4,6-bis(octylthiomethyl)-o-cresol

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a one-generation reproduction toxicity study treatment of parental animals, prior to pairing and throughout gestation and lactation periods at dosages of 15, 50 and 200 mg/kg/day showed no signs of toxicological significance. Reproductive function, as assessed by oestrus cycles, mating performance, pregnancy rate and parturition was not affected by treatment.

Link to relevant study records

Reference

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The study was set up as a two-generation-study (OECD 416). In the absence of findings, it was discontinued on day 21 of the F1 generation.

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: supplied by Harlan Nossan S.r.l., San Pietro al Natisone (UD) Italy
- Age at study initiation: (P) 6-7 weeks and 8 to 9 weeks for males and females, respectively
- Weight at study initiation: the animals used; a total of 110 male and 110 female rats, were within a 25 gram weight range (P)
- Housing: (a) during the pre-mating period in clear polycarbonate cages measuring 59x39x20 cm with a stainless steel mesh lid and floor (Type 4: Techniplast Gazzada S.r.l., Buguggiate, Varese; each cage tray held absorbent material which was inspected daily and changed at least 3 times per week); (b) during the mating period on the basis of one male to one female in clear polycarbonate cages measuring 43x27x15 cm with a stainless steel mesh lid and floor (Type 3: Techniplast Gazzada S.r.l; each cage tray held absorbent material which was inspected and changed daily. The males were re-caged after mating as they were before mating. The mated females were caged in individual, solid bottomed, breeding cages (Type 3: Techniplast Gazzada S.r.l.).
- Diet (ad libitum): commercially available rodent diet (Altromin MT, Altromin-Rieper, Bolzano, Italy)
- Water (ad libitum): potable water
- Acclimation period: at leat 11 days; the male animals arrived on 8 October 1999 and were allocated to groups on 15 October 1999. The females arrived on 3 December 1999 and were allocated to groups on 10 December 1999. The first day of treatment for males was on 21 October 1999. The first day of treatment for females was on 16 December 1999 (the last sacrifice was performed on 3 April 2000).

ENVIRONMENTAL CONDITIONS
The animals were housed in the barriered rodent facility at RTC. Animal room controls were set
- Temperature (°C): 22±3
- Humidity (%): 30±70
- Air changes (per hr): 20
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

peanut oil

Remarks:

a weighed amount of the test substance was suspended in the vehicle and brought to the final volume appropriate for each concentration

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test suspensions were prepared daily at room temperature
- Males: dose volumes were calculated according to individual body weights on the first day of treatment and adjusted according to individual body weights at weekly intervals thereafter.
- Females: dose volumes were calculated according to individual body weights at weekly intervals before pairing and on Days 0, 6, 10 and 15 post-coitum. Thereafter individual dose volumes remained constant

VEHICLE
- Concentration in vehicle: 0, 3, 12.5 or 40 mg/ml, respectively for the 0, 15, 50 and 200 mg/kg bw dose groups
- Amount of vehicle (if gavage): 5 ml/kg bw

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: up to 4 days
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to commencement of treatment the proposed formulation procedure was checked by chemical analysis to confirm that the method was acceptable. Samples of formulations taken during the first and last week of treatment were also analysed for concentration homogeneity and stability. All analytical results were in the acceptable range.

Duration of treatment / exposure:

- Males: a total of approx. 113 days (for 10 weeks prior to pairing, through the mating period and thereafter until the day prior to sacrifice)
- Females: a total of approx. 56 days (for 2 weeks prior to pairing, during the mating period and through to weaning of the offspring)

Frequency of treatment:

Daily

Details on study schedule:

- Selection of parents from F1 generation: since no signs of toxicity were observed, the Sponsor requested that the study be stopped at the first generation after weaning and selection of the F1 generation.

Dose / conc.:

15 mg/kg bw/day (actual dose received)

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: chosen because of the results of 28-day and 90-day toxicity studies, as well as the teratogenicity study performed in rats. In the 28-day oral toxicity study (gavage) the NOEL was 50 mg/kg body weight, with increased alkaline phosphatase and liver weight increase in the 250 and 1000 mg/kg bw dose groups. In the 90-day oral toxicity study in rats (gavage) the NOEL was 10 mg/kg bw, with increased liver weights at 100 and 1000 mg/kg bw. In the rat teratology study 150 mg/kg bw (gavage) represented the overall NOEL, with reduced body weight gain at 300 mg/kg bw of the pregnant dams.
Therefore, since animals were dosed for at least 100 days, 200 mg/kg was considered to yield a maximum tolerated dose, while 15 mg/kg was expected to yield a NOEL with 50 mg/kg to show intermediate toxicity.

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS, DETAILED CLINICAL OBSERVATIONS: Yes
- Mortality: all cages were checked for dead or moribund animals twice daily throughout the study, once in the morning and again in the afternoon. A similar procedure was followed at weekends and Public Holidays except that the female check was carried out at approximately mid-day.
- Clinical signs: all signs were recorded for individual animals. During the treatment period, for both F0 males and females, examination of individual animals for signs of reaction to treatment was carried out daily prior to dosing, immediately after and at approximately 1 hour after dosing. All animals received a more thorough examination once weekly during the entire treatment period.

BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed on the day of allocation, the day treatment commenced, and weekly thereafter and just prior to sacrifice. Females were weighed on the day of allocation, the day treatment commenced and weekly to pairing. During the post-coitum period females were weighed on Days 0, 6, 10, 15, and 20 post-coitum and also on Days 0/1, 4, 8, 12 and 21 post-partum.

FOOD CONSUMPTION:
Food consumption per cage of animals was measured weekly for males from allocation to pairing and for females from the day of allocation to pairing. Individual food consumption of females was measured over the periods of Days 0 to 5, 6 to 9, 10 to 14 and 15 to 19 post-coitum, and Days 0 to 3, 4 to 7, 8 to 11 and 12 to 20 postpartum.

Oestrous cyclicity (parental animals):

- Oestrous cycles and mating performance: vaginal smears were taken daily for 2 weeks prior to pairing starting from the first day of treatment until occurrence of mating. During the mating period each cage was checked each morning for the presence of a copulation plug and a vaginal smear was prepared from each female. This information was used to detect marked anomalies of the oestrus cycle and to determine the precoital interval (the number of nights paired before detection of mating).
-Duration of gestation: gestation periods were taken as the time between the day of successful mating and the commencement of birth (i.e. first detected presence of offspring).

