Registration Dossier

Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
24 August 1993 to 31 May 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study performed according to the OECD guideline 416 with following restrictions: some sperm parameters (e.g. sperm count, motility and morphology) and data on sexual maturation of F1 generation were not reported

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report Date:
1995

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Version / remarks:
26 May 1983
Deviations:
yes
Remarks:
some sperm parameters (e.g. sperm count, motility and morphology) and data on sexual maturation of F1 generation were not reported
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Limit test:
no
Justification for study design:
Not applicable

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Details on test material:
- Receipt date of the lot/batch: 07 July 1992
- Expiration date of the lot/batch: 27 April 1995
- Storage condition of test material: At room temperature

Test animals

Species:
rat
Strain:
Sprague-Dawley
Remarks:
Crl CD (SD) BR strain
Details on species / strain selection:
The rat was chosen because it is a rodent species commonly requested by regulatory authorities and the Sprague-Dawley strain was selected due to the background data available from previous studies performed at the laboratory.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France (76410 Saint-Aubin-les-Elbeuf, France)
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation (P): Approximately 7 weeks
- Weight at study initiation (P): 269 g for males; 213 g for females
- Housing: Housed individually in polycarbonate cages.
- Diet (e.g. ad libitum): A04 C fine ground diet, provided by U.A.R. (91360 Villemoisson-sur-Orge, France), ad libitum
- Water (e.g. ad libitum): Tap water filtered using a 0.22 micron filter (Millipore S.A., 78140 Velizy, France), ad libitum
- Acclimation period: At least 13 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 ± 2°C
- Relative humidity: 50 ± 20%
- Light/dark cycle: 12 hour / 12 hour
- Ventilation: About 13 cycles/hour of filtered, non-recycled air

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
DIET PREPARATION
- Mixing appropriate amounts with (Type of food): The test substance was blended with a small quantity of A04 C fine ground diet (provided by U.A.R., France) using a mortar and pestle (or a Braun mixer on week 8); this premix was then transferred with a Lodige M20 mixer (A.T.R., 75015 Paris, France) with the required total quantity of diet in order to achieve the concentration of 1000 ppm, and then mixed for 10 minutes. This preparation was used as a premix: the premix concentrations (0.5, 2, 15 and 112.5 ppm) were then prepared by dilution of this premix at 1000 ppm with further quantities of untreated diet; homogeneity was then obtained by mixing in a Lodige FM50 mixer (A.T.R., 75015 Paris, France) for 10 minutes.
- Rate of preparation of diet (frequency): The preparations were made once a week (except for the females of the 0.5 ppm group: 3 preparations per day from week 11 onwards).
- Storage temperature of food: Stored at room temperature, in closed opaque plastic bags, pending utilization.
Details on mating procedure:
F0 and F1 parents:
- M/F ratio per cage: 1/1
- Length of cohabitation: Until mating occurred or 3 weeks had elapsed
- Proof of pregnancy: The day when spermatozoa were found in the vagina was designated as Day 0 of pregnancy
- After 3 weeks of unsuccessful pairing replacement of first male by another male with proven fertility.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Sampling frequency and assay method: On weeks 1, 4, 8, 12, 16, 20, 24, 28, 32, 36 and 40 each dietary mixture (control group included) was checked for achieved concentration of the test substance using High Performance Liquid Chromatography with electrochemical detection. Each sample was analysed in duplicate on weeks 1 and 4 and in quadriplate thereafter.

Results: Throughout the study, the results of duplicate analyses revealed a satisfactory concordance between obtained and theoretical concentrations for all groups with few differences for groups 2 (0.5 ppm) and 3 (2 ppm):
- At 0.5 ppm, differences between nominal values were -50% on week 1 (0.252 ppm), -25% on week 8 (first preparation), -23% on week 16 (first preparation), +34% on week 20 (second preparation), +44% on week 24 (first preparation), -25% on week 32, -36% on week 36 and -24% on week 40 (first preparation). Other analysis for this group have shown best values and all measured values were in agreement with homogeneity results.
- At 2 ppm, differences between nominal values were -26% on week 4, +23% on week 12 (first preparation), -22% on week 16 (second preparation) and -19% on week 36 (second preparation). Other analysis for this group have shown best values and all measured values were in agreement with homogeneity results.
Duration of treatment / exposure:
- F0 males: 72 days before mating, during the mating period and until weaning of the F1 offspring.
- F0 females: 72 days before mating, during the mating period, during the pregnancy, during the lactation and until weaning of the F1 offspring.
- F1 males and females: On Day 21 post-partum, F1 pups of each sex were selected and treated at the same dose levels as their parents until the weaning of the F2 offspring.
Frequency of treatment:
Daily in diet
Details on study schedule:
- When the F1 animals were about 13-15 weeks, they were paired.
Doses / concentrationsopen allclose all
Dose / conc.:
0.5 ppm (nominal)
Dose / conc.:
2 ppm (nominal)
Dose / conc.:
15 ppm (nominal)
Dose / conc.:
112.5 ppm (nominal)
No. of animals per sex per dose:
- F0 rats: 30/sex/dose
- F1 rats: 30-31/sex/dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The concentrations in the main study were determined following the results of a preliminary study. In this study, the test substance was administered continuously by oral route (dietary admixture) to F0 male and female Sprague-Dawley rats for 29 days before mating until weaning of F1 pups and to F1 males and females until the age of 8 weeks at the dose levels of 2, 10, 40 and 160 ppm corresponding to 0.12, 0.62, 2.47 and 8.35 mg/kg bw/day in F0 males and 0.19, 0.94, 3.73 and 11.99 mg/kg bw/day in F1 females, respectively. The lowest dose of 2 ppm was not toxic for the F0 and F1 animals, did not induce adverse effects on the reproductive performance of F0 animals and did not impair the development of the F1 offspring. The 10 ppm dose level induced only an increase in thyroid activity, without any other signs of toxicity in F0 or F1 animals or adverse effects on the reproductive performance of F0 animals or development of F1 offspring. The 40 and 160 ppm dose levels were considered to be toxic for F0 and F1 animals (clinical signs, impairment of food consumption and body weight gain, increase in thyroid activity, microscopic changes in pituitary gland and/or hepatocellular hypertrophy). The reproductive capacity of F0 animals and the development of F1 offspring was not affected at 40 ppm, while the 160 ppm dose level induced a decrease in fertility and some mortalities in the F1 offspring. Based on these results, dose levels of 0, 0.5, 2, 15 and 112.5 ppm were chosen for the main study.
- Rationale for animal assignment: (if not random): Before the beginning of the treatment period, the required number of animals was allocated to the groups, according to a computerized stratified procedure (using CITOX internal software), so that the average body weight of each group was similar.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
EXAMINATIONS IN F0 AND F1 GENERATIONS

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked twice a day for mortality and signs of morbidity, except for weekends and public holidays when they were checked at least once a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical signs were observed for each animal at least once a day, at the same approximate daily time.

BODY WEIGHT: Yes
- Time schedule for examinations: Once a week over a 7-day period of treatment until sacrifice

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes
- Time schedule: Once a week over a 7-day period of treatment until sacrifice

PARTURITION:
- F0 and F1 females were allowed to litter normally and rear their progeny to the stage of weaning. Any sign of a difficult or prolonged parturition was noted. The day of parturition was noted. The duration of gestation was calculated from Day 0 of pregnancy.
Oestrous cyclicity (parental animals):
Yes
Sperm parameters (parental animals):
Parameters examined in P/F1/F2 male generations: testis and epididymis were weighed and histopathology was performed.
Litter observations:
STANDARDISATION OF LITTERS
- On Day 4 post-partum, the size of each litter was adjusted by eliminating extra pups by random selection, as nearly as possible, at 4 males and 4 females per litter.
- Whenever, the number of male or female pups prevented having at least 4 of each sex per litter, partial adjustment (for example, 5 males and 3 females) was made. Adjustments were not made for litters of less than 8 pups.

PARAMETERS EXAMINED
- The following parameters were examined in F1 and F2 progeny during the lactation period: Litter size (number and sex of live, dead and cannibalized pups), body weight, clinical signs and pup development (pinna unfolding, hair growth, incisor eruption, eye opening, auricular duct opening, surface righting reflex, cliff avoidance and air righting reflex)

GROSS EXAMINATION OF DEAD PUPS:
- Any gross abnormalities in F1 and F2 pups were noted.
Postmortem examinations (parental animals):
SACRIFICE
- After the weaning of the last F1 litter and of the last F2 litter and after about 18 hours fasting, F0 males and females (except for one F0 female of the 112.5 ppm group) and F1 males and females were asphyxiated using carbon dioxide and killed by exsanguination.
- Throughout the study, moribund animals were killed in the same way.

