3(or 4)-methylbenzene-1,2-diamine

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Remarks:

based on test type (migrated information)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2008-06-17 to 2008-08-11

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted in accordance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

(1995)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3500 (Preliminary Developmental Toxicity Screen)

Version / remarks:

(2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Automatically generated during migration to IUCLID 6, no data available

IUPAC Name:

Automatically generated during migration to IUCLID 6, no data available

Details on test material:

- Name of test material (as cited in study report): TDA-vicinals (ortho-TDA)
- Chemical identity (as cited in study report): 3,4 Diaminotoluene (basically)
- Physical state: solid/brown
- Composition of test material, percentage of components: 3,4-TDA (50%), 2,3-TDA (45%), 2,4-TDA (< 1%)
- Analytical purity: 59.2% 3,4-TDA, 35.2% 2,3-TDA, 2.01 % 2,4-TDA (analyses carried out at GKA Competence Center Analytics, BASF SE, Ludwigshafen, Germany)
- Batch No.: 08.08.07 10:00; K 330
- Date of production: 2007-08-07
- Stability under test conditions: The storage stability under storage conditions was confirmed by reanalysis (Study No. 08L00415)

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 11 to 12 weeks
- Weight at study initiation:
-- mean group body weight for males ca. 315 g
-- mean group body weight for females ca.ca. 226 g
- Fasting period before study: no, but withdrawal of food of about 16 to 20 hours at test ending, before necropsy
- Housing: individually in Makrolon cages, type M III, floor area of about 800 cm2
- Use of restrainers for preventing ingestion (if dermal): no
- Diet (e.g. ad libitum): ground Kliba maintenance diet mouse/rat (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water (e.g. ad libitum): drinking water, ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hrs / 12 hrs

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

(drinking water)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
For preparation of all dosing solutions, the test substance was kept in a water bath at approximately 50-60°C until a soft consistency was attained. Then the specified amount of the test substance was weighed out. After the addition of the intended amount of warmed-up water the preparations were mixed with a magnetic stirrer. Under stirring the preparations were adjusted to pH 6 with HCl. The dosing preparations were made daily.

Details on mating procedure:

At least 13 days after the beginning of treatment, males and females from the same dose group were mated. Each of the male and female animals was mated overnight in a 1:1 ratio for a maximum of 2 weeks. Throughout the mating period, each female animal was paired with a predetermined male animal from the same test group. A vaginal smear was prepared after each mating and examined for sperm. As soon as sperm was detected, pairing of the animals was discontinued. The day on which sperm was detected was denoted gestation day 0.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses of the dosing preparations were carried out at the Analytical Chemistry Laboratory of Experimental Toxicology and Ecology of BASF SE, Ludwigshafen, Germany.
The stability of the test substance in drinking water for a period of 4 hours at room temperature was proven before the start of the administration period.
Homogeneity and concentration control analyses of the test-substance preparations were performed in samples of all concentrations at the start of the administration period. Of each sample, one additional reserve sample was retained. The mean recovery of TDA-vicinals (ortho-TDA) in drinking water ranged between 84.5-103.2% of the nominal concentrations, demonstrating the correctness of the concentrations and the homogeneity of TDA-vicinals (ortho-TDA) in drinking water.

Duration of treatment / exposure:

The duration of treatment covered a 2 week premating period and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period and 4 days of lactation in females.

Frequency of treatment:

Single treatment, daily

Details on study schedule:

The test substance was daily administered by gavage to the parental animals (F0 generation); the treatment lasted up to one day prior to sacrifice.Females being in labor received no treatment. The animals of the control group were treated in the same way as those of the test group, with the vehicle only (drinking water). The daily volume administered was 10 mL/kg bw.
At the end of the study, the animals were sacrificed (males on study day 30, females on study day 49) after a fasting period (withdrawal of food but drinking water available) for at least 16-20 hours.

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 10, 50 and 250 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

Ten animals per sex and dose group were used

Control animals:

yes, concurrent no treatment

Examinations

Parental animals: Observations and examinations:

MORTALITY AND CLINICAL SYMPTOMS, PARENTAL ANIMALS
A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied.
A cageside examination was conducted daily before and about 1 hour after oral treatment for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Littering and lactation behavior of the dams was generally evaluated in the morning in combination with the daily clinical inspection of the dams; on week days (except public holidays) the littering behavior of the dams was further inspected in the afternoon.