Sperm parameters (parental animals):

(Only F0 males) Sperm count in one epididymis and evaluation of sperm viability and motility was performed in control and high-dose males and in one mid-dose male which did not induce pregnancy

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- On Day 4 post-partum, all litters in excess of eight offspring were culled to eight pups (4 males and 4 females, where possible) by random selection, excluding those pups showing signs of ill health. Culled pups were sacrificed on Day 4 post-partum by subcutaneous injection of 'Tanax', necropsied and examined for external and internal abnormalities. Pup sex was confirmed at necropsy. One litter was inadvertently culled to seven pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
The pups (live and dead) were counted, sexed, weighed and examined for external abnormalities as soon as possible after parturition (Day 0 or 1 post-partum). Live pups were identified individually within the litter by toe amputation. All litters were examined daily for dead or abnormal young. The pups were also weighed on Days 4, 8, 12 and 21 post-partum. All pups found dead were given a post-mortem examination.
- 24 males and 24 females were selected for further study. All pups were allocated to 4 groups and treated starting the day after weaning. Clinical observation, post dose observation, food consumption, body weight and post weaning tests were performed. Meanwhile the Sponsor decided to stop the study at the first generation, since no toxicity was observed.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

ASSESSMENT OF PRE-WEANING DEVELOPMENT
All pups in all litters were examined to determine the age at which the following developmental stages were attained:
a) Pinna unfolding, from Day 1 post-partum to 100% occurrence;
b) Hair growth, from Day 3 post-partum to 100% occurrence;
c) Incisor emption (upper), from Day 8 post-partum to 100% occurrence;
d) Startle response to sound, from Day 12 post-partum to 100% occurrence;
e) Eye opening (complete separation of eyelids), from Day 12 post-partum to 100% occurrence;
f) Air righting reflex, once on Day 20 post-partum;
g) Pupil reflex, once on Day 20 post-partum;
h) Testes descent (testes palpable in the scrotum), once on Day 21 post-partum.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: males were killed by carbon dioxide after the birth of the majority of litters and retained for external and internal examinations.
- Maternal animals: all parental animals were killed at weaning (PND 21) by carbon dioxide inhalation. Apparently non pregnant females were sacrificed after at least 25 days post-coitum.

GROSS NECROPSY
- Gross necropsy consisted of and examined for external and internal abnormalities
- The sex of each pup was determined by gonadal inspection and, for each dam, the number of visible implantation sites was recorded

HISTOPATHOLOGY / ORGAN WEIGHTS
- Females: uteri were immersed in a 10% solution of ammonium sulphide to reveal any evidence of implantations. The following tissues were preserved in 10% buffered formol saline: uterus with cervix, ovaries with oviduct, vagina and pituitary
- Males: the following tissues were preserved in 10% buffered formol saline (except testes and one epididymis which were fixed in Bouin's fluid and preserved in 70% alcohol): pituitary, testes, one epididymis, seminal vesicles, prostate, coagulating gland and any Abnormalities. Testes from males which did not induce pregnancy and from 5 concurrent controls were weighed.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 4 days of age.
- All pups found dead in the cage were necropsied.
- Excess pups (i.e., those not selected for the Fl generation) were killed with carbon dioxide on or shortly after Day 21 post-partum
- All animals were killed with carbon dioxide and were subjected to necropsy

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
- Culled pups were sacrificed by intrascapular injection of Tanax on the day of culling and were subjected to a post-mortem examination. The sex of each pup was determined by gonadal inspection.

Statistics:

For the body weights, body weight gain and organ weights the significance of the differences amongst group means was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett's test using a pooled error of variance. The homogeneity of the data were assessed by Bartlett's Test before Dunnett's Test was performed. If the data were found to be inhomogeneous, a modified t test (Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
The non-parametric Kruskal-Wallis analysis of variance was used for litter data.
Intergroup differences between the control and treated groups were assessed by the non-parametric version of the Williams' test.
The criterion for statistical significance was p<0.05.

Reproductive indices:

mating index, copulation index, fertilitiy index

Offspring viability indices:

viability index, lactation index

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs seen during the observations performed at weekly intervals in F0 males and females were limited to common conditions of the skin and fur. No reaction to treatment was seen at the post-dose observations performed immediately and 1 hour after dosing during the entire treatment period except for a few occasions of salivation in the high-dose group.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One control male was found dead and another one was sacrificed for humane reasons during the course of the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Group mean body weights and body weight gain in F0 males were comparable between the treated and the control groups. Statistically significant reductions in body weight were observed on day 8 of treatment in females receiving 15 and 50 mg/kg/day of the test substance when compared to controls. A statistically significant reduction in body weight gain was observed in females receiving the test substance at 50 mg/kg/day on day 8 of treatment. Body weight gain in females receiving the test substance at 15 and 50 mg/kg/day was statistically significantly higher compared to controls on day 15 of treatment. A statistically significant increase in body weight gain was observed on postpartum day 8 for animals of the low and mid-dose groups and on post-partum day 12 for the high-dose group compared to controls. A statistically significant reduction in body weight gain was observed in high-dose females on post-partum Day 0 and was considered to be related to the higher number of pups compared to controls. These occasional differences are not considered to be of toxicological significance.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption of the F0 generation was unaffected by treatment

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

A total of three males, one in the control, one in the mid-dose and one in the high-dose group failed to induce pregnancy.

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

All dams gave birth between days 21 or 22 post-coitum, with the exception of one low-dose female which gave birth prematurely on gestation day 18. This isolated case was considered incidental. The statistically significant reduction observed in gestation periods for high-dose females compared to controls was not considered to be of toxicological significance

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

There were no differences in concentration (million sperm/gr epididymal cauda), viability, motility and morphology of the sperm between the control, the high-dose males and in males which failed to induce pregnancy.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences between the controls and the treated groups in litter size, litter weight, mean pup weight or pup loss throughout the whole lactation period. Lactation index was similar between the control and the treated groups.
Sex ratio of Fl pups: sex ratios of offspring at birth and on Day 21 post-partum did not show any differences between groups. The statistically significant difference in the number of male pups found dead during the weaning period was attributable to the low number of deaths in the high-dose litters compared to controls and was not considered to be a sex related mortality.

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity, fertility

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects up to this dose group

Clinical signs:

no effects observed

Description (incidence and severity):

The abnormalities observed in pups during the post-partum period were incidental with no relation to treatment.

Mortality / viability:

no mortality observed

Description (incidence and severity):

There were no differences between the controls and the treated groups in litter size or pup loss throughout the whole lactation period.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There were no differences between the controls and the treated groups in litter weight or mean pup weight throughout the whole lactation period.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No signs of toxicological significance were observed

Gross pathological findings:

no effects observed

Description (incidence and severity):

No treatment related changes were seen in FI pups which died before weaning. No findings of toxicological significance were seen at the necropsy performed on F1 culled pups. There were no meaningful differences in the incidence of the abnormalities recorded at the necropsy of FI pups at weaning or in selected pups.

Description (incidence and severity):

Pre-weaning development of F1 pups: there were no significant differences between groups in the results obtained from the parameters used to monitor the pre-weaning physical and functional development. One pup in group 2 had bilateral anophthalmia (considered incidental) and therefore was not subjected to the pupil reflex test. Values of eye opening and pupil reflex for this litter were excluded from group mean calculation.

