, Sprague-Dawley rats were administered NP in the diet at concentrations of 0, 200, 650, or 2000 ppm; corresponding to a calculated (in the report) intake of 0, 15, 50 or 150 mg/kg/day (Cunny et al 1997). At the highest dose level, body weight, food consumption, and food utilisation was statistically significantly reduced for both sexes. Haematology, serum chemistry, and ophthalmoscopy findings, oestrous cycle pattern, and spermatogenesis were not affected by treatment. In males, a statistically significant dose-related increase in absolute and relative kidney weight, without accompanying histopathological or clinical-chemical findings was found (actually, a decrease in the occurrence of hyaline droplets was found at the highest dose level). In females of the highest dose group, absolute ovary weight was slightly decreased, without accompanying histopathological changes. Relative ovary weight was not affected. Relative liver weight was increased by 10% in both sexes at the highest dose, and in males only at the next-highest dose level. The NOAEL was considered by the authors to be 50 mg/kg b.w./day.

NP multigeneration study

Further information on repeated dose toxicity can be derived from a good-quality multigeneration study (NIEHS 1998 - quoted in EU-RAR 1998). This study is also described in section 4.3. Groups of 30 male and 30 female Sprague-Dawley rats were exposed to nonylphenol in the diet at concentrations of 0 (control) 200, 650 or 2000 ppm over three generations. Calculated nonylphenol intakes were, respectively, about 0, 15, 50 and 160 mg/kg/day during non-reproductive phases. The F0 generation were exposed for 15 weeks, the F1 and F2 generations from soon after birth to about 20 weeks of age and the F3 generation from birth to about 8 weeks of age.
Evidence of general toxicity was seen in adults of all generations, although there were no treatment-related clinical signs, mortalities or adverse effects on food consumption. At 160 mg/kg/day, bodyweight gain was reduced in comparison with controls in adults across all generations, with the terminal bodyweight being about 10% lower than the controls. Similar reductions in bodyweight gain were also seen at 50 mg/kg/day in F1 females, F2 males and F3 females. Relative kidney weights were increased at 50 and/or 160 mg/kg/day in adult males of the F0, F1 and F2 generations and also at 160  g/kg/day in F1 adult females. Histopathological examination revealed an increase, although often without a convincing dose-response relationship, in the incidence of renal tubular degeneration and/or dilatation in adult males from all generations and all nonylphenol treated groups; similar findings were reported for adult females at 160 mg/kg/day in the F1, F2 and F3 generations and at 15 and 50 mg/kg/day in the F3 generation. These data are shown in table 4a and b.

NPEs 90-day studies

Smyth & Calandra (1969), reported on toxicity studies on alkyl phenol ethoxylates including NPEs. Ninety-day feeding studies in rats have been performed with NPEs with 4, 6, 9, 15, 20, 30, and 40 ethylene oxide units. Test materials were of commercial grade and were added to the diet. The various studies were performed at five different laboratories in the years 1959-65 using individual test protocols. Results are thus not directly comparable. Dose groups consisted of 10 male and 10 female rats, except for one study (Shelanski), where groups consisted of 15 rats of "mixed sexes". The results are presented in Table 5.

In the five studies by Industrial Bio-Test laboratories (NPEs with 4, 6, 15, 20, or 30 ethylene oxide units; doses are shown in Table 5) haematology was studied in 5 rats of each sex from the highest dose level and the control group before treatment and after 11 weeks. All rats were studied for gross pathology. Livers, kidneys, and testes were weighed, and 33 tissues were examined histopathologically from 5 rats of each sex at the highest and control levels. In order to discriminate between poor palatability and toxic effect, 25-day paired-feeding studies were performed at dose levels for which weight gains were shown to be lower than those of the control groups. For NPEs with 4 or 6 ethylene oxide units, effects included growth retardation and increased absolute and relative liver weight. For NPEs with 15 or 20 ethylene oxide units only retarded growth was found. All effects on growth rate were judged to be due to poor palatability of the diets. With respect to the increased liver weights (for NPEs with 4 or 6 ethylene oxide units), no accompanying histopathological findings were found (however, only the highest dose level was examined histopathologically). The liver response was interpreted by the authors as an increase in parenchymatous tissue resulting from increased enzyme activity in relation to metabolism of the test substances. For NPE with 30 ethylene oxide units no effects at all were found.