BODY WEIGHT, PARENTAL ANIMALS
The body weight of the male and female parental animals was determined once a week; body weight change was calculated from these results.
The following exceptions for the females were mentioned:
- During the mating period the females were weighed on the gestation day 0, 7, 14 and 20.
- Females with litter were weighed on the day of parturition (PND 0) and on day post parturition (PND 4).

FOOD CONSUMPTION, PARENTAL ANIMALS
Food consumption was determined once a week (over a period of 7 days) for male and female parental animals, with the following exceptions:
- Food consumption was not determined during the mating period.
- Food consumption of the F0 females with evidence of sperm was determined on gestation day 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined on PND 0 and PND 4.

REPRODUCTION PARAMETERS, PARENTAL MALES
The pairing partners, the number of mating days until vaginal sperm was detected in female animals, and the gestational status of the females were recorded for the F0 breeding pairs.
For the males, the mating index (number of males with confirmed mating X 100 / number of males placed with females) and the fertility index (number of males proving their fertility X 100 / number of males placed with females) were calculated.
Referring to sperm parameters, sperm motility (microscopy), sperm morphology (microscopy after staining with eosin), sperm head count in the cauda epididymis and in the testis (microscopy with MAKLER chamber after homogenization), were evaluated. Sperm motility examinations and the preparation of the specimens for sperm morphology were carried out in a randomized sequence; sperm morphology and sperm head count (cauda epididymis and testis) were evaluated for the control and high-dose test group, only.

REPRODUCTION PARAMETERS, PARENTAL FEMALES
The pairing partners, the number of mating days until vaginal sperm could be detected, and gestational status were recorded.
For the females, the mating index (number of females mated x 100 / number of females placed with males), the fertility index (number of females pregnant X 100 / number of females mated), and the gestation index (number of females with live pups on the day of birth X 100 / number of females pregnant) were calculated.
Referring to delivery data, the total number of pups delivered and the number of liveborn and stillborn pups were noted and the live birth index
(number of liveborn pups at birth X 100 / total number of pups born) was calculated.
Moreover, after sacrifice of the female animals, the implantation sites were counted and the post implantation loss (number of implantations - number of pups delivered X 100 / number of implantations) was calculated for each individual pregnant animal.

NECROPSY, PARENTAL ANIMALS
All F0 parental animals were sacrificed (decapitation under isoflurane anesthesia) for the purpose of necropsy and were subjected to gross pathology; particular attention was given to the reproductive organs.
The weight of the anesthetized animals prior to sacrifice was determined, and following organs were weighed: testes, epididymides and ovaries.
Immediately after organ weighing, the right testis and cauda epididymis were taken from the F0 males of all test groups for evaluation of the sperm parameters (see above).
The following organs/tissues of the parental animals were fixed (4% neutral buffered formaldehyde solution or BOUIN’s solution): all gross lesions, pituitary gland, prostate gland, seminal vesicles with coagulation glands, uterus, oviducts, cervix uteri, vagina, left testis (fixed in BOUIN´s solution), left epididymis (fixed in BOUIN´s solution), and ovaries (fixed in BOUIN´s solution). After fixation, the samples were processed, sectioned and hematoxylin-eosin stained on slides for histopathological assessment by light microscopy. Gross lesions were correlated with histopathological findings. All gross lesions were examined; examination of the left testis, left epididymis and ovaries was done for all males and females of the control and high dose groups, and for males and females of the low and mid dose groups suspected of reduced fertility.

PUPS AND LITTER PARAMETERS
All pups delivered from the F0 dams were examined as soon as possible on the day of birth to determine the total number of pups and the number of liveborn and stillborn pups in each litter. Pups, which died before the first determination of their status on the day of birth, were defined as stillborn pups. Check for dead or moribund pups was made twice daily on week days and once on Saturdays, Sundays or public holidays.
The number and percentage of dead pups on the day of birth (PND 0) and pups dying during the lactation period (PND 0 to PND 4) were determined. The number of live pups/litter was calculated on the day of birth and on PND 4. Furthermore, the viability index (number of live pups on day 4 after birth X 100 / number of liveborn pups on the day of birth) was calculated.
The live pups were examined daily for clinical symptoms including gross morphological findings.
The sex of the pups was determined on PND 0 (anogenital distance) and was confirmed at necropsy of the pups (PND 4); The sex ratio was calculated (number of live male or female pups on PND 0/4 X 100 / number of live male and female pups on PND 0/4).
The pups were weighed one day after birth (PND 1) and on PND 4; body weight change was calculated from these results.
Furthermore the body weights on PND 1 were used for the calculation of "runts" (pups, which weighed less than 25% of the mean weight of the respective control pups).