Dose descriptor:

NOAEL

Remarks:

Postnatal development (up to weaning at postnatal day 21)

Generation:

F1

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects up to this dose group

Reproductive effects observed:

not specified

Table 1: Reproduction parameters

Reproduction parameters		Test groups (mg/kg bw)
		0	15	50	200
Females	Initial group size	24	24	24	24
	Not pregnant	1	0	1	1
	Total litter loss	0	0	1	0
	With live pups at weaning	23	24	22	23
	Pre-coital interval (days)	4.5	3.75	3.17	2.96
	Copulation index (%)	100	100	100	100
	Fertility index (%)	95.8	100	95.8	95.8
Males	Initial group size	24	24	24	24
	Found dead	1	0	0	0
	Humane kill	1	0	0	0
	Failed to induce pregnancy	1	0	1	1
	Copulation index (%)	100	100	100	100
	Fertility index (%)	95.8	100	95.8	95.8

 

Table 2: Implantation and pre-birth loss data from F0 females(group mean data)

Treatment (mg/kg bw)	Total liter size		Implantations/litter		Pre-birth loss /litter				Gestation period (days)
	N	Mean±SD	N	Mean±SD	Number		Percentage		N	Mean±SD
					N	Mean±SD	N	Mean±SD
0	23	14.7±2.6	23	13.6±2.6	23	1.1±1.5	23	7.0±9.6	23	21.9±0.3
15	24	15.1±2.1	24	14.1±2.4	24	1.0±1.1	24	7.0±7.8	23	22.0±0.3
50	22	14.4±2.2	22	13.4±2.6	22	1.0±1.0	22	9.6±11.8	23	21.9±0.3
200	23	14.9±1.8	23	14.0±2.2	23	0.9±0.9	23	6.1±6.3	23	21.6±0.5*
SD: standard deviation; *: p<0.05

 

Conclusions:

Oral administration with the test item to parental F0 animals, prior to pairing and throughout gestation and lactation periods at dosages of 15, 50 and 200 mg/kg/day showed no signs of toxicological significance. Reproductive function, as assessed by oestrus cycles, mating performance, pregnancy rate and parturition was not affected by treatment. These results suggest that a dosage of 200 mg/kg/day can be considered the NOAEL.

Executive summary:

The effects of the test article on reproductive function and fertility were evaluated in male and female rats following oral administration by gavage at dosages of 15, 50 and 200 mg/kg/day. Male parental (F0) rats were treated for ten weeks prior to pairing until the day before sacrifice. Female parental (F0) rats were treated for two weeks prior to pairing, during gestation and lactation periods and up to the day before sacrifice, on or shortly after day 21 post-partum. A concurrent group received the vehicle (peanut oil) at the same dose volume and acted as a control. Each group was comprised of 24 males and 24 females.

There were no mortalities in the treated groups. Two control males died (one was found dead and another was humanely sacrificed) during the course of the study. A total of 3 males failed to induce pregnancy; one each in the control, mid and high-dose groups and consequently the females in the same groups were not pregnant. One mid-dose female had total litter loss on post-partum day 0 and one low-dose female gave birth prematurely on gestation day 18. No treatment-related effects were observed at the post-dose observations. There were no treatment-related clinical signs. There were no changes of toxicological significance in body weight or body weight gain in F0 males. The occasional differences observed in F0 females are not considered to be of toxicological significance. Food consumption of the F0 generation was unaffected by treatment. Oestrus cycle, mating performance, and pregnancy rate were unaffected by treatment. No effects on implantation, pre-birth loss data or gestation length were seen in F0 treated females. Total and live litter size, litter and mean pup weights and pup loss were similar between groups. Sex ratios and lactation index were not affected by treatment. No signs were observed during the post-partum period which could be considered related to maternal treatment. Pre-weaning development did not show any differences between the control and the treated groups. There were no toxicologically relevant necropsy findings in Fl pups which died during the lactation period or in Fl pups which were culled on Day 4 post-partum. No changes were seen at the necropsy of Fl pups sacrificed on or shortly after Day 21 post-partum. No findings of toxicological significance were seen at necropsy of Fl selected pups. No signs of toxicological significance were observed at necropsy of the F0 generation. There were no differences in sperm motility or sperm concentration between the control, the high-dose group or the mid-dose male which failed to induce pregnancy.

In conclusion, oral administration with the test item to parental F0 animals, prior to pairing and throughout gestation and lactation periods at dosages of 15, 50 and 200 mg/kg/day showed no signs of toxicological significance. Reproductive function, as assessed by oestrus cycles, mating performance, pregnancy rate and parturition was not affected by treatment. These results suggest that a dosage of 200 mg/kg/day can be considered the NOAEL.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In an one-generation reproduction toxicity study which was conducted initially according to the OECD Guideline 416, the effects of the test substance on reproductive function and fertility were evaluated in male and female rats (24 animals per sex per group) following oral administration by gavage at dosages of 15, 50 and 200 mg/kg/day. Arachis oil was used as vehicle. Male parental (F0) rats were treated for 10 weeks prior to pairing until the day before sacrifice. Female parental (F0) rats were treated for 2 weeks prior to pairing, during gestation and lactation periods and up to the day before sacrifice, on or shortly after day 21 post-partum. A concurrent group received the vehicle (peanut oil) at the same dose volume and served as control. Mating of the F1 generation was not performed because of overall absence of toxicity at all dose levels.

The dose selection was based on the results of 28-day and 90-day toxicity studies, as well as the teratogenicity study performed in rats. In the 28-day oral toxicity study (gavage) the NOEL was 50 mg/kg body weight, with increased alkaline phosphatase and liver weight increase in the 250 and 1000 mg/kg bw dose groups. In the 90-day oral toxicity study in rats (gavage) the NOEL was 10 mg/kg bw, with increased liver weights at 100 and 1000 mg/kg bw. In the rat teratology study 150 mg/kg bw (gavage) represented the overall NOEL, with reduced body weight gain at 300 mg/kg bw of the pregnant dams. Therefore, since animals were dosed for at least 100 days, 200 mg/kg was considered to yield a maximum tolerated dose, while 15 mg/kg was expected to yield a NOEL with 50 mg/kg to show intermediate toxicity.

 

1) F0 generation

- Fate and mortality of F0 generation: there were no mortalities in the treated groups. Two control males died (one was found dead and another was humanely sacrificed) during the course of the study. A total of three males failed to induce pregnancy; one each in the control, mid and high-dose groups and consequently the females in the same groups were not pregnant. One mid-dose female had total litter loss on post-partum day 0 and one low-dose female gave birth prematurely on gestation day 18.

- Post-dose observations and clinical signs: no treatment-related effects were observed.

- Body weights and body weight gain, food consumption: no changes of toxicological significance in body weight or body weight gain in males. The occasional differences observed in dams are not considered to be of toxicological significance. Food consumption was unaffected by treatment.

- Reproductive parameters: oestrus cycle, mating performance, and pregnancy rate were unaffected by treatment.

- Implantation and pre-birth loss: no effects on implantation, pre-birth loss data or gestation length were seen in F0 treated females.

- Litter data, sex ratios and lactation index: total and live litter size, litter and mean pup weights and pup loss were similar between groups. Sex ratios and lactation index were not affected by treatment.

- Macroscopic examination: no signs of toxicological significance were observed at necropsy of the F0 generation.

- Sperm analysis: there were no differences in sperm motility or sperm concentration between the control, the high-dose group or the mid-dose male which failed to induce pregnancy

 

2) F1 generation

- Pre-weaning clinical signs: no signs were observed during the post-partum period which could be considered related to maternal treatment.

- Pre-weaning development: pre-weaning development did not show any differences between the control and the treated groups.

