2-(1-(2-hydroxy-3,5-di-tert-pentyl-phenyl)ethyl)-4,6-di-tert-pentylphenyl acrylate

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

10 Jul 2013 - 29 Aug 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study

Data waiving:

other justification

Justification for data waiving:

other:

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Version / remarks:

1983

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-(1-(2-hydroxy-3,5-di-tert-pentyl-phenyl)ethyl)-4,6-di-tert-pentylphenyl acrylate

EC Number:

413-850-6

EC Name:

2-(1-(2-hydroxy-3,5-di-tert-pentyl-phenyl)ethyl)-4,6-di-tert-pentylphenyl acrylate

Cas Number:

123968-25-2

Molecular formula:

C37H56O3

IUPAC Name:

2-{1-[2-hydroxy-3,5-bis(2-methylbutan-2-yl)phenyl]ethyl}-4,6-bis(2-methylbutan-2-yl)phenyl prop-2-enoate

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Name of test material (as cited in study report): SUMILIZER GS (F)
- Physical state: granulated powder
- Analytical purity: 98.3%
- Lot/batch No.: 121122
- Stability under test conditions: The stability of the test substance during the experimental period was confirmed based on the results of stability analysis
- Storage condition of test material: at room temperature (actual temperature test facility: 18.5 - 24.1 °C; actual temperature test site: 14.9 - 22.1 °C) in a dark place

Test animals

Species:

rat

Strain:

other: BrlHan: WIST@Jcl (GALAS)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Fuji Breeding Center, CLEA Japan, Inc.
- Age at study initiation: (P) 6 wks
- Weight at study initiation: (P) Males: 137.4 - 175.3 g; Females: 103.3 - 128.3 g
- Housing: 1 animal/cage in stainless-steel cages (W × D × H: 226 × 346 × 198 mm); females during gestation and lactation periods were accomodated in polymethylpentene cages (W × D × H: 220 × 380 × 195 mm)
- Diet: powder diet for experimental animals (CRF-1, Oriental Yeast Co., Ltd.), ad libitum
- Water: well water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.4 - 24.7
- Humidity (%): 38.5 - 96.5* (*Due to lightning, a refrigerator shut down and as a result, humidity of the animal room went to over 75% of the acceptable range. However, as no abnormalities related to this deviation were observed in the clinical signs, it was judged to have no effect on this study.)
- Air changes (per hr): 10-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 27 Aug 2013 To: 11 Mar 2014

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): 10 preparations in total (15 Aug 2013 - 10 Feb 2014), approx. once every 4 weeks
- Mixing appropriate amounts with (Type of food): Diet: Powder diet for experimental animals (CRF-1, Oriental Yeast Co., Ltd.)
Mixed diet with the test substance for stability analysis was prepared at approximately 16 kg for each dose level;
- Storage temperature of food: at room temperature (13.6 - 22.0 °C)

Details on mating procedure:

- M/F ratio per cage: one-to-one
- Length of cohabitation: 14 days
- Proof of pregnancy: vaginal plug or sperm in the vaginal smear on the following morning; the day of confirmed copulation was designated as Day 0 of gestation.
- After successful mating each pregnant female was caged (how): one per cage
- Any other deviations from standard protocol: no

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Upon the first and final preparation of the test substance mixed diet for use in the study, a specified portion was collected from the 3 parts (upper, middle, and lower layers, n=2 each) in the container before use. The concentrations of the test substance were determined by HPLC analysis. According to the results, the mean test substance concentration was confirmed to be within 100±10% of the nominal concentration.

Duration of treatment / exposure:

Administration to F0 (Parental) animals was conducted every day from 6 weeks of age until mating for 10 weeks for both sexes. Administration to males was conducted through the mating period until the day of necropsy. Administration to females was conducted through the mating, gestation, and lactation periods until the day of weaning the F1 offspring (Day 21 of lactation). Total administration period was 14 weeks for males and 17 weeks for females. However, administration to non-delivering females was conducted until 26 days after copulation (day of the necropsy), and that to non-copulation females was conducted until the day of necropsy. Administration to F1 animals was conducted every day from weaning (postnatal Day 21) until mating for 10 to 11 weeks for both sexes.

Frequency of treatment:

continuously in the diet

Details on study schedule:

Selection of parents from F1 generation when pups were 21 days of age.