NECROPSY OF THE PUPS
All surviving pups (after sacrifice on PND 4), all stillborn pups and those pups, which died ahead of schedule, were examined externally, eviscerated and their organs were assessed macroscopically. All pups without any notable findings or abnormalities were discarded after their macroscopic evaluation.

Statistics:

Food consumption, body weight and body weight change (parental animals and pups), number of mating days, duration of gestation, number of pups delivered/litter, implantation sites, and post implantation loss were statistically assessed by simultaneous comparison of all dose groups with the control using the DUNNETT-test (two-sided) for the hypothesis of equal means.

Mating index fertility index, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, and number of litters with affected pups at necropsy were assessed by pairwise comparison of each dose group with the control using FISHER'S EXACT test for the hypothesis of equal proportions.

Males with more that 4% abnormal sperm (90%-Quantile of the control group being equal to 4%) were assessed for statistical significant difference from control using FISHER'S EXACT test

The total number of spermatids/g testis or cauda epididymides was assessed by pairwise comparison of the dose group with the control using the WILCOXON-test (one-sided) for the hypothesis of equal medians; the sperm motility (%) was assessed similarly however with Bonferoni-Holm-Adjustment.

The proportions of affected pups per litter with necropsy observations were assessed by pairwise comparison of the dose group with the control using the WILCOXON-test (one-sided) for the hypothesis of equal medians.

The weight parameters at necropsy were assessed by means of the non-parametric one-way analysis using KRUSKALWALLIS test (two-sided). If the p-value was =< 0.05, a pairwise comparison of each dose group with the control group was done using the WILCOXON test for the hypothesis of equal medians.

Results and discussion

Results: P0 (first parental generation)

Details on results (P0)

MORTALITY AND CLINICAL SYMPTOMS
There were no mortalities in parental animals. Clinical findings assessed as treatment-related were seen at 250 (males and females) and 50 mg/kg bw/day (males). No treatment-related findings were seen at 10 mg/kg bw/day.
MALES:
In the high dose group (250 mg/kg bw/day), almost all males showed reduced activity, salivated and had half closed eyelids after treatment; this was observed most time of the study. Piloerection occurring predominantly after treatment was observed in 5 males whereas one male showed respiration sounds after treatment during the first week of treatment.
In the mid dose group (50 mg/kg bw/day), one male showed reduced activity, half closed eyelids and piloerection after treatment; this was observed temporarily during the administration period. More than half of the males salivated, predominantly after treatment, at several time points in the course of the study.
No treatment-related findings were seen at 10 mg/kg bw/day.
FEMALES:
In the high dose group (250 mg/kg bw/day), more than half of the females treated with 250 mg/kg bw/d had half closed eyelids and salivated after treatment, during premating; one case of piloerection after treatment during the study week 2 (premating period) was observed.
During gestation, more than half of the females treated with 250 mg/kg bw/d had half closed eyelids and salivated after treatment. Reduced activity after treatment in one and discolored feces (light brown) in 4 females treated with 250 mg/kg bw/d were further reported on several days.
During lactation, half-closure of eyelid was observed for only 1 day each in 2 females treated with 250 mg/kg bw/d. One of these females also had discolored feces during lactation and did not properly nurse its offspring on lactation day 1. These findings were related to treatment.
In the mid dose group (50 mg/kg bw/d) salivation after treatment was the only finding seen during premating, gestation and lactation which was considered to be treatment-related; however, salivation rather was related to the mode of application than to the test substance as such. No treatment-related findings were seen at 10 mg/kg bw/day.

BODY WEIGHT, PARENTAL ANIMALS
Treatment–related effects on body weight and body weight gain were seen in the high dose group (250 mg/kg bw/d). Body weights and body weight gain in the mid and low dose groups (50 and 10 mg/kg bw/d) were similar to that of control during the entire study for males and females.
In the high dose group (250 mg/kg bw/d), the mean body weight of males was comparable to that of control during the entire study. However, the mean body weight gain was significantly lower between week 0 and 1. Furthermore, the overall body weight gain (week 0-4) of this test group was also significantly lower (about -46%). This finding was considered to be test substance-related. No treatment-related findings were seen at 50 and 10 mg/kg bw/day.
In the high dose group (250 mg/kg bw/d), the mean body weight of females was significantly decreased during premating weeks 1 and 2, and also significantly lower during the entire gestation period. Same was true for body weight change values between study weeks 0-1 and 0-2, gestation day 7-14 and 14-20 as well as gestation day 0-20. No effects on body weight and body weight change were seen during lactation.
No treatment-related findings were seen at 50 and 10 mg/kg bw/day.