- Necropsy findings F1 pups: there were no toxicologically relevant findings in F1 pups which died during the lactation period or in F1 pups which were culled on Day 4 post-partum. No changes were seen at the necropsy of F1 pups sacrificed on or shortly after Day 21 post-partum. No findings of toxicological significance were seen at necropsy of F1 selected pups.

 

In conclusion, oral administration of the test substance to parental F0 animals, prior to pairing and throughout gestation and lactation periods at dosages of 15, 50 and 200 mg/kg/day showed no signs of toxicological significance. Reproductive function, as assessed by oestrus cycles, mating performance, pregnancy rate and parturition was not affected by treatment. These results suggest that a dosage of 200 mg/kg/day can be considered the NOAEL for rats.

Effects on developmental toxicity

Description of key information

In a prenatal developmental toxicity study with rats conducted according to the OECD Guideline 414, administration of the test substance during organogenesis by oral gavage at a dose level up to 300 mg/kg elicited slight maternal toxicity (slightly reduced body weight gain and - possibly - slightly reduced food consumption) at the high dose level, but no embryotoxicity, and no teratogenicity.

In a second developmental toxicity study performed with rabbits according to OECD guideline 414, the oral administration of the test substance to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) at doses as high as 1000 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity. The NOAEL was 1000 mg/kg body weight.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987 - 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(May 12, 1981)

Deviations:

no

Remarks:

No treatment during gestation days 16-18, as this was not required in the OECD testing guideline 414 version valid at that time. Amount of vehicle higher than recommended (0.5ml instead of 0.4 ml/100g bw/day).

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga GmbH, 8741 Sulzfeld, West Germany
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 180 to 245 g
- Housing: Prior to mating and during mating, the female rats were housed in groups of twenty to twenty-five in communal cages.Mated female rats were individually housed in solid floor macrolone cages of type II with stainless steel lids (dimensions: 260 mm x 200 mm x 140 mm; E. Becker & Co GmbH, 4620 Castrop-Rauxel, West Germany).
- Diet: ad libitium
- Water: ad libitium
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25 °C
- Humidity (%): 40 to 70%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19.03.1987 To: 23.04.1987 (date of last necropsy)

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle (if other than water): Solubility of the test item
- Concentration in vehicle: 0, 10, 30 and 60 mg/ml
- Amount of vehicle (if gavage): 5ml/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples for analysis were taken from dose formulations prepared for each dose level on 27.03.1987 (beginning of treatment), deep-frozen immediately after formulation, and sent to the study sponsor for analysis in dry ice, inadvertently not before 15.07.1987. Based on the analytical data, it was concluded that the test article was sufficiently stable during the application period.

Details on mating procedure:

The male and female animals were mated at a ratio of 1 : 4 in communal cages during the night.
The females were examined on the following morning for the presence of sperm and/or a vaginal plug.
The day on which sperm and/or a vaginal plug were observed was designated day 0 of gestation.

Duration of treatment / exposure:

Day 6 to 15 post-coitum (inclusive)

Frequency of treatment:

daily

Duration of test:

Day 6- 15 with treatment, maintained without treatment until day 20 post-coitum and were sacrificed and examined on that day.

Dose / conc.:

50 mg/kg bw/day (actual dose received)

Dose / conc.:

150 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: Based on the results of a screening study (Report no. 614-380/42,dated March 1986)

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations :signs of ill-health, toxicity, behavioural change, mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: see above

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each inseminated female rat was recorded on days 0, 6 to 15, and 20 post-coitum and evaluated for days 0, 6, 10, 15, and 20 postcoitum.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The food consumption of each inseminated female rat was recorded for days 0 to 6, 6 to 10, 10 to 15, and 15 to 20 post-coitum.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: any abnormalities were recorded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Individual foetal weights and sex of the foetuses

Intra-uterine deaths were classified as follows:
Early resorptions showed decidual or placental tissues only.
Late resorptions showed embryonic or foetal tissue in addition to placental tissue but excluded foetuses dying in utero within approximately two days
prior to the terminal kill. Dead foetuses included only the foetuses dying in utero within approximately the last two days.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: see below

Half of the foetuses from each litter (taking every second foetus in accordance with the position in the uterine horn, if possible) were eviscerated
and preserved in 95 per cent ethanol for determination of skeletal abnormalities (Alizarin staining technique).
The remaining half was fixed in Bouin's fixative for determination of vis ceral abnormalities (Wilson technique).
The uteri of apparently non-pregnant females were immersed in a 10 per cent solution of ammonium sulphide to reveal evidence of implantation (Salewski technique).

Structural deviations were classified as:
Malformations: rare and/or probably lethal e.g. hydrocephaly.
Variations: changes which regularly occur also in control groups and which are not of functional significance.

Statistics:

For body weight, body weight gain, food consumption, and mean foetal weight (overall, males, females) the analysis of variance was performed with one factor TREATMENT followed by the Newman-Keuls test for multiple group comparison. Number of corpora lutea, number of implantation, number of foetuses, preimplantation loss, post-implantation loss, and proportion of male foetuses were statistically analysed using the Kruskal-Wallis-test.
In case of suspected significance (probability > chi square < 0.05), the four groups were compared two by two using the Wilcoxon two-sample test (normal approximation - with continuity correction of 0.5).
All significances found (at least p < 0.05) are indicated in the respective tables.
The statistical evaluation was performed with the standard software package SAS release 6.02.

Indices:

pregnancy indices
pre-implantation loss
post-implantation loss
sex ratio

Historical control data:

Historical control data is included in the report.

Clinical signs:

no effects observed

Description (incidence and severity):

There were no behavioural changes and no clinical observations in any animal of all groups.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In group 4 (300 mg/kg) body weight gain during gestation, particularly during late gestation, was reduced (16 % less than the control group from day 10 to 15 post-coitum, and 8 % less from day 15 to 20 post-coitum). The difference in comparison with the control group was not statistically significant. This reduced body weight gain is considered to be related to treatment with the test item. Body weight gain of groups 2 (50 mg/kg) and 3 (150 mg/kg) was comparable with the concurrent control group.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Mean daily food consumption was slightly lower in group 4 (300 mg/kg) than in the control group, particularly during treatment. In groups 2 (50 mg/kg) and 3 (150 mg/kg) mean daily food consumption was
comparable with the control group.

Description (incidence and severity):

At necropsy unilateral dilatation of the renal pelvis was found in one female of group 3 (150 mg/kg) and two females of group 4 (300 mg/kg). The nature of these findings is considered not to be treatment-related.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

Pre-implantation loss was high in the control group and in group 4 (300 mg/kg). As pre-implantation loss of group 4 (300 mg/kg) was only slightly higher than in the control group (which remains in the upper range obtained from historical background data, this finding is considered to be incidental. In groups 2 (50 mg/kg) and 3 (150 mg/kg) there was no effect on pre-implantation loss. There was no effect of treatment on post-implantation loss, post-implantation loss was lowest in group 4 (300 mg/kg).

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

There was no effect of treatment on pregnancy incidence

Dose descriptor:

NOEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Mean foetal weight was slightly increased in group 4 (300 mg/kg). Differences to the control group were statistically significant. This finding must be considered in relation to the reduced mean number of foetuses per female in the highest dose group and not in relation to treatment. Mean foetal weights of groups 2 (50 mg/kg) and 3 (150 mg/kg) were comparable with the control group.