In the study of NPE with 9 ethylene oxide units by Mellon Institute gross pathology was studied on all rats at sacrifice, livers and kidneys were weighed, and 16 tissues were studied from 12 controls and from 8 rats from the highest dose group, while only 3 tissues were examined in 10 rats on the other two dose levels. Effects included growth retardation and increased relative liver weight at the two highest dose levels (250 or 1250 mg/kg/day). The liver weight increase was accompanied by cloudy swelling, intracellular lipoid, and reduced polysaccharide, while focal hepatic cell necrosis was found at the highest dose level.

In the study by Shelanski of NPE with 9 ethylene oxide units, the 2 lightest male and female rats in each group were sacrificed after 8 weeks. Gross examination was made. On animals from the highest dose group, histopathological examination of 19 tissues was made. After 90 days, all remaining rats were sacrificed and subjected to macroscopic examination. Nine organs were weighed, and histopathological examination was made of 19 tissues from the 2 lightest males and females in each group. In the highest dose group, 11 of 15 rats died during the study. At 0.64% (300 mg/kg/day) and more in the diet, weight gain was retarded. In the two highest dose groups, rats were emaciated. This was judged to be referable to poor palatability by the authors. Food intake, however, was not significantly lower. This apparent contradiction was not discussed by the authors. No histopathological changes indicating toxic effects were seen.

In the Dow studies of NPE with 9 or 40 ethylene oxide units, haematocrit, white blood cell total and differential counts, and haemoglobin were determined on 5 females of the control and the two highest dose levels prior to sacrifice. Six organs were weighed and 8 organs were studied histopathologically. For NPE with 9 ethylene oxide units, at the lowest dose level, 0.1% (100 mg/kg/day), no effects at all were found. At 0.3% (200 mg/kg/day) and above, relative liver weights were high. At the highest dose level, 1.0% (900 mg/kg/day), relative kidney and spleen weights were high, and growth was reduced. In the liver, petecchial areas of central lobular granular degeneration and necrosis were seen. For NPE with 40 ethylene oxide units, no effects were found at or below 0.3% (200 mg/kg/day). At 1% (700 mg/kg/day)and above relative liver weights of male rats were non-significantly heavier than controls, with general cloudy swelling and slight central lobular granular degeneration and necrosis at 3% (2000 mg/kg/day).

Table 5. Results of 90-day feeding studies in rats with nonyl phenol ethoxylates of various ethylene oxide (EO) chain lengths (compiled from Smyth & Calandra 1969)

1) Low kidney weight in females.

2) Cloudy swelling of central hepatic cords, intracellular lipoid, reduced polysaccharide.

3) Focal hepatic-cell necrosis, intracellular lipoid, reduced polysaccharide.

4) Slight petecchial areas of central lobular granular degeneration and necrosis.

5) Increased relative spleen weight in females & increased relative kidney weight in males.

6) Slight central lobular granular degeneration and necrosis with general cloudy swelling.

n.d.) not done (only the highest dose group was examined histopathologically)

*) In the original report, dose was given as % in diet, and was subsequently calculated in mg/kg/day, based on food consumption data, by the rapporteur.

NPE toxicity in dogs

In the same report (Smyth & Calandra 1969), investigations of NPE toxicity in dogs were described (Table 6). Each dosage of NPE with 4, 6, 15, 20 and 30 ethylene oxide units was administered orally in gelatine capsules to 2 male and 2 female dogs. NPE with 9 ethylene oxide units was administered in the diet, a single dog per dose, and 3 dogs in the control group.

1) Focal myocardial necrosis or degeneration (microscopically).

2) Death, grossly detectable focal myocardial necrosis, lung hyperaemia.

Cardiotoxicity in the dog

A number of exploratory/confirmatory experiments, with cardiotoxicity as the endpoint of interest, reported by Smyth & Calandra (1969), are presented in Table 7. The details of these studies are not well-described in the publication.

Cardiotoxicity in other species

Three cats and 3 rabbits were dosed by gavage with 0.7 g/kg of NPE with 20 ethylene oxide units daily for 14 days. These two species did not develop focal myocardial necrosis (Smyth & Calandra 1969).

Nineteen guinea pigs received doses of 1 g/kg of NPE (2 different commercial varieties) with 20 ethylene oxide units daily for 2 or 3 days. Nine of these animals developed myocardial lesions interpreted as early stages of necrosis (Smyth & Calandra 1969).

Rats did not show any heart lesions after 90 days’ feeding at 5 g/kg/day (Smyth & Calandra 1969).