FOOD CONSUMPTION, PARENTAL ANIMALS
Treatment–related effects on food consumption were seen in the high dose group (250 mg/kg bw/d). No changes of toxicological relevance were seen for male and female animals of test groups 1and 2 (10 and 50 mg/kg bw/d)

In the high dose group (250 mg/kg bw/d), food consumption in males was significantly reduced during the premating period. No changes in food consumption were observed in males of the mid and low dose groups (10 and 50 mg/kg bw/d).

In the high dose group (250 mg/kg bw/d), food consumption in females was significantly reduced during the premating period.
During gestation mean food consumption was slightly reduced in females of the high dose group between gestation day 7 and 14. This change was particularly related to the low food consumption of one female which also showed clinical signs of toxicity; the finding was assessed as being related to treatment. No changes in food consumption were observed in this group during lactation.
In the mid dose group (50 mg/kg bw/d), food consumption in females was found to be significantly reduced between study weeks 1 and 2 (premating). The finding however was assessed as being incidental as the value was within the normal range typical for animals in this age and strain of rats. No changes in food consumption were observed in this group during gestation and lactation.
No changes in food consumption were observed in females treated with 10 mg/kg bw/day.

REPRODUCTION PARAMETERS, MALES
For all males, which were placed with F0 females to generate F1 pups, mating was confirmed. Thus the male mating index reached 100% in all test groups including the controls.
Fertility was proven for most of the males within the scheduled mating interval for the F1 litter. Since 2 males of the low dose group (10 mg/kg bw/d) and 2 of the high dose group (250 mg/kg bw/d) did not generate F1 pups (nevertheless the corresponding female to one of the high dose males had implantation sites), the fertility indices were 100%, 80%, 100 % and 90% at 0, 10, 50 and 250 mg/kg bw/d, respectively. The fertility index range (80 to 100%) reflects the normal range of biological variation inherent in the strain of rats used for this study. It has to be noticed that gross and histopathological examinations of the two low dose males cited above and their respective mating females did not reveal relevant morphological correlate for the apparently impaired fertility.
Referring to the sperm parameters, a significant decrease in total spermatids/g testis was reported for the high dose group of 250 mg/kg bw/d (78,000,000 versus 90,000,000 in control; p<= 0.05). Also, motility was reduced in the high dose group but without significance (86% versus 93% in control). Motility in the mid dose group (50 mg/kg bw/d) was significantly reduced (89%, p<= 0.05); however, since no other reproduction or sperm parameter was affected at this dose level, the reduced motility was assessed as being incidental.
In the low dose group (10 mg/kg bw/d), all reproduction and sperm parameters were within negative control range, thus inconspicuous.

REPRODUCTION PARAMETERS, FEMALES
The female mating index was 100% for all groups.
The mean duration until sperm was detected (gestation day 0) amounted to 2.3, 2.5, 2.5 and 2.1 days at the dose levels of 0, 10, 50 and 250 mg/kg bw/d, respectively.
Except for 2 low dose females (10 mg/kg bw/d) and one high dose female (250 mg/kg bw/d) which did no become pregnant, all sperm positive females delivered pups or had implantations in utero.
Similarly as for the males, the female fertility index was 100%, 80%, 100% and 90% at 0, 10, 50 and 250 mg/kg bw/d, respectively.
The mean duration of gestation was 22.2 days in the control and mid dose group (0 and 50 mg/kg bw/d) and 22.2 days in the low dose group (10 mg/kg bw/d). A significantly extended duration of gestation to 23 days was observed in the high dose group (250 mg/kg bw/d); this finding was assessed as being related to treatment.
The gestation index was 78% in the high dose group (250 mg/kg bw/d) whereas it reached 100% in each of the control, the low and the mid dose group. In fact, in the high dose group, one female which was sperm positive, showed implantation sites at necropsy, but delivered no pups. Thus, the decreased gestation index seen at 250 mg/kg bw/d was treatment-related.
The mean number of implantation sites was 14.1, 13.9, 14.0 and 8.6 implants/dam at 0, 10, 50 and 250 mg/kg bw/d, respectively. Thus, the number was significantly lower in the high dose group. This finding was assessed as being related to treatment.
Post implantation loss indicating intrauterine embryo/fetolethality was significantly increased in the high dose group (27.4%) whereas in the remaining two test groups (low and mid), post implantation loss was not affected by the treatment as compared to control.
The mean number of delivered pups was 13.6, 12.6, 13.0 and 7.9 pups/dam at 0, 10, 50 and 250 mg/kg bw/d, respectively. Thus, the mean number of delivered pups was significantly lower in the high dose group; this finding was treatment-related to treatment.
The live birth indices ranged between 89% (high dose group, 250 mg/kg bw/d) and 100% (mid dose group, 50 mg/kg bw/d).
The rate of stillborn pups was comparable between the test groups, however the rate of died pups was statistically significantly increased in the high dose group 3 (250 mg/kg bw/d).