Reduction in number of live offspring:

effects observed, non-treatment-related

Description (incidence and severity):

The mean number of foetuses per dam was slightly lower in group 4 (300 mg/kg). This finding is considered to be related with the slightly reduced mean number of implantations per female and not considered to treatment-related. The mean number of foetuses per dam in groups 2 (50 mg/kg) and 3 (150 mg/kg) was comparable with the control group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex distribution of foetuses did not reveal any compound-related effect.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

An external malformation as micrognathia was found in one foetus of group 2 (50 mg/kg). A further foetus in a second litter of the same dose group showed bilateral anophthalmia. In group 3 (150 mg/kg), one foetus with apodia was observed. In group 4 (300 mg/kg) as well as in the control group no malformations were found.

Skeletal malformations:

no effects observed

Description (incidence and severity):

Skeletal malformations were not detected. The incidence of skeletal variations did not reveal any treatment-related effects.

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no efffects

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 1: Uterine and Implantation data

		Control	50 mg/kg	150 mg/kg	300 mg/kg
		n=24	n=21	n=20	n=20
Corpora Lutea	total	396	354	323	278
	mean	16.5	16.9	16.1	13.9
Implantations	total	296	289	267	202
	mean	12.3	13.8	13.4	10.1
% Pre-Implantation loss		24.0	16.5	14.6	28.8
Live Foetuses	total	277	273	250	193
	mean	11.5	13.0	12.5	9.7
	% of implantations	93.3	94.8	93.5	96.5
Early resorptions	total	18	16	16	9
	mean	0.8	0.8	0.8	0.5
late resorptions	total	1	0	0	0
	mean	0.0	0.0	0.0	0.0
Dead foetuses	total	0	0	1	0
	mean	0.0	0.0	0.1	0.0
Total intrauterine deaths	total	19	16	17	9
	mean	0.8	0.8	0.9	0.5
% Post-Implantation loss		6.1	5.2	6.5	3.5

Table 2: Group Mean Body weight (g)

calculated from animals with live fetuses

Day of gestation	control	50 mg/kg	150 mg/kg	300 mg/kg
0	206.5	204.3	207.3	200.8
6	241.9	239.5	243.5	234.5
10	260.6	257.9	260	252.5
15	290.4	288.6	289.3	277.5
20	345.2	350.2	350.5	327.8

Table 3: Body weight gain (g) for different gestation stages (data for dams with live offspring)
	days 0-6	days 6-10	days 10-15	days 15-20*
control	35	18.8	29.8	54.8
SD	5.7	8.8	6.2	13.9
50mg/kg bw	35.2	18.3	30.7	61.7
SD	7.3	8.4	5.8	9.9
150 mg/kg bw	36.3	16.5	29.3	61.3
SD	8.1	7.3	5.9	9.4
300 mg/kg bw	33.8	18	25	50.3
SD	7	7.3	9.3	21
*Last day of treatment was day 15
SD = Standard Deviation

Table 4: fetal data

	control (n=24)	50 mg/kg (n= 21)	150 mg/kg (n= 20)	300 mg/kg (n= 20)
Number of fetuses	277	273	250	193
mean number of fetuses per female	11.5	13	12.5	9.7
mean litter weight (g)	38.7	43.2	42.6	33.5
mean fetal weight (g) overall	3.33	3.33	3.39	3.51
mean fetal weight (g) males	3.42	3.42	3.51	3.53
mean fetal weight (g) females	3.23	3.25	3.27	3.46
number of males	147	126	124	94
number of females	130	147	126	99
sex ratio in % (males : females)	53.1 : 46.9	46.2 : 53.8	49.6 : 50.4	48.7 : 51.3

Table 5: Malformation, variation data

	control	50 mg/kg	150 mg/kg	300 mg/kg
Number of fetuses examined externally	277	273	250	193
Number of fetuses with external malformations	0	1	1	0
% of fetuses with external malformations	0	0.4	0.4	0
Number of litters with external malformations	0	1	1	0
Number of fetuses examined viscerally	144	141	126	102
Number of fetuses with visceral malformations	0	1	0	0
% of fetuses with visceral malformations	0	0.7	0	0
Number of litters with visceral malformations	0	1	0	0
Number of fetuses examined viscerally	144	141	126	102
Number of fetuses with visceral variations	1	1	0	0
% of fetuses with visceral variations	0.7	0.7	0	0
Number of litters with visceral variations	1	1	0	0
Number of fetuses examined skeletally	133	132	124	91
Number of fetuses with skeletal malformations	0	0	0	0
% of fetuses with skeletal malformations	0	0	0	0
Number of litters with skeletal malformations	0	0	0	0
Number of fetuses examined skeletally	133	132	124	91
Number of fetuses with skeletal variations	133	132	123	91
% of fetuses with skeletal variations	100	100	99.2	100
Number of litters with skeletal variations	24	21	20	18
Total Number of malformed fetuses	0	2	1	0
Total number of litters with malformed fetuses	0	2	1	0
Average % malformed fetuses	0	0.7	0.4	0

Conclusions:

In conclusion, administration of the test substance during organogenesis by oral gavage at a dose level of 300 mg/kg elicited slight maternal toxicity (16% reduction in body weight gain and - possibly - slightly reduced food consumption), but no embryotoxicity, and no teratogenicity. Administration of 50 and 150 mg/kg did not elicit any maternal toxicity, embryotoxicity, or teratogenicity.

Executive summary:

Groups of 25 sexually mature and mated female rats received the test item by oral gavage at dosages of 50, 150, or 300 mg/kg daily for ten consecutive days from day 6 to 15 post-coitum, inclusive. A further group of 25 rats of the same strain which received the vehicle (arachis oil) over the same period served as the control group. The animals were sacrificed on day 20 post-coitum. After conclusion of the present study with including the evaluation of the data, the results of the study can be summarized as follows:

There were no clinical observations and no necropsy findings which might be considered to be related to treatment. At a dose level of 300 mg/kg, maternal body weight gain during gestation was slightly reduced. This is considered to be a slight maternally toxic effect of the compound. Mean daily food consumption was slightly lower in group 4 (300 mg/kg) than in the control group. This finding might be a slightly toxic effect of treatment. There was no effect of treatment on pregnancy incidence. There was no effect of treatment on implantations. Post-implantation loss was not affected by the test item. The mean number of foetuses per dam was slightly reduced in group 4 (300 mg/kg). This finding is considered to be related with the slightly reduced mean number of implantations per female and not to be a compound-related effect. Sex distribution was not affected by treatment. Mean foetal weight of group 4 (300 mg/kg) was slightly increased. This finding is considered to be related with the slightly reduced mean number of foetuses per female in this group and not to indicate any effect of treatment. Nature and incidence of malformations and variations did not reveal any compound-related effect.