Doses / concentrations

Remarks:

Doses / Concentrations:
2000, 7000, and 20000 ppm
Basis:
nominal in diet
equivalent to 146.5, 510.1, and 1490.4 mg/kg bw/d for F0 (P) males and 175.8, 621.0, and 1817.5 mg/kg bw/d for F0 (P) females; 203.8, 705.2, and 2067.7 mg/kg bw/d for F1 males, and 224.3, 786.2, and 2279.4 mg/kg bw/d for F1 females, respectively.

No. of animals per sex per dose:

F0 (P): 24
F1: 46 (control), 42 (2000 ppm), 47 (7000 ppm), 44 (20000 ppm)

Control animals:

yes, plain diet

Details on study design:

The dose levels were selected based on the results of a "Dose Range-Finding Study for One-Generation Reproduction Toxicity Study of SUMILIZER GS (F) in Rats". In that study, the test substance at 0, 2000, 7000, and 20000 ppm was administered orally via the diet to male and female rats [BrlHan:WIST@Jcl (GALAS)] from 2 weeks before mating, and through the mating period until the day of the necropsy in males, and through the mating, gestation, and lactation periods until the day of the weaning of F1 offspring (Day 21 of lactation) in females. In the 20000 ppm group (chemical intake [mean]; males before mating period 1377.65 mg/kg/day, females before mating period 1609.97 mg/kg/day, gestation period 1390.89 mg/kg/day, lactation period 3318.18 mg/kg/day), no death occurred and no changes due to administration were observed in the clinical observation, body weight, food consumption, reproductive function in either sex, or fertility, parturition, or nursing behavior in females or development of offspring (weight gain). Based on these results, the high dose level was set at 20000 ppm, and the middle and low dose levels were set at 7000 and 2000 ppm, respectively, with a common ratio of about 3.

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
F0 (P) animals were observed from cage-side once a day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: observation by palpation at least once a week

BODY WEIGHT: Yes
- Time schedule for examinations:
Body weights were measured on the first day of dosing. Thereafter, they were measured once a week until the necropsy, including the day of the necropsy for males and until copulation for females. The copulated females were weighed on Days 0 (day of copulation), 7, 14, and 20 of gestation and on Days 0 (day of parturition), 4, 7, 14, and 21 of lactation (day of the necropsy). Moribund animals were measured on the day of the necropsy. For males, body weight gain was calculated on the basis of the body weight on the first day of dosing during the pre-mating and through the mating periods until the day of necropsy. For females, body weight gain was calculated on the basis of the body weight on the first day of dosing during the pre-mating period, on Day 0 of gestation during the gestation period, and on Day 0 of lactation during the lactation period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption was measured once a week from the first day of dosing to the week of the necropsy for males, except for the period of cohabitation with a female, and it was measured once a week from the first day of dosing through the start of mating for females. For copulated females, food consumption was measured from Days 0 to 7, 7 to 14, and 14 to 20 of gestation and from Days 0 to 4, 4 to 7, 7 to 14, and 14 to 21 of lactation. The gross weight of the diet was measured for each cage, and daily food consumption of each animal was calculated by dividing the difference of the measured gross weights by the number of days between the measurements.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

Oestrous cyclicity (parental animals):

For all females (F0 (P) female: 14 weeks of age), a vaginal smear was collected with a swab every day in the morning for 2 weeks beginning at 9 weeks after the start of dosing before mating period and from the day after the start of mating until copulation was confirmed in the mating period. The smear was stained with Giemsa, and the stage of the estrous cycle was examined under a microscope. The vaginal smear of the same animal was collected on a single piece of slide glass. The stage of the estrous cycle was classified into diestrus (D), proestrus (P), estrus (E), or metestrus (M).
Estrous cycle: Average number of days from estrous (E) to the next estrous (E)
Count of estrus: Count of estrous (E) during the examination period

Sperm parameters (parental animals):

Parameters examined in [P/F1] male parental generations:
[P]: testis weight, epididymis weight at scheduled necropsy
[F1]: testis weight, epididymis weight on PND 91

Litter observations:

STANDARDISATION OF LITTERS
On postnatal Day 4, the litter size was standardized to 8 (4/sex/litter) by random removal of F1 pups. Even when the number of either males or females per litter was less than 4, the litter size was adjusted to 8 by leaving more than 4 males/females. Litter with less than 8 pups was maintained as it is. Pups culled at the litter size adjustment were euthanized by overdosing with pentobarbital sodium (stock solution 0.05 to 0.1 mL/body). Then, they were fixed and preserved in a 10 vol% phosphate buffered formalin solution.