GROSS NECROPSY
- A complete macroscopic examination was performed on all parental animals (F0 and F1) including those found dead or killed prematurely during the study.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The following organs were weighed: Adrenals, pituitary gland, spleen, epididymides, prostate, testes, liver, seminal vesicles with coagulating glands, thyroids with parathyroids, ovaries, uterus and kidney
- The following tissues were prepared for microscopic examination: Adrenals, pituitary gland, spleen, epididymides, prostate, testes, liver, seminal vesicles with coagulating glands, thyroids with parathyroids, ovaries, uterus and kidney
Postmortem examinations (offspring):
SACRIFICE
- F1 and F2 pups not selected on Day 4 post-partum and F1 and F2 pups whose mother died during lactation were killed by asphyxiation using carbon dioxide.
- F1 and F2 pups killed between Day 22 and Day 25 post-partum were asphyxiated using carbon dioxide and killed by exsanguination.

GROSS NECROPSY
- A complete macroscopic examination was performed on all F1 and F2 pups including those found dead or killed prematurely during the study.

HISTOPATHOLOGY / ORGAN WEIGHTS
- The following organs were weighed: Adrenals, pituitary gland, spleen, epididymides, prostate, testes, liver, seminal vesicles with coagulating glands, thyroids with parathyroids, ovaries, uterus and kidney
- The following tissues were prepared for microscopic examination: Adrenals, pituitary gland, spleen, epididymides, prostate, testes, liver, seminal vesicles with coagulating glands, thyroids with parathyroids, ovaries, uterus and kidney
Statistics:
- Mean values were compared by one-way variance analysis and Dunnett's test.
- Percentage values were compared by Fisher's exact probability test.
Reproductive indices:
- Male mating index: (Number of males able to mate / Number of paired males) * 100
- Female mating index: (Number of mated females / Number of paired females) * 100
- Male fertility index: (Number of pregnant female partners / Number of males able to mate) * 100
- Female fertility index: (Number of pregnant females / Number of mated females) * 100
- Gestation index: (Number of females with live born pups / Number of pregnant females) * 100
- Live birth index: (Number of live born pups / Number of delivered pups) * 100
Offspring viability indices:
- Viability index on day 4 pp: (Number of live pups on day 4 pp / Number of live born pups) * 100
- Viability index on day 21 pp: (Number of live pups on day 21 pp / Number of live born pups on day 4 pp) * 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs attributable to treatment were observed during the study.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
no mortality observed
Description (incidence):
No deaths occurred in males or in females during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males:
- The mean body weight gain of males was similar in the control, 0.5,2, and 15 ppm groups.
- In the 112.5 ppm group, the body weight gain was significantly lower than that of the control group from the third week of treatment. When considering the period from Day 1 to Day 135, the body weight gain of males given 112.5 ppm was 134 g vs. 406 g in controls (difference statistically significant; p < 0.001). On Day 135, the mean absolute body weight of males was 40% lower than that of control males (403 g vs. 672 g; p < 0.001). This decrease in body weight gain was attributed to the treatment.

Females:
- The mean body weight gain of females was similar in the control and the 0.5, 2, and 15 ppm groups during the premating, pregnancy and the lactation periods.
- In the 112.5 ppm group, it was lower than that of the control group during the premating period (body weight gain from Day 1 to Day 71: 80 g vs. 129 g; p < 0.001), during the pregnancy (body weight gain from day 0 to day 20 of the pregnancy: 92 g vs. 145 g; p < 0.001). During the lactation, the body weight gain of females was unaffected. The decrease in body weight gain during the premating and pregnancy periods was attributed to the treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males:
- The mean food consumption of males was similar in the control and the 0.5, 2, and 15 ppm groups.
- In the 112.5 ppm group, it was significantly lower than that of the control group from Day 1 to Day 135 (differences from the control group ranging from 3 to 45%). This decrease in food consumption was attributed to the treatment.

Females
- The mean food consumption of females was similar in the control and the 0.5, 2, and 15 ppm groups during the premating, pregnancy and lactation periods.
- In the 112.5 ppm group, it was significantly lower than that of the control group from the third week of treatment of the premating period (reduction by 13 to 30%). during the pregnancy (reduction by about 30%) and during lactation (reduction by 22 to 33%). This decrease in food consumption was attributed to the treatment.

Compound Intake
- The mean achieved dose levels of the test substance were 0.03, 0.12, 0.90 and 5.88 mg/kg bw/day in males and 0.04, 0.16, 1.23 and 7.83 mg/kg bw/day in females for the 0.5, 2, 15 and 112.5 ppm concentrations, respectively.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Statistically significant lower mean body autopsy weight was noted in the males and females given 112.5 ppm (-40% and -20%. respectively) when compared to controls.
- Statistically significant differences from controls in mean absolute and relative organ weights were noted in the high dose group as shown in Table 7.8.1/1.
- The higher mean thyroid weights noted in the F0 males and females given 112.5 ppm, as well as the lower adrenal weights noted in the females of the same group, were considered to be related to treatment with the test substance. The other above-mentioned organ weight differences from controls noted in the high dose F0 males and females were not correlated with any relevant histological findings which could explain these differences. They were most probably secondary to the lower body autopsy weight noted for these animals.
- Minor, although statistically significant, lower (-7%) mean relative liver weight was noted in the females given 0.5 ppm, when compared to controls. This difference from controls was considered not to be treatment-related, as it was not recorded in the intermediate dose group.
- No other apparent organ weight differences from controls were noted in the different treated groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Enlargement of the thyroid glands was noted in all males and females given 112.5 ppm, together with reddish colour for 2 of these males and 15 of these females. These changes were considered to be related to treatment with the test substance.
- Small adrenals were noted in 1/30 males given 15 ppm and in 27/30 males and 21/30 females given 112.5 ppm. This was considered to be treatment-related for the high dose males and females, although no microscopic changes that could explain this macroscopic observation were noted for the high dose males. The low incidence noted in the 15 ppm group was considered to be of no toxicological importance.
- Reddish pituitary gland was noted in 3/30 males and 1/30 females given 112.5 ppm. No correlated histological finding was noted that could explain this change of colour. However, it was considered to be related to treatment with the test substance.
- Small spleen was found for 1/30 control males and for 29/30 males and 22/30 females given 112.5 ppm. No relevant histological changes were noted in the spleen of the high dose animals which could be correlated to this gross finding. For the high dose animals, this was considered to be related to the low body weight.
- Small ovaries were noted in 1/30 females (suspected of infertility) given 15 ppm and in 4/30 females given 112.5 ppm. Except for one high dose female, the absolute and relative ovary weights were comparatively similar to the lowest control values. Furthermore, no correlated microscopic findings were noted, that could explain this gross finding. Thus, it was considered to be of no toxicological importance.
- Small kidneys were noted in 26/30 males given 112.5 ppm. No histological findings were noted that could explain this macroscopic change. However, as retardation of kidney maturity was noted for 1/28 high dose F1 females, the relationship to treatment for the small kidneys noted in the high dose F0 males cannot be ruled out.
- The other macroscopic findings noted were those which are commonly recorded in untreated laboratory rats of this strain and age and none was considered to bear a relationship to treatment with the test substance.
Neuropathological findings:
not examined
Description (incidence and severity):
not applicable
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
THYROID GLANDS
- Morphological changes indicative of an increase in thyroid activity (slight to marked presence of small follicles, slight to moderate reduction of colloid content, and slight follicular cell hypertrophy) were noted in the thyroid glands of the F0 animals of the control (14/30 males, 3/30 females), 2 ppm (16/30 males) and 15 ppm (23/30 males, 2/30 females) groups. The slightly higher incidence together with similar intensity) noted for the F0 males given 15 ppm were considered to be related to treatment with the test substance. Incidence and severity of these morphological changes noted for the F0 males given 2 ppm were comparatively similar to those noted for respective controls. Consequently, the thyroid activity noted for these treated animals given 2 ppm was considered to be of no toxicological importance.
- For all the high dose males and females of the F0 generation a higher severity of all these above-mentioned histological findings was noted, when compared to respective controls, as follows: moderate to very large number of small follicles, moderate to severe decrease in follicular colloid contents, slight to marked follicular cell hypertrophy.
- For the F0 generation: minimal to moderate follicular cell hyperplasia was noted for 24/30 males and 26/30 females, nodular follicular cell hyperplasia was noted for 3/30 males.
- Minimal to slight vascular ectasia was also noted in the thyroid glands of all the high dose F0 animals of both sexes, versus 1/30 F0 control males and none of the F0 control females. All these changes noted for the F0 animals given 112.5 ppm were indicative of a moderate to severe increased thyroid activity, and were well-correlated with the higher thyroid weights as well as the thyroids enlargement and/or reddish colouration noted at necropsy.
- In addition, minimal to moderate fibrosis of the capsule, sometimes together with mononuclear cell aggregation, was noted in the thyroid glands of 9/30 high dose F0 males, 3/30 high dose F0 females versus none of their respective controls. All these changes noted in the thyroid glands of the F0 animals given 112.5 ppm were considered to be treatment-related.