In conclusion, administration of the test article during organogenesis by oral gavage at a dose level of 300 mg/kg elicited slight maternal toxicity (slightly reduced body weight gain and - possibly - slightly reduced food consumption), but no embryotoxicity, and no teratogenicity. Administration of 50 and 150 mg/kg did not elicit any maternal toxicity, embryotoxicity, or teratogenicity.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Qualifier:

according to guideline

Guideline:

other: Regulation 440/2008 laying down test methods pursuant to Regulation (EC) No 1907/2006 Part B: Methods for the determination of toxicity and other health effects: Prenatal Developmental Toxicity Study; Official Journal of the European Union, No. L 142

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 15-18 weeks
- Weight at study initiation: Based on the pregnant animals the body weight on GD 0 varied between 3452 – 4485 g.
- Fasting period before study: no
- Housing: During the acclimatization and study period, the rabbits were housed singly in Type 4X03B700CP cages supplied by TECNIPLAST Deutschland GmbH, Hohenpeißenberg, Germany (floor space 4264 mm², internal height 450 mm).
- Diet: pelleted Kliba maintenance diet rabbit and guinea pig “GLP”, ad libitum
- Water: ad libitum from water bottles
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-22
- Humidity (%): 45-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 0.5% Sodium carboxymethyl cellulose suspension in deionized water (with 10 mg/100 mL Cremophor EL)

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance preparations were prepared at the beginning of the administration period and thereafter at intervals, which took into account the period of established stability. The preparations were kept at room temperature. For the test substance preparation, the specific amount of the test substance was weighed, topped up with 0.5% CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL) in a calibrated beaker and intensely mixed with a magnetic stirrer. Before and during administration, the preparations were kept homogeneous with a magnetic stirrer.

VEHICLE
- Concentration in vehicle: 1.0, 3.0 and 10.0 g/100ml
- Amount of vehicle (if gavage): 10 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

PROCEDURE: The analytical investigations of the test substance preparations were carried out as a separate study. The study was carried out in compliance with the Principles of Good Laboratory Practice. Samples of the test substance preparations were sent once (at the beginning of administration) to the analytical laboratory for verification of the concentrations. The samples taken for the concentration control analyses were also used to verify the homogeneity of the samples of the low- and high-concentrations each (100 and 1000 mg/kg bw/d). Three samples (one from the top, middle and bottom) were taken for each of these preparations from the preparation vessel with a magnetic stirrer running.
RESULTS: The stability of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL) over a period of a maximum of 7 days was demonstrated prior to the start of the study. The homogeneous distribution of the test substance in the vehicle (0.5% CMC suspension in deionized water [with 10 mg/100 mL Cremophor EL]) was demonstrated. The results of the analyses of the test substance preparations in 0.5% CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL) confirmed the correctness of the prepared concentrations. The measured concentrations of the samples of treatment groups corresponded to the expected values within the limits of the analytical method, i.e. were above 90% and below 110% of the nominal concentrations.

Details on mating procedure:

After an acclimatization period of at least 5 days, the does were fertilized by means of artificial insemination. A synthetic hormone (0.2 mL), which stimulates release of LH and FSH from the anterior pituitary lobe (Receptal®) was injected intramuscularly to the female rabbits about 1 hour before insemination. The ejaculate samples used for the artificial insemination were obtained from male New Zealand White rabbits of the same breed as the females. Each female was inseminated with the sperm of a defined male donor as documented in the raw data. The male donors were kept under conditions (air conditioning, diet, water) comparable to those of the females participating in this study.

Duration of treatment / exposure:

GD 6-28

Frequency of treatment:

Daily

Duration of test:

The test item was administered daily as an aqueous preparation to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28). Sacrifice occurred on GD 29.

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: During the acclimatization period the animals were assigned to the different test groups according to a randomization plan (NIJENHUIS and WILF) and on the basis of their body
weights.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
A cage-side examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. If such signs occurred, the animals were examined several times daily (GD 0-29). During the administration period (GD 6-28) all animals were checked daily for any abnormal clinical signs before the administration as well as within 5 hours after the administration.

BODY WEIGHT: Yes
All animals were weighed on GD 0, 2, 4, 6, 9, 11, 14, 16, 19, 21, 23, 25, 28 and 29. The body weight change of the animals was calculated based on the obtained results. Furthermore, the corrected body weight gain was calculated after terminal sacrifice (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6).

FOOD CONSUMPTION: Yes
The consumption of food was recorded daily during GD 0-29.

POST-MORTEM EXAMINATIONS: Yes
On GD 29, the surviving does were sacrificed in randomized order by intravenous injection of pentobarbital (Narcoren®; dose 2 mL/animal) and later, fetuses were removed from the uterus. All prematurely dead or sacrificed females were examined following the same procedures as for females sacrificed on schedule with the exception that no uterine weights were determined. After the does had been sacrificed, they were necropsied and were assessed by gross pathology in randomized order to minimize bias. The uteri and the ovaries were removed and the following data were recorded:
- Weight of the unopened uterus
- Number of corpora lutea
- Number and distribution of implantation sites classified as:
• Live fetuses
• Dead implantations:
a) Early resorptions (only decidual or placental tissues visible or according to SALEWSKI from uteri from apparently non-pregnant animals and the empty uterus horn in the case of single horn pregnancy)
b) Late resorptions (embryonic or fetal tissue in addition to placental tissue visible)
c) Dead fetuses (hypoxemic fetuses which did not breathe spontaneously after the uterus had been opened)
After the weight of the uterus had been determined, all subsequent evaluations of the does (except of gross pathology including organ weights) and the gestational parameters were conducted by technicians unaware of treatment group in order to minimize bias. For this purpose animal numbers were encoded.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter

At necropsy each fetus was weighed and examined macroscopically for external findings. Furthermore, the viability of the fetuses and the condition of placentas, umbilical cords, fetal membranes, and fluids were examined. Individual placental weights were recorded. After the fetuses had been sacrificed, the abdomen and thorax were opened in order to examine the organs in situ before they were removed. The heart and the kidneys were sectioned in order to evaluate the internal structure. The sex of the fetuses was determined by examination of the gonads in situ.
After these examinations, the heads of approximately one half of the fetuses per doe (and the heads of any fetus which revealed severe findings during the external examination, e.g. anophthalmia, microphthalmia or hydrocephalus) were severed from the trunk. These heads were fixed in BOUIN’s solution and were, after fixation, processed and evaluated according to WILSON’s method (WILSON and WARKANY). About 10 transverse sections were prepared per head. After the examination the heads were discarded. All fetuses (including those without heads) were skinned and fixed in ethyl alcohol. After fixation for approx. 1-5 days, the intact fetuses were removed from the fixative and a transversal incision was made into the frontal/parietal head bones. The two halves of the calvarium were cautiously bent outward and the brain was thoroughly examined. Subsequently, these fetuses were placed back into the fixative for further fixation.