2-year oral administration

NPEs with 4 and 9 ethoxylene oxide units have been administered orally to rats and dogs over periods of 2 years (Smyth & Calandra 1969). Results are shown in Table 8.

rats

Groups of 35 male and 35 female Sprague-Dawley rats, with 5 male and 5 female rats in addition in the highest and control groups received NPE with 4 ethoxylene oxide units at 3 dose levels plus control. After 12 months 5 rats of each sex from the highest dose and control group; and 3 of each sex from the two lower-dosage groups were sacrificed. After 24 months all rats were sacrificed. Livers, kidneys, and testes were weighed. Histopathological examination of 28 tissues was done on 5 rats of each sex in the highest dose and control group. At 200 mg/kg/day females showed a reduced weight gain after 12 months, but not after 24 months. At 1000 mg/kg/day, this effect was also found in male rats.

NPE with 9 ethoxylene oxide units was administered to 3 dose groups plus a control group of 36 male and 36 female Carworth-Elias rats for 2 years. Sixteen rats of each sex were interim sacrificed, at 6 and 12 months. At sacrifice livers and kidneys were weighed, and 11 tissues were histopathologically examined. There was no difference between control and treated rats in any observation made.

No increased frequency of tumours was reported in either rat study.

dogs

NPE with 4 ethoxylene oxide units was administered for 2 years to groups of 3 male and 3 female Beagle dogs in gelatine capsules in dosages 40, 200, and 1000 mg/kg/day. An untreated control group was present.. Haematology and blood and urinary clinical-chemical parameters were measured repeatedly during the study. At sacrifice, liver, kidneys, spleen, heart, brain and testes were recorded, and 28 tissues were examined microscopically. At 200 mg/kg/day, and more pronounced at 1000 mg/kg/day, there was a moderate elevation in serum alkaline phosphatase and relative liver weight, without histopathological changes.

NPE with 9 ethoxylene oxide units was administered in the diet in concentrations of 0, 0.03, 0.09, and 0.27%, corresponding to 0, 8.5, 28, and 88 mg/kg/day (author’s calculation), to groups of 3 male and 3 female Beagle dogs for 2 years. Haematology and blood and urinary clinical-chemical parameters were measured repeatedly. At sacrifice liver, kidneys, and heart was weighed, and 21 tissues were examined histopathologically. NPE with 9 ethoxylene oxide units caused an increased relative liver weight at 0.27% in the diet (88 mg/kg/day) without accompanying histopathological findings.

.

ALP: alkaline phosphatase

Dermal contact

No data have been found.

Oestrogenic effects of NP and NPE

Some alkyl phenols have been implicated in the hypothesis that low-level exposure can disrupt the human endocrine system, that is, that alkyl phenols may act as endocrine disrupters. Alkyl phenols, including NP, have been shown in laboratory studies to mimic the effects of oestrogen in vitro and in vivo (Lee & Lee 1996, Odum et al. 1997).

The oestrogenic effect of nonylphenol and nonylphenol ethoxylates in fish and Daphnids has been studied by a number of authors. Generally the work shows that nonylphenol and nonylphenol ethoxylates do exhibit oestrogenic activity. For nonylphenol ethoxylates the activity was found to increase with decreasing chain length, with nonylphenol showing the greatest activity. (EU-RAR 1998).

The oestrogenic activity of nonylphenol has been investigated in a number of studies using either recombinant yeast, oestrogen sensitive human breast tumour MCF-7 cells, or a rodent uterotrophic assay response. None of these assays have been validated as an internationally accepted toxicity test method, although the MCF-7 and uterotrophic assays have been established for a number of years as standard assays for oestrogenic activity. It should be noted that the significance to human health of oestrogenic activity detected in these assays has yet to be established. (EU-RAR 1998).

NP effects in rodent uterotrophic assay

The oestrogenic activity of nonylphenol in mammals has been assessed in several studies using an assay based upon the uterotrophic response in the rat. Although not stated, it is assumed that the studies have been performed with commercial grade NP which is the branched type.

In the first study, five groups of immature (aged 20 - 22 days) female rats (six in each group) of a Wistar derived strain received single oral gavage doses of nonylphenol in corn oil on each of three consecutive days (ICI 1996 - quoted in EU-RAR 1998). The dose levels ranged from 9.5 to 285 mg/kg/day. Vehicle and positive (oestradiol benzoate 8 µg/kg, by subcutaneous route) groups were included. One day after the final dose the females were killed and the uterus was removed from each animal and weighed. Absolute uterus weight and bodyweight related uterus weight were statistically significantly increased, in a dose-dependent manner, at levels of 47.5 mg/kg/day and above. The NOAEL was 9.5 mg/kg/day. The uterine response seen in the positive control group was much greater than that of the nonylphenol groups, although a direct comparison of potency is not possible given the differing exposure routes. Similar data from the same laboratory have also been presented in peer-review literature (Odum et al. 1997). This latter report also included oral positive control groups (17ß-oestradiol, 10-400 µg/kg), which indicated that oestradiol was about 1000 times more potent in this assay than nonylphenol.