ADRENALS
- Uni- or bilateral minimal to moderate atrophy of the cortex (zonae fasciculata and reticularis) was noted in the adrenal glands of 27/30 F0 females given 112.5 ppm. This change was well correlated with the lower absolute and relative weights noted for these females and were considered to be related to treatment with the test substance.
- Cortical cell atrophy was not found in the adrenals of any high dose F0 male, nor was it observed in the adrenals of the F0 females given 15 ppm.
- For the F0 generation, uni- or bilateral minimal to slight accumulation of ceroid pigment was noted in the cortical cells (zona reticularis) of the adrenals of 2/30 control males and 17/30 control females, of 4/30 males and 14/30 females given 15 ppm, and in 16/30 males and 16/30 females given 112.5 ppm. The incidence and severity of this finding was similar between the males of the control and 15 ppm groups, and between the females of the control, 15 and 112.5 ppm groups. The moderately higher incidence of ceroid pigment accumulation noted in the adrenal cortex of the F0 males given 112.5 ppm was considered to be related to treatment with the test substance.

LIVER
- Slight to severe hepatocellular hypertrophy (sometimes with giant multinucleated hepatocytes) was noted in the liver of all F0 males and females given 112.5 ppm. Hepatocellular hypertrophy was considered to be treatment-related and is contradictory with the low absolute and relative liver weights noted for these F0 animals. Hepatocellular hypertrophy was not found in any F0 animals given 15 ppm.
- Minimal to slight multifocal hepatocellular degeneration/necrosis was noted for 6/30 high dose F0 males. Minimal to moderate perilobular steatosis was noted in the liver of 2/30 F0 control males and
1/30 F0 control females (minimal intensity only), 2/30 F0 males given 15 ppm (minimal to slight intensity), and of 5/30 F0 males and 19/30 F0 females given 112.5 ppm (minimal to moderate intensity). The hepatic cell degeneration/necrosis, as well as the higher incidence and/or severity of steatosis noted for the F0 animals of both sexes given 112.5 ppm, was considered to be treatment-related.
- For the F0 generation, slight multifocal coagulative hepatocellular necrosis was noted in 1/30 females given 15 ppm and a large area of coagulative necrosis was observed in the left lateral liver lobe of 1/30 males given 112.5 ppm. All these findings can be seen spontaneously in the liver of untreated laboratory rats of this strain and age. Consequently, the reported low incidence was considered to be of no toxicological importance.

PITUITARY GLAND
- Minimal to severe decrease in the number of acidophil cells was noted in the routinely stained
(Hematoxylin-eosin) pituitary sections of all F0 males and females given 112.5 ppm, which were killed on completion of the study (for those animals found dead or killed during the study, fixation artifacts prevent a correct histological examination and, therefore, were not evaluated), versus none of their respective controls. This was considered to be related to treatment with the test substance.
- In the F0 generation, minimal to moderate presence of vacuolated cells was noted in the pituitary of all control males, in all males given 15 ppm, in all males and 29/30 females given 112.5 ppm (versus none of the control females). For the males, the severity of this finding was similar between the control and 15 ppm groups but was higher for the males given 112.5 ppm. Consequently, the higher intensity of vacuolated cells noted in the pituitary gland of the F0 males given 112.5 ppm, as well as the presence of similar finding for the F0 and F1 females of the same group were considered to be related to treatment with the test substance.

SPLEEN
- For the F0 generation, minimal to slight haemosiderosis was noted in the spleen of 8/30 control males and 11/30 control females, and in 14/30 males and 15/30 females given 112.5 ppm.
- The slightly higher incidence (together with similar severity) of this finding in the F0 males given 112.5 ppm was considered to be fortuitous and of no toxicological importance.

FEMALE GENITAL ORGANS
- Morphological changes indicative of a regular oestrous cycle was noted in all F0 control females and in 21/30 F0 females.
- The remaining 9/30 high dose F0 females showed morphological changes indicative of pseudopregnancy (early phase to late phase). The reported high incidence of pseudopregnancy noted in the females given 112.5 ppm was considered to be related to treatment with the test substance.
- For the treated F0 females of the low and intermediate dose groups which were suspected of infertility, evidence of regular oestrous cycle was noted as follows: In the F0 generation, for 2/2 females given 0.5 ppm, 1/2 females given 2 ppm and 1/2 females given 15 ppm.
- The few remaining females (i.e. 1 F0 female given 2 ppm and 1 F0 female given 15 ppm) showed signs indicative of pseudopregnancy. The reported low incidence of these findings was considered to be of no toxicological importance, as they can be seen spontaneously in untreated laboratory rats of this strain and age.

MALE GENITAL ORGANS
- No relevant microscopic findings were noted in the testes, the epididymides, prostate and seminal vesicles for the F0 males of the low, intermediate and high dose groups, which were suspected of infertility.
- Slightly to markedly small seminiferous tubules and minimal to marked absence of spermatogenesis in testes, together with aspermia in epididymides were noted for 5/32 males found dead or killed prematurely between Days 2 and 25 of the treatment period. These findings, which are characteristics of sexual immaturity, are expected findings in young laboratory rats of this age. Changes indicative of sexual immaturity were also noted in the prostate and seminal vesicles for 4 of these young males.

MAMMARY GLAND
- Minimal vacuolation of the acinar and/or ductular epithelium was noted in the mammary gland for the F0 generation in 1/30 control females and 1/31 females given 15 ppm. This finding was noted with minimal to marked intensity in the mammary gland of 9/30 F0 females given 112.5 ppm. The higher incidence and/or severity of acinar and/or ductular epithelial cell vacuolation noted for the F0 females given 112.5 ppm was considered to be related to treatment with the test substance.

KIDNEYS
- Minimal to moderate mineralization of urothelium and/or urinary gravel in the renal pelvis were noted in the kidneys of 12/26 males given 112.5 ppm and of the examined female of the same group. No microscopic examination was performed in the control animals. The high incidence of urothelial mineralization and/or urinary gravels noted in the renal pelvis of the high dose F0 animals was considered to be related to treatment with the test substance.
- Minimal focal urothelium hyperplasia, associated with urothelial mineralization and urinary gravels in pelvis, was noted in the kidneys of one of these high dose male. In addition, uni- or bilateral, minimal to slight leucohistiocytic pyelitis was noted in the kidneys of 14/26 high dose males. These changes are most probably secondary to the presence of urinary gravels and/or mineralization of the urothelium.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
- Morphological changes indicative of a regular oestrous cycle was noted in all F0 control females and in 21/30 F0 females.
- The remaining 9/30 high dose F0 females showed morphological changes indicative of pseudopregnancy (early phase to late phase). The reported high incidence of pseudopregnancy noted in the females given 112.5 ppm was considered to be related to treatment with the test substance.
- For the treated F0 females of the low and intermediate dose groups which were suspected of infertility, evidence of regular oestrous cycle was noted as follows: In the F0 generation, for 2/2 females given 0.5 ppm, 1/2 females given 2 ppm and 1/2 females given 15 ppm.
- The few remaining females (i.e. 1 F0 female given 2 ppm and 1 F0 female given 15 ppm) showed signs indicative of pseudopregnancy. The reported low incidence of these findings was considered to be of no toxicological importance, as they can be seen spontaneously in untreated laboratory rats of this strain and age.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- No relevant microscopic findings were noted in the testes, the epididymides, prostate and seminal vesicles for the F0 males of the low, intermediate and high dose groups, which were suspected of infertility.
- Slightly to markedly small seminiferous tubules and minimal to marked absence of spermatogenesis in testes, together with aspermia in epididymides were noted for 5/32 males found dead or killed prematurely between Days 2 and 25 of the treatment period. These findings, which are characteristics of sexual immaturity, are expected findings in young laboratory rats of this age. Changes indicative of sexual immaturity were also noted in the prostate and seminal vesicles for 4 of these young males.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
MATING
- Males: The mating index of males was similar in the control, 0.5, 2 and 15 ppm groups (between 93 and 100%). In the 112.5 ppm group, it was lower, i.e. 77% as 23 out of 30 males were able to mate.
- Females: The mating index was 100% in the control, 0.5,2 and 15 ppm groups. In the 112.5 ppm group, it was 83.3% as only 25 out of 30 females were able to mate. This decrease in mating index was attributed to treatment.

FERTILITY
- Males: The fertility index in males was considered to be similar in the control, 0.5, 2 and 15 ppm groups. In the 112.5 ppm group, it was slightly lower (87% vs. 100% in controls). This decrease was attributed to treatment.
- Females: The fertility index was similar in the control, 0.5, 2 and 15 ppm groups (100, 93.3, 93.3 and 93.3%, respectively) and the number of implantation sites was also similar (16.2, 16.7, 15.4 and 16.0 in the control, 0.5, 2 and 15 ppm, respectively). In the 112.5 ppm group, it was slightly lower i.e. 88.0% as 22 out of the 25 mated females were pregnant. Moreover, the number of implantation sites was lower than that of the control group (12.2 vs. 16.2; p < 0.001). This reduction in the number of implantation sites as well the decrease in the fertility index were attributed to treatment.