Statistics:

- Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (twosided) for the hypothesis of equal means: Food consumption*, body weight, body weight change, corrected body weight gain (net maternal body weight change), carcass weight, weight of unopened uterus, number of corpora lutea, number of implantations, number of resorptions, number of live fetuses, proportions of preimplantation loss, proportions of postimplantation loss, proportions of resorptions, proportion of live fetuses in each litter, litter mean fetal body weight, litter mean placental weight
- Pairwise comparison of each dose group with the control group using FISHER'S EXACT test (one-sided) for the hypothesis of equal proportions: Female mortality, females pregnant at terminal sacrifice, number of litters with fetal findings
- Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians: Proportions of fetuses with malformations, variations and/or unclassified observations in each litter

* For the parameter food consumption the "mean of means" was calculated and can be found in the relevant summary tables. The "mean of means" values allow a rough estimation of the total food consumption during different time intervals (pretreatment, treatment and entire study); they are not exactly precise values, because the size of the intervals taken for calculation differs. For the "mean of means" values no statistical analysis was performed.

Indices:

conception rate, preimplantation loss, postimplantation loss

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

One female of the low-dose group (No. 43 - 100 mg/kg bw/d) had a vaginal hemorrhage after treatment on GD 27. Furthermore, one high-dose female (No. 76 - 1000 mg/kg bw/d) had diarrhea on the day of scheduled sacrifice (GD 29). In total, reduced defecation was observed in nine control, ten low-dose, ten mid-dose and nine high-dose females (0, 100, 300 and 1000 mg/kg bw/d). No defecation was observed in three control females, five low-dose, four mid-dose and four high-dose females. Incidence and distribution of these findings do not indicate a relationship to the test substance. There were no further clinical findings in the other does in this study.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One female of the mid-dose group (No. 71 - 300 mg/kg bw/d) died after gavage error on GD 17.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

The mean body weights and the average body weight gain of the low-, mid- and high-dose groups (100, 300 and 1000 mg/kg bw/d) were generally comparable to the concurrent control group throughout the entire study period. Mean carcass weights and the corrected body weight gain (terminal body weight on GD 29 minus weight of the unopened uterus minus body weight on GD 6) were comparable among all test groups.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The food consumption of the low-, mid- and high-dose rabbits (100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control (0 mg/kg bw/d) throughout the entire study period.

Description (incidence and severity):

The mean gravid uterus weight of the rabbits of test groups 1-3 (100, 300 or 1000 mg/kg bw/d) was not influenced by the test substance. The differences between these groups and the control group showed no dose-dependency and were assessed to be without biological relevance.

Description (incidence and severity):

Some spontaneous findings were noted in individual females of all test groups (0, 100, 300 and 1000 mg/kg bw/d). These gross findings were:
• watery feces combined with no feces in rectum in control doe No. 5, low-dose doe No. 37 and high-dose doe Mo. 77,
• just watery feces in control doe No. 15, low-dose doe No. 42, mid-dose does Nos. 57, 64, 67 and one high-dose doe No. 76.
An additional finding was noted. It was associated with unscheduled maternal death or sacrifice:
• findings after gavage error (thoracic cavity filled with blood) in doe No. 71 (died on GD 17).

Number of abortions:

effects observed, non-treatment-related

Description (incidence and severity):

Two mid-dose females (Nos. 60 [GD 26] and 67 [GD 29] - 300 mg/kg bw/d) were sacrificed after abortion ahead of schedule. Spontaneous abortions in single does are not uncommon findings in the strain of rabbits used for this study. Thus, this was considered to be a spontaneous incident.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The conception rate was 76% in the mid-dose group (300 mg/kg bw/d), 84% in the control group, 92% in the high-dose group (1000 mg/kg bw/d) and 96% in the low-dose group (100 mg/kg bw/d). There were no test substance-related and/or biologically relevant differences between the different test groups in conception rate, in the mean number of corpora lutea and implantation sites or in the values calculated for pre- and post-implantation losses, the number of resorptions and viable fetuses. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age.
One dead fetus was found at cesarean section of high-dose doe No. 97 (1000 mg/kg bw/d) which is a rare finding but may occur spontaneously in this rabbit strain. As there was no evidence whatsoever for any potential developmental toxicity of the test item in this study at large, this is considered to be an incidental finding.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: no adverse effects reported

Fetal body weight changes:

no effects observed

Description (incidence and severity):

The mean placental weights in test groups 1, 2 and 3 were not influenced by the test substance and were comparable to the control value. The mean fetal weights of test groups 1, 2 and 3 were not influenced by the test substance and did not show any biologically relevant differences in comparison to the control group.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

The sex distribution of the fetuses in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) was comparable to the control fetuses. Any observable differences were without biological relevance.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

One external malformation was recorded for a control fetus. There were no external malformations in any test substance-treated fetuses.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Skeletal malformations were detected in single fetuses of test groups 0 and 2 (0 and 300 mg/kg bw/d). No statistically significant differences between the test substance-treated groups and the control were noted. The overall incidences were well within the historical control range of the test facility.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Soft tissue malformations occurred in single fetuses of test groups 0, 2 or 3 (0, 300 or 1000 mg/kg bw/d). The distribution of the findings about the test groups does not indicate an association to the treatment and no statistically significant differences between the groups were noted. The total incidence of soft tissue malformations in treated animals did not differ significantly from the control group and was comparable to the historical control data.

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects reported

Abnormalities:

not specified

Developmental effects observed:

not specified

SUMMARY OF MATERNAL DATA

Table 1. Mean maternal body weights during gestation (g)

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
DAY 0	MEAN	3739	3770	3788	3732
	S.D.	182.9	168.9	211.1	145
	N	21	24	21	23
DAY 2	MEAN	3850	3881	3891	3826
	S.D.	192.8	180.5	221.3	156.2
	N	21	24	21	23
DAY 4	MEAN	3903	3926	3937	3879
	S.D.	178.1	177.3	215.7	159.2
	N	21	24	21	23
DAY 6	MEAN	3955	4009	4020	3949
	S.D.	221	201.2	247.4	187.5
	N	21	24	21	23
DAY 9	MEAN	4007	4065	4067	3986
	S.D.	228.2	214.6	247.5	188.6
	N	21	24	21	23
DAY 11	MEAN	4044	4108	4102	4015
	S.D.	232.7	218.6	257.1	190.9
	N	21	24	21	23
DAY 14	MEAN	4130	4181	4166	4092
	S.D.	241.2	217.2	263	195.4
	N	21	24	21	23
DAY 16	MEAN	4179	4222	4211	4118
	S.D.	225.3	227.7	276.5	179.9
	N	21	24	21	23
DAY 19	MEAN	4154	4213	4184	4083
	S.D.	241.6	246.5	293.7	203
	N	21	24	21	23
DAY 21	MEAN	4150	4214	4164	4082
	S.D.	250.8	241.4	281.7	222.6
	N	21	24	21	23
DAY 23	MEAN	4165	4202	4150	4110
	S.D.	255.6	240.8	270.1	192.4
	N	21	24	21	23
DAY 25	MEAN	4180	4195	4160	4123
	S.D.	253.5	233	291.3	174.1
	N	21	24	21	23
DAY 28	MEAN	4188	4208	4203	4142
	S.D.	258.6	245.1	294	198.3
	N	21	24	20	23
DAY 29	MEAN	4198	4217	4243	4152
	S.D.	276.7	277.1	294.9	201.1
	N	21	24	19	23

Table 2. Mean maternal body weight change during gestation (g)