SELECTION OF F1 ANIMALS AT WEANING
As for F1 animals, 2 males and 2 females were selected from each litter upon weaning (postnatal Day 21) and subjected to administration. When the original test animal died, stock animals were used as replacements and the experiment was performed with the specified number of animals. Two males and 2 females were selected from each litter (litters 1 and 2). The subsequent observation and test were carried out using litter 1 and the clinical observation as well as body weight and food consumption measurements were performed using litter 2, and the chemical intake was calculated.

PARAMETERS EXAMINED
All newborns were examined for the number of offspring (live or stillborn), weighed, sexed, and examined for external anomalies. The birth index was calculated for each litter based on the numbers of live newborns and implantation sites.

GROSS EXAMINATION OF DEAD PUPS
Stillborns were fixed and preserved in a 10 vol% phosphate buffered formalin solution.

Postmortem examinations (parental animals):

On Day 98 of dosing, males were euthanized by exsanguination from the lateral iliac artery under anesthesia by intraperitoneal injection of pentobarbital sodium (30 mg/kg bw), and the head, thoracic and abdominal organs/tissues were examined macroscopically. On Day 21 of lactation, dams were euthanized by exsanguination in the same manner as the males, and their head, thoracic and abdominal organs/tissues were examined macroscopically. Then, the number of implantation sites was counted after removing the uterus. Non-delivery females were necropsied 26 days after copulation. Non-copulated females were necropsied 13 days after the mating period (day of the necropsy in males).
The following organs/tissues of all animals were fixed and preserved in 10 vol% phosphate buffered formalin. However, the testis and epididymis of the terminal necropsy animals were fixed in Bouin’s solution and preserved in a 10 vol% phosphate buffered formalin solution:
pituitary, testis, epididymis, seminal vesicle, coagulating gland, prostate (ventral lobe), ovary, fallopian tube, uterus (including cervical region), vagina, gross lesions.
Besides, one animal of the control group showed paralytic gait due to paralysis of both hind legs from Day 89, which resulted in a significant decrease in body weight and food consumption as it became difficult to ingest food and water normally. Although a follow-up observation was carried out, as these conditions continued without recovery, this animal was judged appropriate for necropsy under moribund conditions from the standpoint of animal welfare. The necropsy was performed on Day 92. In addition to the above-stated organs and tissues, abnormal lesions including the liver, spleen, pancreatic lymph node, renal lymph nodes, and medial iliac lymph nodes as well as the brain and spinal cord suspected being relevant to paralytic gait were fixed and preserved in a 10 vol% phosphate buffered formalin solution.

Postmortem examinations (offspring):

At necropsy on postnatal Day 21 (weaning day) and postnatal Day 91, the following organs of both sexes were weighed. The relative organ weights (ratio to body weight) were calculated based on each body weight at necropsy. The gross weight of the bilateral organs was weighed individually (separately left and right) and calculated as a sum. Postnatal Day 21 (weaning day): brain, thymus, and spleen. Postnatal Day 91: testis, epididymis, ovary, and uterus (including cervical region).

On postnatal Day 21 (weaning day), pups in litters 3 or 4 were euthanized by exsanguination from the lateral iliac artery under anesthesia by intraperitoneal injection of pentobarbital sodium (stock solution 0.1 to 0.5 mL/body), and their heads, thoracic, and abdominal organs/tissues were examined macroscopically. On postnatal Day 91, pups in litter 1 were euthanized by exsanguination from the lateral iliac artery under anesthesia by intraperitoneal injection of pentobarbital sodium (30 mg/kg), and their heads, thoracic and abdominal organs/tissues were examined macroscopically. The organs/tissues removed for organ weight (including pups in litters 3 or 4) and gross lesion were fixed and preserved in 10 vol% phosphate buffered formalin. The testes and epididymis of the terminal necropsy animals were fixed in Bouin’s solution and preserved in 10 vol% phosphate buffered formalin solution. On postnatal Days 99 to 103, pups in litter 2 were necropsied in the same manner as the pups in litter 1.