GESTATION
- The mean duration of gestation was similar in the control and treated groups.

Effect levels (P0)

Key result
Dose descriptor:
NOAEL
Effect level:
2 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive performance
Remarks on result:
other: equivalent to 0.12 mg/kg bw/day

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
- Males: No clinical signs attributed to the treatment were observed in males of the 0.5, 2 and 15 ppm groups. In the 112.5 ppm group, hypoactivity was noted in 6 males associated mainly with piloerection in 3 of them and dyspnea in 4 of them. These clinical signs were attributed to treatment.
- Females: No clinical signs attributed to the treatment were observed in females of the 0.5, 2 and 15 ppm groups. In the 112.5 ppm group, piloerection was noted in 11 females associated mainly with hypoactivity and hypothemia in 7 of them, dyspnea in 4 of them. These clinical signs were attributed to treatment.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality:
mortality observed, treatment-related
Description (incidence):
- Males: No deaths occurred in males of the control, 2 and 15 ppm groups. In the 0.5 ppm group, one male was found dead on Day 7. This death was considered not to be treatment-related. In the 112.5 ppm group, 6 males were found dead on Days 4, 5, 25 or 135 and 2 were killed for poor clinical condition on Day 77 or 86. The deaths were attributed to treatment.
- Females: No deaths occurred in females of the control and 0.5 ppm groups. In the 2 ppm group, one female was found dead on Day 5 and in the 15 ppm group, one female was killed on Day 3. These deaths were considered not to be treatment-related. In the 112.5 ppm group, during the premating period, 2 females were found dead on Days 4 and 58; 6 were killed for poor clinical condition on Days 69, 75, 77, 78 or 112; an additional female was also killed for poor clinical condition but was replaced). During pregnancy, one female was found dead on Day 17. During lactation, one female was killed on Day 2 post-partum for incomplete delivery. Two other females were found dead on Day 121 or 133. These deaths were attributed to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Males: The mean body weight gain of males was similar in the control, 0.5, 2 and 15 ppm groups. In the 112.5 ppm group, the absolute body weight was lower than that of the control group from the first day of treatment (43 vs. 58 g; p < 0.001). The body weight gain was lower than that of the control group from the first week of treatment. When considering the period from Day 1 to Day 141, the body weight gain of males given 112.5 ppm was 170 g vs. 610 g in controls (difference statistically significant; p < 0.001). On Day 141, the mean absolute body weight of males was 67% lower than that of control males (219 g vs. 668 g; p < 0.001). This decrease in body weight gain was attributed to treatment.
- Females: Over the entire study duration, the mean body weight gain of females was similar in the control, 0.5, 2, and 15 ppm groups during the premating, pregnancy and the lactation periods. In the 112.5 ppm group, the absolute body weight was lower than that of the control group from the first day of treatment (44 vs. 57 g; p < 0.001). The body weight gain was lower than that of the control group during the premating period (body weight gain from Day 1 to Day 78: 67 g vs. 261 g; p < 0.001), during the pregnancy (body weight gain from Day 0 to Day 20 of the pregnancy: 45 g vs. 160 g; p < 0.001). During the lactation, the body weight gain of females was similar to that of the control group. The decrease in body weight gain during the premating and pregnancy periods was attributed to treatment.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
- Males: The mean food consumption of males was similar in the control, 0.5, 2, and 15 ppm groups. In the 112.5 ppm group, it was significantly lower than that of the control group from Day 8 to Day 141 (differences from the control group ranging from 32 to 59%). This decrease in food consumption was attributed to treatment.
- Females: The mean food consumption of females was similar in the control, 0.5, 2, and 15 ppm groups during the premating, pregnancy and lactation periods. In the 112.5 ppm group, it was significantly lower than that of the control group from the fourth week of treatment of the premating period (reduction by 32 to 45%). during the pregnancy (reduction by 48 to 59%) and during lactation (reduction by about 51%). This decrease in food consumption was attributed to treatment.

- Compound intake: The mean achieved dose levels of the test substance were 0.04, 0.16, 1.24 and 12.02 mg/kg bw/day in males and 0.05, 0.21, 1.64 and 15.64 mg/kg/day in females for the 0.5, 2, 15 and 112.5 ppm concentrations, respectively.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Statistically significant and markedly lower mean autopsy body weight was noted for the males and females given 112.5 ppm (-69% and -60%, respectively) when compared to controls.
- Statistically significant differences in mean absolute and/or relative organ weights were noted as shown in Table 7.8.1/1
- The higher mean thyroid weights noted in the males and females given 112.5 ppm, as well as the lower adrenal weights noted in the females of the same group, were considered to be treatment-related (see Microscopic examination).
- The other organ weight differences from controls noted in the high dose males and females were not correlated with any relevant histological changes which could explain these differences. They were most probably secondary to the lower body weight noted for these high dose animals.
- No other apparent organ weight differences from controls were noted in the different treated groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Thyroid gland enlargement was noted for 26/32 males and 24/31 females given 112.5 ppm and considered to be treatment-related.
- Small liver was noted for 4/31 females given 112.5 ppm which were killed prematurely during the study. This finding is most probably related to the small size of these animals. Small liver was also noted for 1/30 males given 0.5 ppm: as this finding was not recorded in any males of the 2 intermediate and high dose groups, it was considered to bear no relationship to treatment with the test substance. Liver enlargement was noted for 2/30 males given 2 ppm. The individual absolute and relative liver weights of these animals were similar to or slightly higher than the control values. Furthermore, liver enlargement was not recorded for the males given 15 ppm. Consequently, the enlarged liver noted for the males given 2 ppm was considered to be of no toxicological importance.
- Small adrenals were noted for 1/32 males and 5/31 females given 112.5 ppm. This was considered to be treatment-related for the high dose F1 animals of both sexes; although relevant microscopic changes were noted for the females only (see Microscopic examination). Blackish adrenals were noted for another female which was killed prematurely. It was considered to bear no relationship to treatment, as it was correlated with sinusal congestion as the result of poor bleeding.
- Macroscopic changes were noted among the 9 males and 16 females given 112.5 ppm, which were found dead or killed prematurely during the study. They are as follows: reddish pituitary gland for 1 female and enlarged and/or reddish coloured penis in 2 males (these changes were considered to be of no toxicological importance as they are most probably secondary to post mortem stasis and/or autolysis); small spleen, for 4 males and 10 females, small kidneys for 1 male and 6 females [these macroscopic findings were not associated with any relevant finding (except for few high dose females where lymphoid depletion was noted in spleen)]; small thymus for 1 male and 4 females, cryptorchidism for 1 male, killed prematurely on Day 86 of the treatment period (the small organs might be related to the small body weights recorded for those animals to treatment). The relationship of cryptorchism to treatment, which was noted for 1 high dose male, was considered unlikely.
- Small spleen was also noted for 1/31 females given 15 ppm, which was killed prematurely on Day 3 of treatment period. The low incidence of this finding in the 15 ppm group was considered to be of no toxicological importance.
- The few other macroscopic findings noted were those which are commonly recorded in untreated laboratory rats of this strain and age. Furthermore, they were noted either with a similar incidence to that of controls or showed no dose-related incidence. They were, thus, considered not to be treatment-related.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
THYROID GLANDS
- Morphological changes indicative of an increase in thyroid activity (slight to marked presence of small follicles, slight to moderate reduction of colloid content, and slight follicular cell hypertrophy) were noted in the thyroid glands of the F1 animals of the control (10/30 males, 6/30 females), 2 ppm (15/30 males, 2/31 females) and 15 ppm (21/30 males, 10/30 females) groups. The slightly higher incidence together with similar intensity noted for the F1 males and females given 15 ppm were considered to be related to treatment with the test substance. Incidence and severity of these morphological changes noted for the F1 males and females given 2 ppm were comparatively similar to those noted for respective controls. Consequently, the thyroid activity noted for these treated animals given 2 ppm was considered to be of no toxicological importance.
- For 27/32 high dose males and 28/31 high dose females of the F1 generation, a higher severity of all these above-mentioned histological findings was noted, when compared to respective controls, as follows: moderate to very large number of small follicles, moderate to severe decrease in follicular colloid contents, slight to marked follicular cell hypertrophy.
- For the F1 generation: minimal to slight follicular cell hyperplasia was noted for 19/31 males and 22/30 females, nodular follicular cell hyperplasia was noted for 2/31 males and 2/30 females, follicular cell adenoma was noted for 1/31 males.
- Minimal to slight vascular ectasia was also noted in the thyroid glands of 25/31 F1 males and 20/30 F1 females given 112.5 ppm, versus none of their respective controls. All these changes noted for the F1 animals given 112.5 ppm were indicative of a moderate to severe increased thyroid activity, and were well-correlated with the higher thyroid weights as well as the thyroids enlargement and/or reddish colouration noted at necropsy.
- In addition, minimal to moderate fibrosis of the capsule, sometimes together with mononuclear cell aggregation, was noted in the thyroid glands of 1/32 high dose F1 females, versus none of their respective controls. All these changes noted in the thyroid glands of the F1 animals given 112.5 ppm were considered to be treatment-related.