GROSS PATHOLOGY, PARENTAL ANIMALS
Gross pathology revealed no treatment-related abnormalities.

ORGAN WEIGHTS, PARENTAL ANIMALS
Compared to control (set to 100%), the mean terminal body weight for males and females was significantly decreased in the high dose group of 250 mg/kg bw/d (94% and 93%, respectively). The mean terminal body weight for the males of the mid and low dose group (50 and 10 mg /kg bw/d) was similar to control (100%, respectively); the mean terminal body weight for the females of the mid and low dose group (50 and 10 mg /kg bw/d) was similar to control (97 and 101%, respectively). The finding was considered to be treatment-related.
Compared to control (set to 100%), the mean absolute testes weight was significantly increased in all 3 treated groups, reaching 111%, 111% and 112% of control, at 10, 50 and 250 mg/kg bw/d, respectively. Since no histopathological correlates could be evidenced for the testes, the increase in absolute organ weight reported above was considered to rather be incidental than treatment-related.
Compared to control (set to 100%), the mean relative testes and epididymides weight were significantly increased in the low and the high dose groups (10 and 250 mg/kg bw/d) and increased without statistical significance in the mid dose group (50 mg/kg bw/d). For the testes, the values were 111% (p <= 0.01), 111% and 120% (p <= 0.01) at 10, 50 and 250 mg/kg bw/d, respectively. For the epididymides, the values were 111% (p <= 0.05), 106% and 113% (p <= 0.01) at 10, 50 and 250 mg/kg bw/d, respectively. Since no histopathological correlates could be evidenced for the testes and epididymides, and since a clear-dose relationship is missing, the increase in relative organ weights reported above was considered to rather be incidental than treatment-related.

HISTOPATHOLOGY, PARENTAL ANIMALS
Histopathology revealed no treatment-related abnormalities. No histopathological correlates could be evidenced for the changes in weight of the testes and epididymides reported above.

Effect levels (P0)

1

Dose descriptor:

NOAEL

Remarks:

(Systemic Toxicity)

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No treatment-related effects were seen.

2

Dose descriptor:

LOAEL

Remarks:

(Systemic Toxicity)

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: - Salivation was observed, which however was considered as related to the mode of application; - Decreased mean food consumption was not considered to be an adverse effect as no changes in body weight could be evidenced at this dose level.

3

Dose descriptor:

LOAEL

Remarks:

(Systemic Toxicity)

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Eyelid halfclosure after treatment, reduced activity and piloerection were reported as treatment-related effects.

4

Dose descriptor:

LOAEL

Remarks:

(Systemic Toxicity)

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Eyelid halfclosure after treatment, reduced activity, piloerection, discolored feces, decreased food consumption, lowered body weight and lowered body weight gain were reported as treatment-related effects

5

Dose descriptor:

NOAEL

Remarks:

(Reproductive Performance and Fertility)

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were seen.

6

Dose descriptor:

LOAEL

Remarks:

(Reproductive Performance and Fertility)

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Decreased number of spermatids/g testis and decreased number of implantation sites were reported as treatment-related effects.

Results: F1 generation

Details on results (F1)

PUPS AND LITTER PARAMETERS
In the high dose group (250 mg/kg bw/d) the mean number of delivered pups per dam was significantly decreased (7.9, p<= 0.01).when compared to the control (13.6). In the mid and low dose groups (50 and 10 mg/kg bw/d), the mean number of delivered pups per dam was within control range (respectively 13 and 12.6). In the high dose group (250 mg/kg bw/d) the rate of liveborn pups also was decreased when compared to the control (89% versus 95%); in the mid and low dose groups, values of 100 and 98% respectively were reported. The findings reported for the high dose group were treatment-related.
Considering pup viability, a significant increased pup mortality was observed during lactation (PND 0 to 4) in the high dose group (250 mg/kg bw/d).In fact, one pup died on day 0 (PND 0) and further 4 between day 0 and day 4 (PND 4). Thus, a total of 5 dead pups was reached in the high dose group versus 1 in each of the control and the low dose group; no pup died in the mid dose group. Thus, the viability index was 99%, 99%, 100% and 91% (p<= 0.05) at 0, 10, 50 and 250 mg/kg bw/day, respectively. The finding reported for the high dose group was treatment-related.
The pups showed no adverse clinical signs up to scheduled sacrifice on PND 4.
The sex distribution and sex ratios of live pups on the day of birth (PND 0) and on PND 4 did not show biologically relevant differences between control and test groups.
Mean pup body weights and pup body weight changes in the treated groups (10, 50 and 250 mg/kg bw/d) were comparable to the concurrent control values; the observable differences between the test groups were assessed as being spontaneous in nature and without any biological relevance (see table). The numbers of runts were 0, 16, 1 and 6 at 0, 10, 50 and 250 mg/kg bw/d, respectively. Thirteen of the 16 runts counted in the low test group (10 mg/kg bw/d) belonged to one litter. Since no dose-response relationship was evident, and since runts were mostly clustered in individual litters, the findings were not considered to be treatment-related.