Statistics:

Statistical analysis was performed for the following data, except for the sex ratio, using a computer system (Provantis®, Instem LSS Limited). SAS 9.1.3 (SAS Institute Japan Ltd.) was used for the sex ratio. A two-tailed test was used, and levels of p<0.01 and p<0.05 were considered significant in either case. The mean and standard deviation were calculated by multiple comparisons test in each group. Bartlett’s test was performed to compare the variances among the control and test article groups. If variance of the data was homogeneous, Dunnett’s multiple comparisons test was performed between the control and each test substance group. If variance of the data was heterogeneous, Steel's test was performed between the control and each test substance group. Non-parametric data such as incidence were calculated, and Wilcoxon’s rank sum test was performed between the control and each test substance group. Other non-parametric data were analyzed by Chi-square test. The data of offspring before weaning were analyzed on the basis of litter mean values, except for the sex ratio. The following data were excluded from the analysis: body weights, body weight gain, and food consumption from non-pregnant females after copulation.
(1) Multiple comparisons test
Body weight, body weight gain, food consumption, estrous cycle length, count of estrous cycles, number of days required for successful copulation, organ weight (including relative organ weight), gestation length, number of implantations, number of offspring, and number of live offspring.
(2) Wilcoxon’s rank sum test
Birth index, viability index on Day 4, weaning index, incidence of live newborns with external anomalies, and sexual maturation (vaginal opening, preputial separation).
(3) Chi-square test
Copulation index, fertility index, gestation index, and sex ratio (Number of live male offspring / Number of live female offspring).

Reproductive indices:

Number of days required for successful copulation: Number of days from the start of mating to the detection of copulation
Copulation index (%): (Number of copulation pairs/Number of mated pairs) × 100
Fertility index (%): (Number of pregnant females/Number of copulated females) × 100

Offspring viability indices:

Viability index on Day 4 (%): (Number of offspring alive on Day 4 / Number of offspring born alive) × 100
Weaning index (viability index on Day 21) (%): (Number of live weanlings / Number of live offspring after culling) × 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not specified

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No test substance-related mortality or abnormal clinical signs were observed in males or females of any group throughout the observation period, including the gestation and lactation periods of pregnant females.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
No test substance-related changes were observed in the body weight or body weight gain in either sex of any treated group. In the 20000 ppm group, body weight gain decreased significantly in females on Day 14 of lactation. However, body weight gain in females was almost comparable to that in the control group before mating throughout the gestation period. This change was considered to be a transient change and judged to be of no toxicological significance. In the 7000 ppm group, body weight gain decreased significantly in males on Days 22 and 29 of dosing; however, it was judged to be incidental because it was a transient change without dose-dependency. In the 2000 ppm group, body weights were comparable to those in the control group in males and females throughout the observation period, including the gestation and lactation periods of pregnant females, with no significant differences in body weight or body weight gain.
No test substance-related changes were observed in the food consumption in either sex of any treated group. In the 20000 ppm group, food consumption increased significantly in females on Days 8 to 15 and Days 15 to 22 of dosing. In the 7000 ppm group, food consumption increased significantly in females on Days 0 to 7 of gestation. These differences were not considered to be test substance-related because the increases were very slight and the gain in food consumption was not considered to be toxicologically important. In the 2000 ppm group, food consumption was comparable to that in the control group in males and females throughout the observation period, including the gestation and lactation periods of pregnant females, with no significant differences in food consumption.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
The mean test substance intakes before mating period (or nurturing period) in the 2000, 7000, and 20000 ppm groups were 146.5, 510.1, and 1490.4 mg/kg bw/day for F0 (P) males and 175.8, 621.0, and 1817.5 mg/kg bw/day for F0 (P) females, 203.8, 705.2, and 2067.7 mg/kg bw/day for F1 males and 224.3, 786.2, and 2279.4 mg/kg bw/day for F1 females, indicating that the exposure increased dose-proportionally among the treated groups. The mean intakes before mating period in F1 animals were about 1.3 to 1.4-fold for both sexes compared with those in F0 (P) animals, reflecting the higher intake in the growth period after weaning. The mean intake during the gestation period was almost equal to that in the later stage before mating period, whereas the mean intake during the lactation period was more than 2-fold of that during the gestation period in F0 (P) females.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No test substance-related changes were observed in the estrous cycles in females of any treated group. Almost all females had a normal estrous cycle of 4 to 5 days in each group, and no significant differences were found in the mean estrous cycle length. One female in the 2000 ppm group showed irregular estrous cycles due to continuous diestrus. However, it was judged to be incidental, because continuous diestrus is seen spontaneously in this strain, and the corresponding female showed estrus in the mating period, with copulation, and conception as described in the next section.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No test substance-related changes were observed in the mating ability or fertility of any treated group. Almost all mating pairs of each group copulated during the first estrus cycle after the start of mating (except: 1 female of the 2000 ppm group, No. 927), and no significant differences were observed in the number of days required for successful copulation. The copulation indices were 100.0, 95.8, 100.0, and 100.0% for the control, 2000, 7000, and 20000 ppm groups, respectively, and the fertility indices were 95.8, 91.3, 100.0, and 91.7% for the control, 2000, 7000, and 20000 ppm groups, respectively. The number of non-pregnant females was 1, 2, and 2 for the control, 2000, and 20000 ppm groups, respectively.