In a similar assay, groups of ten ovariectomised female Sprague-Dawley rats were dosed once daily for three consecutive days with ethanol/oil suspensions of nonylphenol at levels of 0 (vehicle control), 30, 100 and 300 mg/kg/day (Chemical Manufacturers Association 1997b - quoted in EU-RAR 1998). Positive control groups received ethynyloestradiol in ethanol at levels of 10, 30 and 80 µg/kg/day according to the same dosing regimen. The route of administration was not stated. One day after the final dose the females were killed and the uterus was removed from each animal and weighed. Uterus weights at 300 mg/kg/day were significantly increased (1.5-fold) in comparison with the vehicle control group. A slightly greater response (a 2-fold increase) was seen in the 30 and 80 µg/kg/day positive control groups.

In another uterotrophic assay, groups of three immature (aged 20-21 days) Sprague-Dawley rats each received a single intraperitoneal injection of nonylphenol at dose levels of 0, 1, 2 or 4 mg/animal (approximately 25, 50 or 100 mg/kg) (Lee and Lee 1996). Oestradiol, administered by the same route, served as a positive control. The animals were killed 24 hours later and each uterus was removed, weighed and analysed for protein and DNA content and peroxidase (thought to be a uterotrophic marker enzyme) activity. There was a dose-dependent and statistically significantly increase in uterine weight at all levels, with associated increases in uterine protein and DNA content and uterine peroxidase activity. In further experiments, the uterotrophic activity of nonylphenol was found to be blocked by the co-administration ICI 182,780, an oestrogen antagonist, providing evidence that the effect of nonylphenol is mediated through the oestrogen receptor. Also, the potency was compared with oestradiol; in this assay oestradiol was found to be about 1000 - 2000 times more potent than nonylphenol.

Overall, these in vitro and in vivo studies show that nonylphenol has oestrogenic activity of a potency that is between 3 to 6 orders of magnitude less than that of oestradiol. The oestrogenic effect of NP appears to be mediated through the oestrogen receptor since its action can be blocked by oestrogen antagonists. The structure of the aliphatic side-chain (nonyl) is believed to be highly important for oestrogenic activity. Linear NP is not oestrogenic, while one or more of the branched-chain isomers may be able to mimic oestradiol in binding to the oestrogen receptor. (Odum et al. 1997).

NP oestrogenic effect in 28- and 90-day study

In the 28-day and 90-day studies of NP described in section 4.2 sexual organ morphology, oestrous cycle pattern, and spermatogenesis were not found to be affected by treatment at any dose level. In the 90-day study the absolute, but not relative, ovary weight in the highest dose group was slightly decreased without accompanying histopathological changes.