NECROPSY OF THE PUPS
Necropsy of all pups revealed no abnormalities. Only one case of hydronephrosis was reported for the mid dose group (50 mg/kg bw/d), which clearly was incidental in nature and not treatment-related.

Effect levels (F1)

1

Dose descriptor:

NOAEL

Remarks:

(Developmental Toxicity)

Generation:

F1

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No treatment-related effects were seen.

2

Dose descriptor:

LOAEL

Remarks:

(Developmental Toxicity)

Generation:

F1

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Increased post implantation loss, decreased number of delivered pups and decreased viability index of the pups were reported as treatment-related effects.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

MEAN BODY WEIGHT DATA
Parental Males, mean body weights (g) ± SD (N = number of animals)
Week	Dose groups (mg/kg bw/d)
	0 (control)	10	50	250
0	313.8±10.45 (10)	316.5±9.05 (10)	315.8±9.90 (10)	316.4±7.88 (10)
1	328.1±15.01 (10)	330.8±7.11 (10)	329.2±13.46 (10)	316.4±9.70 (10)
2	337.4±16.26 (10)	343.5±6.21 (10)	340.9±16.75 (10)	326.4±10.22 (10)
3	347.2±17.84 (10)	352.6±7.47 (10)	352.2±18.96 (10)	333.0±12.10 (10)
4	360.5±19.27 (10)	362.5±8.45 (10)	364.3±24.66 (10)	341.8±15.08 (10)
*, p ≤ 0.05; **, p ≤ 0.01
Parental Females, mean body weights (g) ± SD (N = number of animals)
Week	PREMATING
	Dose groups (mg/kg bw/d)
	0 (control)	10	50	250
0	226.8±8.29 (10)	229.1±8.34 (10)	224.3±10.28 (10)	226.9±7.00 (10)
1	229.6±11.56 (10)	233.1±6.74 (10)	228.2±11.00 (10)	218.3±10.28* (10)
2	237.7±11.50 (10)	239.6±7.28 (10)	237.6±11.28 (10)	226.7±7.42* (10)
*, p ≤ 0.05; **, p ≤ 0.01
Day	GESTATION
	Dose groups (mg/kg bw/d)
	0 (control)	10	50	250
0	242.2±10.04 (9)	241.0±5.73 (8)	237.3±12.05 (10)	227.4±6.10** (8)
7	273.8±10.40 (9)	272.4±8.79 (8)	267.0±11.42 (10)	259.5±4.88* (8)
14	306.5±10.71 (9)	300.6±11.10 (8)	295.8±13.11 (10)	276.0±13.41** (8)
20	376.0±17.42 (9)	366.0±19.98 (8)	358.9±21.74 (10)	312.7±30.63** (8)
*, p ≤ 0.05; **, p ≤ 0.01
Day	LACTATION
	Dose groups (mg/kg bw/d)
	0 (control)	10	50	250
0	274.7±19.53 (10)	278.2±17.71 (8)	273.7±16.64 (10)	261.8±12.62(8)
4	291.0±12.40 (10)	293.9±13.94 (8)	288.1±15.45 (10)	276.0±10.09(8)
*, p ≤ 0.05; **, p ≤ 0.01