ORGAN WEIGHTS (PARENTAL ANIMALS)
In females, significant decreases in the absolute and relative weights of the ovaries were noted in the 20000 ppm group. The slightly lower ovary weights were unlikely to be toxicologically significant because 1) the decrease was very slight, 2) no effect was seen on fertility or reproductive function, 3) there was no change in the ovary weight of the F1 generation (breeding for 13 weeks), 4) similar decreases did not occur at 20000 ppm in the dose-finding study. Moreover, there were no changes in ovary weights or its histopathology at 20000 ppm (1430 mg/kg bw/day) in a 3-month subacute diet toxicity study. In males, there were no significant differences in the absolute weights or relative weights in the treated groups as compared to those in the control group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
There were no changes in any treated animal (except: 1 male of the control group).

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

>= 20 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

2

Dose descriptor:

NOAEL

Effect level:

>= 1 817.5 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: highest dose tested

3

Dose descriptor:

NOAEL

Effect level:

>= 1 490.4 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: highest dose tested

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

not specified

Details on results (F1)

VIABILITY (OFFSPRING)
All parameters in each treated group, including the numbers of stillborn and live newborn offspring, sex ratio, and birth index were comparable to those in the control group, with no significant differences.

CLINICAL SIGNS (OFFSPRING)
No test substance-related mortality or abnormal clinical signs were observed in males or females of any treated group. Before weaning, death was observed sporadically in a few pups in all groups, including the control group, mainly up to postnatal Day 4. As for the death after the litter size adjustment on postnatal Day 4, one dead female pup (No. 951-6) was observed in the 7000 ppm group from postnatal Day 5, and it was cannibalized by the dam. In addition, 2 dead female pups (Nos. 978-8 and 979-5) were observed in the 20000 ppm group on postnatal Days 16 and 21, respectively. Among dead pups in the 20000 ppm group, decrease in locomotor activity, bradypnea, pale skin, and abdominal distention were observed in 1 female pup (No. 979-5) from postnatal Day 20, no clinical signs were observed in the other female pup (No. 978-8). As a result of the necropsy, enlargement of the liver was observed in 1 female pup (No. 978-8), and small thymus and spleen were observed in the other female pup (No. 979-5). However, these findings were judged to be not test substance-related because of their low occurrence (only in a few cases). No external anomaly was observed in these pups. Other findings included a black patch on the back in 1 pup in the 7000 ppm group on postnatal Day 0, and the pup was cannibalized by its dam on postnatal Day 1. However, this finding was judged to be not test substance-related because there was no dose-dependency in its incidence. In addition, viability index on Day 4 and weaning index were comparable to those in the control group, with no significant differences. After weaning, there were no death and test substance-related abnormal clinical signs in males and females of any group throughout the observation period. Loss of teeth was observed in 1 male in the control group from postnatal Day 95 until necropsy (display; Days 75 to 80). Small testis was observed in 1 male in the 7000 ppm group from postnatal Day 36 until necropsy (display; Days 16 to 71). However, these findings were judged to be not test substance-related because there was no dose-dependency in their incidences.
The following changes occurred incidentally:
Malocclusion (deformity of upper incisor) was observed in 2 males in the 7000 and 20000 ppm groups, respectively, and was observed in 1 female in the 7000 ppm group from postnatal Day 36. However, this change was judged to be not test substance-related because it was considered that the animals' upper incisor was hooked in clearance of the feeder, which caused the malocclusion (deformity of upper incisor). Of these animals, as 1 male with decreased locomotor activity, nasal noise, and reddish tear from postnatal Day 41 showed signs of deterioration, this animal was necropsied on the next day due to moribund condition. As a result of necropsy, fracture of the nasal bone was observed. All of these changes were considered to have occurred incidentally due to the upper incisor being hooked in the feeder and were not considered to be test substance-related.