ADRENALS
- Uni- or bilateral minimal to moderate atrophy of the cortex (zonae fasciculata and reticularis) was noted in the adrenal glands of 17/28 F1 females given 112.5 ppm. This change was well correlated with the lower absolute and relative weights noted for these females and were considered to be related to treatment with the test substance.
- Cortical cell atrophy was not found in the adrenals of any high dose F1 male, nor was it observed in the adrenals of the F1 females given 15 ppm.
- For the F1 generation, uni- or bilateral minimal to slight accumulation of ceroid pigment was noted in the cortical cells (zona reticularis) of the adrenals of 4/30 control females, 1/30 males and 3/31 females given 15 ppm and in 18/32 males and 6/28 females given 112.5 ppm. The incidence and severity of this finding was similar between the males of the control and 15 ppm groups, and between the females of the control, 15 and 112.5 ppm groups. The moderately higher incidence of ceroid pigment accumulation noted in the adrenal cortex of the F1 males given 112.5 ppm was considered to be related to treatment with the test substance.

LIVER
- Slight to severe hepatocellular hypertrophy (sometimes with giant multinucleated hepatocytes) was noted in the liver of 28/32 F1 males and 29/31 F1 females given 112.5 ppm. Hepatocellular hypertrophy was not observed for the remaining 4 F1 high dose males and 2 F1 high dose females, which were found dead or killed prematurely between Day 2 and Day 5 of the treatment period. Hepatocellular hypertrophy was considered to be treatment-related and is contradictory with the low absolute and relative liver weights noted for these F1 animals. Hepatocellular hypertrophy was not found in any F1 animals given 15 ppm.
- Minimal steatosis was noted only in 1/30 males given 15 ppm and 1/31 females given 112.5 ppm (mainly centrilobular) and was considered to be of no toxicological importance.
- For the F1 generation, minimal focal or multifocal coagulative hepatocellular necrosis was noted for 2/30 males given 15 ppm and a large area of coagulative necrosis was noted in the right median liver lobe for 1/31 females given 15 ppm. In addition, minimal focal hepatocellular vacuolation was noted in the liver of 1/32 males given 112.5 ppm. All these findings can be seen spontaneously in the liver of untreated laboratory rats of this strain and age. Consequently, the reported low incidence was considered to be of no toxicological importance.

PITUITARY GLAND
- Minimal to severe decrease in the number of acidophil cells was noted in the routinely stained
(Hematoxylin-eosin) pituitary sections of all F1males and females given 112.5 ppm, which were killed on completion of the study (for those animals found dead or killed during the study, fixation artifacts prevent a correct histological examination and, therefore, were not evaluated), versus none of their respective controls. This was considered to be related to treatment with the test substance.
- In the F1 generation, minimal to slight presence of vacuolated cells in 28/30 control males, in 28/30 males given 15 ppm and all females (15/15) given 112.5 ppm and which were killed on completion of the study, versus none of the control females. The same finding was noted in 21/21 F1 high dose males, which were killed at term, with minimal to slight intensity for 18/21 males and moderate intensity for 3/21 males. Consequently, the higher intensity of vacuolated cells noted in the pituitary gland of the F1 males given 112.5 ppm, as well as the presence of similar finding for the F0 and F1 females of the same group were considered to be related to treatment with the test substance.

SPLEEN
- For the F1 generation, minimal to slight haemosiderosis was noted in the spleen of 3/30 control males, 11/30 control females, and in 4/29 males and 5/29 females given 112.5 ppm.
- For the F1 generation, slight to marked extramedullary haemopoiesis was noted in 1/30 males and 1/30 females of the control group (slight intensity) and in 7/29 males and 3/29 females given 112.5 ppm. In the high dose group, 4 out of these 7 males and 2 out of these 3 females were killed early in the study, i.e. between Day 2 and 4 of the treatment period. Moderate to marked extramedullary haemopoiesis is an expected finding in young laboratory rats. Therefore, the higher incidence and severity of this finding noted in the high dose group was considered to be of no toxicological importance.
- Minimal to slight lymphoid depletion was noted in the spleen of 4/29 F1 females given 112.5 ppm, vs. none of the controls. All these females were killed prematurely during the study. Consequently, the lymphoid depletion was considered to be related to the poor clinical condition of the animals and to be of no toxicological importance.

FEMALE GENITAL ORGANS
- Morphological changes indicative of a regular oestrous cycle was noted in all F1 control females, in 19/29 F1 females given 112.5 ppm (for 2 other F1 females, found dead during the study, histological evaluation was not possible due to severe autolysis).
- Immaturity was noted for 2/29 F1 females which were found dead or killed prematurely between Day 2 and Day 4 of the treatment period, and is an expected finding in animals of that age. Immaturity was also noted for 1/30 F1 female given 2 ppm and 1/31 F1 female given 15 ppm, which were found dead or killed prematurely on Days 5 and 3, respectively.
- Morphological changes indicative of pregnancy was noted for another high dose F1 female which was killed prematurely on Day 107 of the treatment period. The remaining 7/29 high dose F1 females showed morphological changes indicative of pseudopregnancy (early phase to late phase). The reported high incidence of pseudopregnancy noted in the females given 112.5 ppm of F1 generations was considered to be related to treatment with the test substance.
- For the treated F1 females of the low and intermediate dose groups which were suspected of infertility, evidence of regular oestrous cycle was noted as follows: In the F1 generation, for 5/5 females given 0.5 ppm, 2/4 females given 2 ppm and 5/5 females given 15 ppm.
- The few remaining females (i.e. 2 F1 females given 2 ppm) showed signs indicative of pseudopregnancy. The reported low incidence of these findings was considered to be of no toxicological importance, as they can be seen spontaneously in untreated laboratory rats of this strain and age.

MALE GENITAL ORGANS
- Unilateral or bilateral marked to severe degeneration of seminiferous tubules and inhibition of spermatogenesis in testes together with aspermia in epididymides were noted for 1/30 F1 males given 0.5 ppm and 2/31 F1 males given 112.5 ppm. This was considered to be the cause of infertility which was suspected for the low dose male and 1/2 of the high dose males. The reported low incidence noted in these groups was considered to be of no toxicological importance, as it might be seen spontaneously in untreated laboratory rats of this strain and age.
- No relevant microscopic findings were noted in the testes, the epididymides, prostate and seminal vesicles for the other F1 males of the low, intermediate and high dose groups, which were suspected of infertility.
- Slightly to markedly small seminiferous tubules and minimal to marked absence of spermatogenesis in testes, together with aspermia in epididymides were noted for 5/32 males found dead or killed prematurely between Days 2 and 25 of the treatment period. These findings, which are characteristics of sexual immaturity, are expected findings in young laboratory rats of this age. Changes indicative of sexual immaturity were also noted in the prostate and seminal vesicles for 4 of these young males.

MAMMARY GLAND
- Minimal vacuolation of the acinar and/or ductular epithelium was noted in the mammary gland, for the F1 generation in 1/30 control females and 1/30 females given 15 ppm. This finding was noted with minimal to marked intensity in the mammary gland of 8/31 F1 females given 112.5 ppm. The higher incidence and/or severity of acinar and/or ductular epithelial cell vacuolation noted for the F1 females given 112.5 ppm was considered to be related to treatment with the test substance.