MEAN BODY WEIGHT CHANGE DATA
Parental Males, mean body weight change (g) ± SD (N = number of animals)
Week	Dose groups (mg/kg bw/d)
	0 (control)	10	50	250
0 - 1	14.4±6.87 (10)	14.3±5.14 (10)	13.3±6.87 (10)	0.0±5.69** (10)
1 - 2	9.3±5.24 (10)	12.7±5.58 (10)	11.7±4.28 (10)	10.0±3.05 (10)
2 - 3	9.8±5.04 (10)	9.1±6.28 (10)	11.3±5.46 (10)	6.6±5.67 (10)
3 - 4	13.3±4.24 (10)	10.0±5.12 (10)	12.0±6.67 (10)	8.8±5.72 (10)
0 - 4	46.8±13.83 (10)	46.1±13.31 (10)	48.4±18.76 (10)	25.5±11.43** (10)
*, p ≤ 0.05; **, p ≤ 0.01
Parental Females, mean body weight change (g) ± SD (N = number of animals)
Week	PREMATING
	Dose groups (mg/kg bw/d)
	0 (control)	10	50	250
0 - 1	2.8±5.38 (10)	4.0±3.88 (10)	3.9±4.95 (10)	-8.7±9.54** (10)
1 - 2	8.1±8.06 (10)	6.6±7.07 (10)	9.4±5.79 (10)	8.5±8.17 (10)
0 - 2	10.9±6.87 (10)	10.6±8.61 (10)	13.4±7.36 (10)	-0.2±6.29** (10)
*, p ≤ 0.05; **, p ≤ 0.01
Day	GESTATION
	Dose groups (mg/kg bw/d)
	0 (control)	10	50	250
0 - 7	31.6±3.22 (9)	31.4±6.73 (8)	29.7±5.42 (10)	32.0±5.44 (8)
7 - 14	32.7±2.98 (9)	28.2±2.68 (8)	28.7±5.16 (10)	16.5±12.86** (8)
14-20	69.5±7.60 (9)	65.4±14.09 (8)	63.1±13.06 (10)	36.7±18.84** (8)
0-20	133.8±10.54 (9)	125.0±15.06 (8)	121.6±15.35 (10)	85.3±28.38** (8)
*, p ≤ 0.05; **, p ≤ 0.01
Day	LACTATION
	Dose groups (mg/kg bw/d)
	0 (control)	10	50	250
0 - 4	16.3±11.68 (10)	15.6±13.38 (8)	14.4±10.54 (10)	14.2±12.54(8)
*, p ≤ 0.05; **, p ≤ 0.01

MEAN FOOD COMSUMPTION DATA
Parental Males, mean food consumption (g/animal/day) ± SD (N = number of animals)
Week	Dose groups (mg/kg bw/d)
	0 (control)	10	50	250
0 - 1	19.3±2.43 (10)	20.3±1.63 (10)	19.5±1.61 (10)	15.4±1.18** (10)
1 - 2	19.7±1.66 (10)	20.2±1.27 (10)	20.6±2.05 (10)	18.0±0.64* (10)
0 - 2	19.5±0.30 (2)	20.2±0.08 (2)	20.0±0.72 (2)	16.7±1.86 (2)
*, p ≤ 0.05; **, p ≤ 0.01
Parental Females, mean food consumption (g/animal/day) ± SD (N = number of animals)
Week	PREMATING
	Dose groups (mg/kg bw/d)
	0 (control)	10	50	250
0 - 1	16.0±1.29 (10)	15.4±0.74 (10)	14.4±1.25 (10)	10.0±2.6** (10)
1 - 2	17.5±1.09 (10)	16.7±1.27 (10)	15.4±1.37** (10)	14.0±0.97** (10)
0 - 2	16.7±0.99 (2)	16.0±0.91 (2)	14.9±0.73 (2)	12.0±2.81 (2)
*, p ≤ 0.05; **, p ≤ 0.01
Day	GESTATION
	Dose groups (mg/kg bw/d)
	0 (control)	10	50	250
0 - 7	20.8±1.27 (9)	20.3±1.82 (8)	19.8±1.59 (10)	19.7±1.32 (8)
7 - 14	23.5±1.33 (9)	22.3±2.42 (8)	22.1±2.25 (10)	20.4±3.52* (8)
14-20	24.4±1.48 (9)	24.5±1.68 (8)	24.4±2.17 (10)	22.1±2.94 (8)
0-20	22.9±1.88 (3)	22.4±2.12 (3)	22.1±2.26 (3)	20.7±1.25 (3)
*, p ≤ 0.05; **, p ≤ 0.01
Day	LACTATION
	Dose groups (mg/kg bw/d)
	0 (control)	10	50	250
0 - 4	30.5±3.54 (10)	28.2±6.0 (8)	31.2±6.6 (10)	26.4±6.37 (8)
*, p ≤ 0.05; **, p ≤ 0.01