BODY WEIGHT (OFFSPRING)
No test substance-related changes were observed in the body weights in either sex of any treated group. In all treated groups, birth weights and postnatal body weights in males and females were almost comparable to those in the control group before weaning and through the nurturing period, with no significant differences.

FOOD CONSUMPTION (OFFSPRING)
No test substance-related changes were observed in the food consumption in either sex of any treated group. In the 7000 ppm group, food consumption decreased significantly in females on postnatal Days 21 to 28; however, it was judged to be incidental because it was a transient change without dose-dependency.

SEXUAL MATURATION (OFFSPRING)
No test substance-related changes were observed in the sexual maturation in either sex of any treated group. In the 7000 ppm group, incidence of preputial separation in males was significantly delayed on postnatal Day 44; however, it was judged to be incidental because it was a transient change without dose-dependency, and there was no significant difference the next day. In the 2000 ppm group, incidence of vaginal opening in females was significantly higher on postnatal Days 35 and 36; however, it was judged to be incidental because it was a transient change without dose-dependency. In all treated groups, no significant differences were found in body weights in males or females on the corresponding days.

ORGAN WEIGHTS (OFFSPRING)
No test substance-related changes were observed in the organ weight in either sex of any treated group. In all treated groups, absolute and relative weights in both sexes were almost comparable to those in the control group on weaning Day (postnatal Day 21) and postnatal Day 91, with no significant differences.

ESTROUS CYCLE (OFFSPRING)
No test substance-related changes were observed in the estrous cycles in females of any treated group. All females in each group had a normal estrous cycle of 4 to 5 days, and no significant differences were found in the mean estrous cycle length. Moreover, there were no females with irregular estrous cycles in any group, including the control group.

NECROPSY (OFFSPRING)
No test substance-related abnormal findings were found in either sex of any treated group, animals necropsied at weaning, postnatal Day 91 and end of the nurturing period. In the animals necropsied at weaning, dilatation of the renal pelvis was observed in 2, 3, and 1 females in the control, 2000, and 7000 ppm groups, respectively; however, it was judged to be incidental, because such findings are seen spontaneously in rats and there was no dose-dependency in their incidences. In the animals necropsied on postnatal Day 91 and the end of the nurturing period, dilatation of the renal pelvis was observed in 1, 2, 1, and 5 males and 1, 4, 3, and 3 females in the control, 2000, 7000, and 20000 ppm groups, respectively; however, it was judged to be incidental, because such findings are seen spontaneously in rats. Absence of the testis and epididymis (unilateral) was observed in 1 male in the 7000 ppm group; however, the change was not considered to be test substance-related because it was found in the background data and there was no dose-dependency in its incidence. Cyst of the uterus was observed in 1 female in the 20000 ppm group; however, it was judged not to be treatment-related because only one case was observed of the incidence. Loss of tooth was observed in 1 male in the control group. Malocclusion was observed in 1 male and 1 female in the 7000 ppm group, and fracture of the nose and a small thymus were observed in 1 moribund male animal in the 20000 ppm group.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 20 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: highest dose tested

2

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 2 279.4 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: highest dose tested

3

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

>= 2 067.7 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: highest dose tested

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

Table 1: Absolute and relative ovary weights in F0 (P) dams

Ovary Weights (Rat)
		Ovaries (mg)	Ovaries Ratio (×10^-3%)	Ovary (R) (mg)	Ovary (R) Ratio (×10^-3%)	Ovary (L) (mg)	Ovary (L) Ratio (×10^-3%)
Control	Mean SD N	96.02 13.97 23	33.61 4.14 23	48.76 9.15 23	17.04 2.72 23	47.26 8.19 23	16.57 2.76 23
2000 ppm	Mean SD N	94.97 12.60 21	33.41 4.93 21	48.29 7.61 21	16.98 2.85 21	46.68 8.38 21	16.42 3.15 21
7000 ppm	Mean SD N	92.78 10.70 24	32.63 3.79 24	47.83 9.54 24	16.82 3.37 24	44.95 4.97 24	15.81 1.73 24
20000 ppm	Mean SD N	85.82* 11.17 22	30.27* 3.81 22	43.08 6.93 22	15.20 2.30 22	42.64 6.47 22	15.07 2.32 22

* [d - Test: Dunnett 2 Sided p < 0.05]

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