KIDNEY
- Mineralization of urothelium was noted in the renal pelvis of 2/30 control females (minimal), 1/30 males and 2/30 females given 15 ppm (minimal or slight). This finding was noted with a minimal to moderate intensity for 9/27 males and 10/29 females given 112.5 ppm. In addition, urinary gravels were noted in the renal pelvis of 5/27 males and 2/29 females given 112.5 ppm. These changes were considered to be related to the treatment with the test substance.
- Minimal or slight focal urothelium hyperplasia, associated with urothelial mineralization or urinary gravels in pelvis, was noted for 1/30 males given 15 ppm and 2/31 females given 112.5 ppm.
- Marked pyelonephritis or slight leucohistiocytic pyelitis were noted in the kidneys of 1/29 females given 112.5 ppm. These inflammatory changes might be secondary to the urinary gravels noted in the renal pelvis of this animal.
- Minimal to slight mineralization of the papilla and/or medulla was noted in the kidneys of 1/30 control males and 10/30 control females, 1/30 males and 7/31 females given 15 ppm, and 3/27 males and 6/29 females given 112.5 ppm which were killed at end of the treatment period. The same finding was noted in 1/2 examined kidneys of 1/29 females given 112.5 ppm, which was killed prematurely on Day 2 of treatment period, but with moderate intensity. As this finding was noted with similar incidence and severity between the F1 animals of the treated and control groups, which were killed on completion of the treatment period, the moderate intensity noted for the high dose F1 female killed prematurely was considered to be fortuitous and to bear no relationship to treatment with the test substance.
- Morphological changes indicative of moderate retardation of renal maturity were also noted in the examined kidney of this high dose female. This includes: numerous small, compact glomeruli together with small tubules showing basophilism with a few mitoses mainly at the cortico-medullary junction. The retardation of renal maturity noted for the F1 high dose female was considered to be related to treatment with the test substance (see F2 pups).
- In addition, for the F1 generation only, a lower incidence of mononuclear cell aggregation (0 ppm: 15/30 males, 9/30 females; 15 ppm: 18/30 males, 6/31 females; 112.5 ppm: 0/27 males, 0/29 females), tubular basophilia (0 ppm: 15/30 males, 8/30 females; 15 ppm: 16/30 males, 7/31 females; 112.5 ppm: 1/27 males, 0/29 females) and accumulation of acidophilic globules in the cortical tubular epithelium (0 ppm: 23/30 males; 15 ppm: 28/30 males; 112.5 ppm: 0/27 males) was noted in the high dose males and females when compared to controls and animals given 15 ppm. The absence or very low incidence of these above-mentioned findings in the high dose F1 animals might be related to retardation of renal maturity, and was, therefore, considered to be related to treatment with the test substance.
- The few other microscopic findings noted in the treated animals were those which are commonly recorded spontaneously in untreated laboratory rats of this strain and age. Furthermore, their incidence, severity and morphological characteristics were similar (or not dose-related) to those noted in the control animals. Thus, none of these changes was considered to bear a relationship to treatment with the test substance.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:
effects observed, treatment-related
Description (incidence and severity):
- Morphological changes indicative of a regular oestrous cycle was noted in all F1 control females, in 19/29 F1 females given 112.5 ppm (for 2 other F1 females, found dead during the study, histological evaluation was not possible due to severe autolysis).
- Immaturity was noted for 2/29 F1 females which were found dead or killed prematurely between Day 2 and Day 4 of the treatment period, and is an expected finding in animals of that age. Immaturity was also noted for 1/30 F1 female given 2 ppm and 1/31 F1 female given 15 ppm, which were found dead or killed prematurely on Days 5 and 3, respectively.
- Morphological changes indicative of pregnancy was noted for another high dose F1 female which was killed prematurely on Day 107 of the treatment period. The remaining 7/29 high dose F1 females showed morphological changes indicative of pseudopregnancy (early phase to late phase). The reported high incidence of pseudopregnancy noted in the females given 112.5 ppm of F1 generations was considered to be related to treatment with the test substance.
- For the treated F1 females of the low and intermediate dose groups which were suspected of infertility, evidence of regular oestrous cycle was noted as follows: In the F1 generation, for 5/5 females given 0.5 ppm, 2/4 females given 2 ppm and 5/5 females given 15 ppm.
- The few remaining females (i.e. 2 F1 females given 2 ppm) showed signs indicative of pseudopregnancy. The reported low incidence of these findings was considered to be of no toxicological importance, as they can be seen spontaneously in untreated laboratory rats of this strain and age.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
- Unilateral or bilateral marked to severe degeneration of seminiferous tubules and inhibition of spermatogenesis in testes together with aspermia in epididymides were noted for 1/30 F1 males given 0.5 ppm and 2/31 F1 males given 112.5 ppm. This was considered to be the cause of infertility which was suspected for the low dose male and 1/2 of the high dose males. The reported low incidence noted in these groups was considered to be of no toxicological importance, as it might be seen spontaneously in untreated laboratory rats of this strain and age.
- No relevant microscopic findings were noted in the testes, the epididymides, prostate and seminal vesicles for the other F1 males of the low, intermediate and high dose groups, which were suspected of infertility.
- Slightly to markedly small seminiferous tubules and minimal to marked absence of spermatogenesis in testes, together with aspermia in epididymides were noted for 5/32 males found dead or killed prematurely between Days 2 and 25 of the treatment period. These findings, which are characteristics of sexual immaturity, are expected findings in young laboratory rats of this age. Changes indicative of sexual immaturity were also noted in the prostate and seminal vesicles for 4 of these young males.
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
Mating
- Males: The mating index of males was similar in the control, 0.5, 2 and 15 ppm groups. In the 112.5 ppm group, it was decreased (52%) as only 12 out of the 23 males paired were able to mate.
- Females: The mating index of females was similar in the control, 0.5, 2 and 15 ppm groups (between 93.5 and 100%). In the 112.5 ppm group, only 13 females mated (7 out of 31 died or were killed before to be mated and only 13 out of the 24 remaining were able to mate). This decrease in mating index was attributed to treatment.

Fertility
- Males: The fertility index of males was similar in the control, 0.5, 2 and 15 ppm groups (between 82 and 96%). In the 112.5 ppm group, it was lower (67%) as only 8 females became pregnant.
- Females: The fertility index was similar in the control, 0.5, 2 and 15 ppm groups (82.8, 83.3, 89.7 and 86.2%, respectively) and the number of implantation sites was also similar (17.1, 15.7, 16.5 and 16.6 in the control, 0.5, 2 and 15 ppm, respectively). In the 112.5 ppm group, the fertility index was lower i.e. 53.8% as 7 out of the 13 mated females were pregnant. Moreover, the number of implantation sites was lower than that of the control group (6.6 vs. 17.1; p < 0.001). This reduction in the number of implantation sites as well the decrease in the fertility index were attributed to treatment.

Gestation
- The mean duration of gestation was similar in the control, 0.5, 2 and 15 ppm groups. In the 112.5 ppm group, only 6 females delivered (the last pregnant female had total foetal resorption in uterine horns) and their mean duration of gestation of females that delivered was slightly higher (23.0 days vs. 21.5; p < 0.001) as 1 female delivered on Day 22, 4 on Day 23 and 1 on Day 24. This increase in the mean duration of gestation was attributed to treatment. One female had an incomplete delivery.

Details on results (P1)

None

Effect levels (P1)

Key result
Dose descriptor:
NOAEL
Effect level:
2 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
reproductive performance
Remarks on result:
other: equivalent to 0.12 mg/kg bw/day

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
- No clinical signs were noted in F1 pups during lactation.
- Pup physical development: The physical development of pups as assessed by the following parameters: pinna unfolding, hair growth, tooth eruption and eye opening did not show any treatment-related differences between control and treated groups. Auditory canal opening was delayed in the 112.5 ppm group (difference from controls statistically significant). This finding was in line with the retardation of development and correlated with the decreased pup body weight.
Dermal irritation (if dermal study):
not examined
Description (incidence and severity):
not applicable
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- Data at birth: The mean number of live born pups was similar in the control, 0.5, 2 and 15 ppm groups (14.4, 15.6, 13.7 and 14.3 respectively). In the 112.5 ppm group, the mean number of live born pups was lower than that of the control group (11.0 vs. 14.4; p < 0.01) as a consequence of the lower number of implantation sites. This decrease was attributed to the treatment.
- Pup viability: The viability index on Day 4 post-partum was similar in the control and treated groups (92.1, 92.7, 82.8, 93.5 and 94.4% in the control, 0.5, 2, 15 and 112.5 ppm groups respectively). The lower viability noted at 2 ppm (82.8; p < 0.001) was considered to be incidental as the variations in the indices were not dose-related. On Day 21 post-partum, the lactation index was considered unaffected by treatment (80.2, 75.4, 72.1, 67.9 and 81.0% in the control, 0.5, 2, 15 and 112.5 ppm groups, respectively).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Pup body weight: The mean body weight of pups was similar in the control, 0.5,2 and 15 ppm groups. In the 112.5 ppm group, it was significantly lower than that of the control group from Day 1 to Day 21 post-partum (except on Day 4 - not significant). On Day 21 post-partum, the mean body weight of pups was 25.5% lower than that of control pups. This body weight retardation observed at 112.5 ppm was attributed to treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
- Statistically significant lower mean body autopsy weight was noted in the male and female pups from the 112.5 ppm group (-22% for both sexes), when compared to controls.
- Differences from controls in mean absolute and relative organ weights were noted as shown in Table 7.8.1/1. The higher mean thyroid weights from controls noted in the high dose group was considered to be related to treatment with the test substance (see Microscopic examination). The other mentioned lower mean absolute and/or higher mean relative organ weights were in line (about 20% in increase or decrease) with the lower (-22%) mean body autopsy weight noted for these high dose pups.
- No other apparent organ weight differences from controls were noted in the different treated groups.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
- Macroscopic examination of pups culled on Day 4 post-partum and pups dead during lactation: No macroscopic changes attributable to treatment were noted.
- Enlargement of thyroid glands was noted in 9/31 male pups and 5/34 female pups of the 112.5 ppm group. Reddish colour was noted in 2/31 males and 1/34 females of this group. These macroscopic changes were considered to be related to treatment with the test substance (see Microscopic examination).
- Enlargement of thyroid glands, together with a serous cyst in the right gland, was noted for 1/59 female pups of the 0.5 ppm group. Enlargement of adrenal glands was noted for 1/34 female pups of the 112.5 ppm group. However, for both these female pups, these above-mentioned enlargements were not correlated with a higher absolute and/or relative weight. Furthermore, the enlargement of adrenal glands, noted in 1/34 female pups of the 112.5 ppm group is in contradiction with the small adrenals noted in the high dose F0 parents. Consequently, these enlargements were considered to be of no toxicological importance.
- The other macroscopic findings noted were those which are commonly recorded in laboratory rat pups. Furthermore, they were not encountered in the high dose group. They were, thus, considered to bear no relationship to treatment with the test substance.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
THYROID GLAND
- Slight to severe decrease in colloid contents, together with slight to marked follicular cell hypertrophy were noted in the thyroids of 12/15 male pups and 13/17 female pups of the 112.5 ppm group. In addition, minimal follicular cell hyperplasia was also noted in the thyroids for 7/12 of these males and 7/13 of these females.
- These morphological changes, which are indicative of an increased thyroid activity were considered to be related to treatment with the test substance. They were well-correlated with the high thyroid weights and the thyroids enlargement and/or reddish colouration noted at necropsy for some of these animals.
- These microscopic findings were not found in the thyroids of any male or female pups of the low (0.5 ppm) or intermediate (2 and 15 ppm) dose groups.
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- Pup reflex development: The reflex development of pups as assessed by the following parameters: surface righting, cliff avoidance and air righting did not show any treatment-related differences between control and treated groups.