SUMMARY OF FEMALE REPRODUCTION AND DELIVERY DATA
Parameters examined			Dose groups (mg/kg bw/d)
			0			10				50		250
Females on study			10			10				10		10
Mating Index (%)			100			100				100		100
Mating days until gestation day 0			2.3			2.5				2.5		2.1
Pregnant females (Fertility Index as %)			10 (100)			8 (80)				10 (100)		9 (90)
Duration of gestation (days)			22.2			22.3				22.2		23.0*
Mean implantation sites/dam			14.1			13.9				14.0		8.6**
Mean post implantation loss/dam			0.5			1.3				1.0		1.6
Post implantation loss as %			3.5			11.6				6.8		27.4*
Females with liveborn pups (%)			10 (100)			8 (100)				10 (100)		7 (78)
Females with stillborn pups (%)			2 (20)			2 (25)				0 (0)		2 (25)
Females with all stillborn pups (%)			0			0				0		1(13)
Mean pups delivered/dam			13.6			12.6				13.0		7.9**
Liveborn (Live Birth Index as %)			129 (95)			99 (98)				130 (100)		56 (89)
Stillborn (as %)			7 (5.1)			2 (2.0)				0 (0.0)		7 (11)
*, p ≤ 0.05; **, p ≤ 0.01
SUMMARY OF LITTER AND PUP DATA
Parameters examined				Dose groups (mg/kg bw/d)
				0			10		50			250
Total number of litter				10			8		10			8
Litter with liveborn pups (%)				10 (100)			8 (100)		10 (100)			7 (88)
Litter with stillborn pups (%)				2 (20)			2 (25)		0 (0)			2 (25)
Litter with all stillborn pups (%)				0 (0)			0 (0)		0 (0)			1 (13)
Total number of pups delivered/group				136			101		130			63
Pups liveborn (%)				129 (95)			99 (98)		130 (100)			56 (89)
Pups stillborn (%)				7 (5.1)			2 (2.0)		0 (0.0)			7 (11)
Pups died (%)				0 (0.0)			0 (0.0)		0 (0.0)			5** (7.9)
Pups sacrificed moribund				0 (0.0)			0 (0.0)		0 (0.0)			0 (0.0)
Pups cannibalized/accidentally died, sacrificed because of maternal death				1 (0.7)			1 (1.0)		0 (0.0)			0 (0.0)
Dead Pups, day 0 (%)				0 (0.0)			0 (0.0)		0 (0.0)			1 (1.8)
Dead Pups, day 1 - 4 (%)				1 (0.8)			1 (1.0)		0 (0.0)			4 (7.1)
Surviving Pups day 0-4 (Viability Index as %)				128 (99)			98 (99)		130 (100)			51* (91)
Sex ratio day 0 (%live males/%live females)				51.2/48.8			49.5/50.5		53.8/46.2			66.1/33.9
Sex ratio day 4 (%live males/%live females)				51.6/48.4			50/50		53.8/46.2			68.6/31.4
Mean pup body weights in g (Number of pups)		Dose groups (mg/kg bw/d)
day	Sex	0			10			50			250
1	Males (M)	6.7±0.42 (10)			6.7±1.42 (8)			6.7±0.48 (10)			6.1±0.59 (6)
1	Females (F)	6.5±0.42 (10)			6.5±1.46 (8)			6.3±0.50 (10)			5.5±0.76 (6)
1	M & F	6.6±0.42 (10)			6.6±1.45 (8)			6.5±0.48 (10)			5.9±0.62 (6)
4	Males (M)	10.5±0.75 (10)			10.5±2.6 (8)			10.3±1.14 (10)			9.3±0.93 (6)
4	Females (F)	10.2±0.74 (10)			10.1±2.63 (8)			10.0±1.25 (10)			8.6±1.08 (6)
4	M & F	10.4±0.73 (10)			10.3±2.62 (8)			10.2±1.17 (10)			9.1±0.94 (6)
Runts
day	Sex
1	M & F	0			16 (8 M & 8 F)			1 ( 0 M & 1 F)			6 ( 3M & 3F)
Mean pup body weight changes in g		Dose groups (mg/kg bw/d)
day	Sex	0			10			50			250
1 - 4	Males (M)	3.8±0.44 (10)			3.7±1.22 (8)			3.6±0.73 (10)			3.3±0.46 (6)
1 - 4	Females (F)	3.7±0.44 (10)			3.7±1.19 (8)			3.7±0.83 (10)			3.1±0.46 (6)
1 - 4	M & F	3.8±0.43 (10)			3.7±1.19 (8)			3.7±0.75 (10)			3.2±0.44 (6)
*, p ≤ 0.05; **, p ≤ 0.01