Niehs three-generation study of NP

The effects of nonylphenol on fertility and reproductive performance have been investigated in a comprehensive, good-quality multigeneration study, conducted in compliance with GLP (NIEHS 1998 - quoted in EU-RAR 1998). The overall study design was based on the OECD two-generation reproduction toxicity study guideline, with an extension to include the production of an F3 generation. This study has previously been described in the present report in relation to long term toxicity. Groups of thirty male and thirty female Sprague-Dawley rats were exposed to nonylphenol via incorporation in the diet at concentrations of 0 (control) 200, 650 or 2000 ppm over three generations. Calculated nonylphenol intakes were, respectively, about 0, 15, 50 and 160 mg/kg/day during non-reproductive phases and rising to around 0, 30, 100 and 300 mg/kg/day during lactation. Nonylphenol exposure commenced for the F0 generation at about 7 weeks of age and continued until study termination when the F3 generation were about 8 weeks old. F0 animals were mated (one male with one female) within each dose group to produce the F1 generation, selected F1 animals were similarly mated to produce the F2 generation and selected F2 animals were mated to produce the F3 generation. For the F0 generation and retained F1, F2 and F3 animals, clinical signs of toxicity, bodyweights and food consumption were reported. Oestrous cycles were monitored prior to mating. At the necropsy of adult animals, sperm samples were taken (but not from the F3 generation) for analysis of density, motility (using a computer assisted sperm motion analysis system, only conducted on control and high dose group males) and morphology, a number of organs were weighed and selected organs were sampled for histopathology. Additionally, testicular spermatid counts were made. Parameters assessed in the young offspring included litter size, bodyweights, survival, gross appearance, ano-genital distance, sexual development and, for animals killed at weaning, gross appearance of organs at necropsy and reproductive organ weights. There was evidence of general toxicity in adults of all generations, seen as a reduction in bodyweight gain at 50 and 160 mg/kg/day and histopathological changes in the kidneys at all dose levels. Considering the reproduction-related parameters, there were no adverse effects on fertility or mating performance. However, several other parameters were affected. Oestrous cycle length was increased by about 15% in the F1 and F2 females at 160 mg/kg/day, in comparison with controls. The timing of vaginal opening was accelerated by 1.5-7 days at 50 mg/kg/day and by 3-6 days at 160 mg/kg/day in females of the F1, F2 and F3 generations. Also, absolute ovarian weights were decreased at 50 mg/kg/day in the F2 generation and at 160 mg/kg/day in the F1, F2 and F3 generations; however, no effect on ovarian weight was apparent in the F1 and F3 generations when analysed as an organ-to-bodyweight ratio. In males, changes in sperm endpoints were seen only in the F2 generation; epididymal sperm density was decreased by about 10% at 50 and 160 mg/kg/day and spermatid count was decreased by a similar amount at 160 mg/kg/day. However, there may have been methodological problems with the epididymal sperm density measurements, because the density in all F2 generation groups, including controls, was considerably greater (by about 25-40%) than reported for the F0 and F1 generation males; the age of each generation was similar at necropsy, so major differences in the sperm density would not be expected.

NP effects on the foetus

In a study performed according to OECD guideline 414, of GLP quality, female rats were administered corn oil solutions of NP (presumably by gavage) at dose levels of 0, 75, 150, and 300 mg/kg/day from gestational day 6 to 15. Females were killed on day 20 of gestation, and foetuses were examined. At the highest and second-highest dose levels, maternal toxicity was evident, with mortality of two females. No maternal toxicity was found at 75 mg/kg/day. Post-implantation loss, litter size, foetal weights, and incidence of foetal abnormalities was not affected, even at dose levels which caused maternal toxicity (EU-RAR 1998).

NPE effects on the foetus

Meyer et al. (1988) administered NPE with 9 or 30 ethylene oxide units (NPE 9, NPE 30) to pregnant rats from gestational day 6 to 15. Doses of NPE 9 were 50, 250, or 500 mg/kg/day by gavage. A satellite group received 500 mg/kg/day by gavage on gestational day 1-20, and two further groups received 50 or 500 mg/kg/day dermally on gestational day 6-15. NPE 30 was given at 50, 250, or 1000 mg/kg/day by gavage on gestational day 6-15, with a satellite group receiving 1000 mg/kg/day on gestational day 1-20. The rats were killed on day 21 and the foetuses examined. NPE 30 did not cause any toxic effects on dams or foetuses. NPE 9, at 500 mg/kg, caused an increase in extra ribs in the foetuses. This dose caused a decreased weight gain in the dams. 

NP appears to be negative in the Ames test (Salmonella typhimurium), however, a study of sufficient quality has not been found (EU-RAR 1998). In an in vitro mammalian cell gene mutation test performed according to the OECD test guideline 476, and of GLP quality, and confirmed by a second independent experiment, NP was found non-mutagenic. The dose level may not have been high enough (EU-RAR 1998).

An NPE with unknown ethylene oxide chain length was found negative in the Ames test, using Salmonella typhimurium (CIR 1983). NPEs with 9 or 30 ethylene oxide units have been found negative in the Ames test (Salmonella typhimurium) (Meyer et al. 1988).

4.5 Carcinogenic effects

For nonylphenol, no data have been found (EU-RAR 1998).

NPEs with 4 and 9 ethoxylene oxide units have been administered orally to rats for 2 years (Smyth & Calandra 1969). The group size was 35 of each sex for NPE with 4 ethylene oxide units, and 5 of each sex were interim sacrificed at 12 months. Histopathological examination was done on 28 tissues on 5 rats of each sex from the control and highest dose group.

For NPE with 9 ethylene oxide units the group size was 36 of each sex, with 16 rats of each sex interim sacrificed at 6 and 12 months. Histopathology was performed on 11 tissues and all neoplasms.

No increased frequency of tumours was reported in either study.