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

None

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
other: F1 pups
Effect level:
15 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
organ weights and organ / body weight ratios
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: equivalent to 0.9 mg/kg bw/day

Results: F2 generation

General toxicity (F2)

Clinical signs:
no effects observed
Description (incidence and severity):
- Pup clinical observations: No clinical signs were noted in pups during lactation.
- Pup physical development: The physical development of pups as assessed by the following parameters: pinna unfolding, hair growth, tooth eruption, eye opening and auditory canal opening did not show any treatment-related differences between control, 0.5, 2 and 15 ppm groups. In the 112.5 ppm group, no clear conclusion could be drawn due to a very low number of pups surviving on the day of testing (between 3 and 7).
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, treatment-related
Description (incidence and severity):
- Data at birth: The mean number of live born pups was similar in the control, 0.5, 2 and 15 ppm groups (16.2, 14.5, 15.0 and 15.5, respectively). In the 112.5 ppm group, when considering the 5 females having a complete delivery, the mean number of live born pups was markedly lower than that of the control group (4.4 vs. 16.2; p < 0.01) as a consequence of the lower number of implantation sites as well as post-implantation loss. This decrease was attributed to treatment.
- Pup viability: The viability index on Day 4 post-partum was similar in the control and treated groups (83.8, 88.5, 85.6 and 87.9% in the control, 0.5,2 and 15 ppm groups, respectively). In the 112.5 ppm group, it was 36.4% (8 out of 22 pups were surviving on Day 4 post-partum). This decrease was attributed to treatment. On Day 21 post-partum, the lactation index was considered to be similar in the control, 0.5, 2 and 15 ppm groups (82.6, 83.6.78.2 and 83.8% respectively). In the 112.5 ppm group, it was 37.5% as only 3 pups were surviving on Day 21 post-partum. This decrease was attributed to treatment.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
- Pup body weight: The mean body weight of pups was similar in the control, 0.5,2, 15 ppm groups. In the 112.5 ppm group, it was significantly lower than that of the control group from Day 1 to Day 21 post-partum. On Day 21 post-partum, the mean body weight of pups was 77.7% lower than that of control pups. This body weight retardation was attributed to treatment.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
- Only one male and one female F2 pups of the high dose group were killed and weighed on Day 22 or 23 post-partum. When compared to mean control values, markedly lower body autopsy weight (-70% and -81%, respectively) together with moderately to markedly lower individual absolute organ weights (including the thyroid glands) were noted for these animals. These individual absolute lower values were considered to be related to the low autopsy body weight.
- Higher individual relative thyroid weight was noted for the high dose male and female pup when compared to mean value of respective controls (+136% and +107%, respectively). As these weight values were not associated with any relevant histological changes, this was considered to be of no toxicological importance.
- The slight to marked differences from controls noted for the other relative organ weights were considered to be due to the low body weight recorded for both these F2 pups.
- No apparent differences in mean organ weights were noted between the F2 pups of the other treated (low and intermediate dose) and control groups.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
- Only one male and 2 female F2 pups of the high dose group were killed on Day 24 (postpartum) of the study. No macroscopic findings were noted for these 3 animals.
- The few macroscopic findings noted for the F2 pups of the other treated (low and intermediate dose) groups were noted with a similar incidence to that recorded in the control F2 pups or showed no dose-related incidence. Consequently, they were considered to bear no relationship to treatment with the test substance.
Histopathological findings:
effects observed, treatment-related
Description (incidence and severity):
THYROID GLAND
- No histological changes indicative of an increased thyroid activity were noted in the thyroid glands of the 0.5 ppm, 2 ppm, 15 ppm, and 112.5 ppm F2 pups, when compared to control F2 pups.

KIDNEY
- Morphologic changes indicative of retardation of maturity were noted in the kidneys of 1/21 control females pups and of the examined male and female F2 pups of the 112.5 ppm group: these changes consisted of small, compact glomeruli together with small tubules showing basophilism, mainly at the cortico-medullary junction and medulla. Mitoses were noted in the basophilic tubular epithelium. These changes were noted with higher intensity for both 112.5 ppm F2 pups (marked or severe) when compared to the control F2 female (moderate). They were not recorded for the remaining control F2 pups (20/20 males and 20/21 females) and for the 15 ppm F2 pups (22 males and 22 females).
- Immature kidneys noted for both these F2 high dose pups were considered to be related to treatment with the test substance. The same can be said for the high dose F1 female which was killed prematurely on Day 2 of treatment.
Other effects:
no effects observed
Description (incidence and severity):
- Macroscopic examination of pups culled on day 4 post-partum and pups dead during lactation: No macroscopic changes attributable to treatment were noted.

Developmental neurotoxicity (F2)

Behaviour (functional findings):
no effects observed
Description (incidence and severity):
- Pup reflex development: The reflex development of pups as assessed by the following parameters: surface righting, cliff avoidance and air righting did not show any treatment-related differences between control, 0.5, 2 and 15 ppm groups. In the 112.5 ppm group, no clear conclusion could be drawn due a very low number of pups surviving on the day of testing (between 3 and 7).

Developmental immunotoxicity (F2)

Developmental immunotoxicity:
not examined

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
other: F2 pups
Effect level:
15 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
viability
body weight and weight gain
organ weights and organ / body weight ratios
histopathology: non-neoplastic
Remarks on result:
other: equivalent to 0.9 mg/kg bw/day

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Table 7.8.1/1: Changes in organ weights

 

Differences (%) from controls in mean

F0 Parents

F1 Pups

F1 Parents

Absolute weight

Relative weight

Absolute weight

Relative weight

Absolute weight

Relative weight

Organ

M

F

M

F

M

F

M

F

M

F

M

F

Thyroid glands

+316

+540

+412

+514

+75

+50

+92

+85

+218

+356

+920

+986

Adrenal glands

-51

-56

-11

-48

-15 (NS)

-11 (NS)

+9 (NS)

+17 (NS)

-62

-72

+22

-25

Liver

-41

-23

-17

-17

-250

-58

-10

+5 (NS)

Pituitary gland

-21

-18

+33

-50

-44

+100

+50

Spleen

-68

-49

-46

-38

-24

-24

-81

-73

-151

-50

Ovaries

-31

-15

-19

-57

-19

+8 (NS)

Uterus

-19

-25

-30

+95

Testes

-11

+49

-22

-17

+166

Epididymides

-16

+41

+23

-33

+115

Prostate

-37

+26

-45

+76

Seminal vesicles

-24

+26

+23

-40

+88

Kidneys

-75

-64

-8

M: Male; F: Female; NS: Not statistically significant

Applicant's summary and conclusion

Conclusions:
Under the experimental conditions, the test substance was not toxic for F0 and F1 parent animals up to 2 ppm and did not induce adverse effects on the reproductive performance of F0 and F1 animals, nor impaired the development of F1 and F2 off-springs up to 15 ppm. The dose of 15 ppm was toxic for the F0 (males only) and F1 parental animals, as shown by minimal increased thyroid activity. The 112.5 ppm dose level was severely toxic for F0 and F1 parents, impaired their fertility and disturbed the development of F1 and F2 litters. Nevertheless, all findings on reproduction correlated with severe systemic parental toxicity, so that a primary effect on reproduction is excluded. Consequently, the No Observable Adverse Effect Level (NOAEL) of the test substance was considered to be 2 ppm (equivalent to 0.12 mg/kg bw/day) for parental toxicity and at 15 ppm (equivalent to 0.9 mg/kg bw/day) for reproductive toxicity.
Executive summary:

A two-generation reproduction toxicity study was performed in rats according to the OECD guideline 416 and in compliance with GLP to evaluate the potential toxic effects of the test substance on the reproductive performance of F0 and F1 parents and on growth and development of the respective F1 and F2 off-springs.

The test substance was given continuously to 5 groups of F0 Sprague-Dawley rats (30/sex/dose) by admixture to the diet at the dose levels of 0.5, 2, 15 and 112.5 ppm. The F0 control animals (30 males and 30 females) received the untreated diet only. After 72 days of treatment in males and females, the F0 male and female rats were paired. Treatment was continued in males until weaning of the F1 offspring and in females during the same period. F0 animals were observed daily for clinical signs. Food consumption and body weight were measured at designated intervals. The F0 females were allowed to deliver normally. The F1 litters were examined daily for clinical signs, viability and physical and reflex development during lactation. Pup body weights were recorded on Days 1, 4, 7, 14 and 21 postpartum. On Day 21 post-partum 30 to 31 F1 pups of each sex were selected and treated at the same dose levels as their parents. They were paired when they were 12 to 14 weeks old. F1 animals were observed daily for clinical signs. Food consumption and body weight were measured at designated intervals. The F1 females were allowed to deliver normally. The F2 litters were examined daily for clinical signs, viability, physical and reflex development during lactation. Pup body weights were recorded on Days 1, 4, 7, 14 and 21 postpartum. Between Days 22 and 25 postpartum, pups were killed. Macroscopic examination of F0 males and females, F1 males and females, F1 and F2 pups was performed. Organ weights and microscopic examination were performed on a range of tissues.

F0 clinical parental data

- No clinical signs attributable to treatment and no deaths were observed.

- In the 112.5 ppm group, the mean food consumption was significantly reduced in males (differences from the control group ranging from 3 to 45%) and in females (differences from the control group ranging from 13 to 33%).

- In the 112.5 ppm group, the body weight gain of males was significantly lower than that of the control group from the third week of treatment. At the time of sacrifice, the mean absolute body weight of males was 40% lower than that of control males. In females, it was reduced during the premating period and during the pregnancy.

- The mean achieved dose levels of the test substance were 0.03, 0.12, 0.90 and 5.88 mg/kg bw/day in males and 0.04, 0.16, 1.23 and 7.83 mg/kg bw/day in females for the 0.5, 2, 15 and 112.5 ppm concentrations, respectively.

F0 reproductive data

- In the 112.5 ppm group, the mating index was lower than that of controls (77% in males and 83.3% in females).

- In the 112.5 ppm group, the fertility index was lower than that of controls (87% in males and 88.0% in females vs 100% in controls) and the number of implantation sites was 12.2 vs. 16.2 in controls.

- The mean duration of gestation was similar in the control and all treated groups.

F1 litter data

- In the 112.5 ppm group, the mean number of live born pups was lower than that of controls (11.0 vs. 14.4 in controls) as a consequence of the lower number of implantation sites.

- The viability index on Day 4 and Day 21 post-partum was similar in the control and all treated groups.

- In the 112.5 ppm group, the mean body weight of pups was significantly lower than that of controls: on Day 21 post-partum, the mean body weight of pups was 25.5% lower than that of control pups.

- The physical development of pups as assessed by the following parameters: pinna unfolding, hair growth, tooth eruption and eye opening and the reflex development of pups as assessed by the following parameters: surface righting, cliff avoidance and air righting did not show any treatment-related differences between control and treated groups. The opening of the auditory canal opening was delayed in the pups of the 112.5 ppm group. This finding was in line with the retardation of development and correlated with the decreased pup body weight.

- No clinical signs were noted in pups during lactation.

- No macroscopic changes attributable to treatment were noted in pups culled on day 4 post-partum and pups dead during lactation.

F1 clinical data

- In the 112.5 ppm group, hypoactivity, piloerection and dyspnea were noted in some males and females.

- In the 112.5 ppm group, 6 males and 4 females were found dead between Days 3 and 135 and 2 males and 6 females were killed for poor clinical condition between Days 2 and 112. During pregnancy, one female was also found dead on Day 17 and during lactation, one female was killed on Day 2 post-partum for incomplete delivery.

- In the 112.5 ppm group, the mean food consumption was reduced in males and females (differences from the control group ranging from 32% to 59%).

- In the 112.5 ppm group, the mean body weight gain was lower than that of controls, from the first week of treatment, in males and females. At the time of sacrifice, the mean absolute body weight of males was 67% lower than that of control males. In females, the mean body weight gain was lower than that of the control group during the premating period (67 g vs. 261 g), during the pregnancy (45 g vs. 160 g).

- The mean achieved dose levels of the test substance were 0.04, 0.16, 1.24 and 12.02 mg/kg bw/day in males and 0.05, 0.21, 1.64 and 15.64 mg/kg/day in females for the 0.5, 2, 15 and 112.5 ppm concentrations, respectively.

F1 reproductive data

- In the 112.5 ppm group, the mating index was markedly reduced in males (52%) and in females (54.2%).

- In the 112.5 ppm group, the fertility index was lower in males (67% vs. 82% in controls) and in females (53.8% vs. 82.8% in controls) and the number of implantation sites was 6.6 vs. 17.1 in controls.

- In the 112.5 ppm group, the mean duration of gestation was slightly increased (23.0 days vs. 21.5 days in controls).

F2 litter data

- In the 112.5 ppm group, the mean number of live born pups was markedly lower than that of controls (4.4 vs. 16.2 in controls) as a consequence of the lower number of implantation sites.

- In the 112.5 ppm group, the viability index was 36.4% on Day 4 post-partum (vs. 83.8% in controls) and 37.5% on Day 21 post-partum (vs. 82.6% in controls).

- In the 112.5 ppm group, the mean body weight of pups was significantly lower than that of controls: on Day 21 post-partum, it was 77.7% lower than that of control pups.

- The physical development of pups as assessed by the following parameters: pinna unfolding, hair growth, tooth eruption, eye opening and auditory canal opening and the reflex development of pups as assessed by the following parameters: surface righting, cliff avoidance and air righting did not show any treatment-related differences between control, 0.5, 2 and 15 ppm groups. In the 112.5 ppm group, due to a very low number of pups no clear conclusion could be drawn.

- No clinical signs were noted in pups during lactation.

- No macroscopic changes attributable to treatment were noted on pups culled on Day 4 postpartum and pups dead during lactation.

Pathology in F0 and F1 generations

- Low autopsy body weight was noted in the F0 and F1 parents, F1 and F2 pups of the 112.5 ppm group, together with high thyroid weights and low absolute and/or high relative weights in many organs, related to the small size of the high dose animals in all generations.

- Thyroid enlargement, sometimes with reddish colour, was noted in all F0 parents, almost all F1 parents and some F1 pups of the 112.5 ppm group. Small adrenals, small spleen, small kidneys, reddish pituitary gland were noted for some of the high dose F0 and F1 animals, and small liver was noted for some prematurely killed F1 parents given 112.5 ppm.

- Morphological changes indicative of a moderate to severe increased thyroid activity were noted for the F0 and F1 parents and F1 pups of the 112.5 ppm group, together with nodular follicular cell hyperplasia and/or follicular cell adenoma in the thyroids of few of these F0 and F1 parents. Minimal increased thyroid activity was noted for the F0 (males only) and F1 parents given 15 ppm. This was the only microscopic change observed at this dose-level. Hepatocellular hypertrophy was noted in the liver of almost all F0 and F1 parents given 112.5 ppm, together with hepatocellular degeneration/necrosis and perilobular steatosis in the liver of some F0 parents. In addition, in F0 and F1 parents given 112.5 ppm, treatment-related changes were noted as follows: decreased number of acidophil cells and/or increased number of vacuolated cells in pituitary gland (pars distalis), vacuolation of acinar and/or ductular cells in mammary glands for the females, cortical adrenal atrophy for females and increased ceroid pigment accumulation in adrenal cortex for the males, mineralization of urothelium and/or urinary gravels in kidneys for both sexes. Retardation of kidney maturity was noted for few F1 parents and the 2 examined F2 pups.

Under the experimental conditions, the test substance was not toxic for F0 and F1 parent animals up to 2 ppm and did not induce adverse effects on the reproductive performance of F0 and F1 animals, nor impaired the development of F1 and F2 off-springs up to 15 ppm. The dose of 15 ppm was toxic for the F0 (males only) and F1 parental animals, as shown by minimal increased thyroid activity. The 112.5 ppm dose level was severely toxic for F0 and F1 parents, impaired their fertility and disturbed the development of F1 and F2 litters. Nevertheless, all findings on reproduction correlated with severe systemic parental toxicity, so that a primary effect on reproduction is excluded. Consequently, the No Observable Adverse Effect Level (NOAEL) of the test substance was considered to be 2 ppm (equivalent to 0.12 mg/kg bw/day) for parental toxicity and at 15 ppm (equivalent to 0.9 mg/kg bw/day) for reproductive toxicity.