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
DAYS 0 TO 2	MEAN	112.0	111.0	103.1	94.5
	S.D.	39.79	64.59	46.00	65.91
	N	21	24	21	23
DAYS 2 TO 4	MEAN	52.8	45.4	45.8	52.8
	S.D.	42.34	28.31	37.45	36.84
	N	21	24	21	23
DAYS 4 TO 6	MEAN	52	83	83.7	70.3
	S.D.	69.01	43.68	45.36	38.48
	N	21	24	21	23
DAYS 6 TO 9	MEAN	52	56.5	46.7	36.7
	S.D.	72.71	53.51	27.01	50.77
	N	21	24	21	23
DAYS 9 TO 11	MEAN	37	42.8	35	28.9
	S.D.	39.88	23.34	39.26	35.75
	N	21	24	21	23
DAYS 11 TO 14	MEAN	86.1	73.2	63.9	77.7
	S.D.	109.04	72.3	83.4	50.77
	N	21	24	21	23
DAYS 14 TO 16	MEAN	48.3	40.6	45.1	25.7
	S.D.	92.72	42.58	52.58	62.31
	N	21	24	21	23
DAYS 16 TO 19	MEAN	-25.2	-8.4	-27	-35.6
	S.D.	67.55	58.64	99.45	62.83
	N	21	24	21	23
DAYS 19 TO 21	MEAN	-3.1	0.5	-20.4	-0.6
	S.D.	68.48	46.17	73.34	44.6
	N	21	24	21	23
DAYS 21 TO 23	MEAN	14.3	-11.6	-13.4	27.6
	S.D.	51.53	42.69	70.71	57.89
	N	21	24	21	23
DAYS 23 TO 25	MEAN	15.2	-7.2	9.3	13.7
	S.D.	59.19	76.2	58.62	51.19
	N	21	24	21	23
DAYS 25 TO 28	MEAN	8.4	12.6	21	18.6
	S.D.	86.43	54.02	63.48	63.72
	N	21	24	20	23
DAYS 28 TO 29	MEAN	9.9	8.8	21.5	10.5
	S.D.	43.51	53.00	55.33	53.89
	N	21	24	19	23
DAYS 0 TO 6	MEAN	216.7	239.3	232.6	217.5
	S.D.	69.6	90.14	79.49	93.4
	N	21	24	21	23
DAYS 6 TO 28	MEAN	233.1	198.9	180.1	192.7
	S.D.	247.14	137.88	187.64	153.82
	N	21	24	20	23
DAYS 0 TO 29	MEAN	459.8	447	446.9	420.7
	S.D.	257.91	190.95	198.92	171.34
	N	21	24	19	23

Table 3. Mean gravid uterine weights and net maternal body weight chage (g)

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
GRAVID UTERUS	MEAN	504.6	477.2	471.1	477.6
	S.D.	101.26	105.44	96.14	78.62
	N	21	23	19	23
CARCASS	MEAN	3693.7	3741.5	3771.7	3674.7
	S.D.	214.08	257.33	296.03	240.88
	N	21	23	19	23
NET WEIGHT CHANGE FROM DAY 6	MEAN	-261.5	-268.7	-260.7	-274.5
	S.D.	229.39	167.12	199.24	160.95
	N	21	23	19	23

Table 4. Summary of maternal necropsy observations

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
FEMALES EXAMINED	N	25	25	25	25
NOTHING ABNORMAL DETECTED	N	23	23	20	23
	%	92	92	80	92
FINDINGS AFTER GAVAGE ERROR	N	0	0	1	0
	%	0	0	4	0
WATERY FECES	N	2	2	3	2
	%	8	8	12	8
RECTUM: NO FECES	N	1	1	3	1
	%	4	4	12	4
PARTICULAR FINDINGS ON IMPLANTS IN DAMS WHICH ABORTED	N	0	0	2	0
	%	0	0	8	0

SUMMARY OF REPRODUCTION DATA

Table 5. Summary of reproduction data

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Females mated	N	25	25	25	25
Pregnant	N	21	24	22	23
	%	84	96	88	92
Aborted	N	0	0	2	0
Premature births	N	0	0	0	0
Dams with viable fetuses	N	21	24	19	23
Dams with all resorptions	N	0	0	0	0
Female Mortality	N	0	0	3	0
	%	0	0	12	0
Pregnant at terminal sacrifice	N	21	24	19	23
	%	84	96	76	92
Corpora Lutea	Mean	10.1	9.9	10.9	10.6
	SD	2.33	2.21	1.37	1.93
	TOTAL	213	237	207	243
Implantation Sites	Mean	9.8	9.1	9.6	10.0
	SD	2.32	2.47	2.48	2.10
	TOTAL	205	218	183	231
Preimplantation Loss	Mean	3.7	7.8	11.8	5.0
	SD	7.41	14.59	19.60	9.38
Postimplantation Loss	Mean	6.2	3.7	5.6	5.2
	SD	11.04	5.80	9.23	7.30

Table 6. Summary of reproduction data (continued)

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Pregnant at terminal sacrifice	N	21	24	19	23
Resorptions: Total	Mean	0.7	0.4	0.5	0.5
	SD	1.45	0.58	0.7	0.73
	TOTAL	15	9	9	12
	Mean %	6.2	3.7	5.6	4.9
	SD	11.04	5.8	9.23	6.98
Resorptions: Early	Mean	0.3	0.2	0.4	0.3
	SD	0.66	0.41	0.68	0.54
	TOTAL	7	5	7	6
	Mean %	3.5	2.1	4.6	2.6
	SD	7.28	4.25	9.25	5.64
Resorptions: Late	Mean	0.4	0.2	0.1	0.3
	SD	1.07	0.38	0.32	0.62
	TOTAL	8	4	2	6
	Mean %	2.7	1.6	1.0	2.3
	SD	7.59	3.74	3.10	5.43
Dead Fetuses	N	0	0	0	1

Table 7. Summary of reproduction data (continued)

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Dams with viable fetuses	N	21	24	19	23
Live Fetuses	Mean	9.0	8.7	9.2	9.5
	SD	1.83	2.33	2.48	1.97
	Total	190	209	174	218
	Mean %	93.8	96.3	94.4	94.8
	SD	11.04	5.80	9.23	7.30
Females	Mean	4.3	4.0	4.4	5.5
	SD	1.68	1.60	1.89	1.83
	Total	90	95	84	126
	Mean %	44.2	43.8	47.7	55.1
	SD	17.36	15.75	17.31	16.62
Males	Mean	4.8	4.8	4.7	4.0
	SD	1.45	1.94	2.47	1.78
	Total	100	114	90	92
	Mean %	49.6	52.4	46.6	39.7
	SD	14.53	15.12	20.58	17.68
Percent live females		47.4	45.5	48.3	57.8
percent live males		52.6	54.5	51.7	42.2

Table 8. Mean placental and fetal body weights (on a litter basis)

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
PLACENTAL WEIGHTS (g)
of all viable Fetuses	MEAN	5.2	5.0	4.9	4.9
	S.D.	0.89	0.81	1.00	0.77
	N	21	24	19	23
of Male Fetuses	MEAN	5.2	5.1	4.9	5.1
	S.D.	0.89	0.74	0.73	0.85
	N	21	24	18	22
of Female Fetuses	MEAN	5.1	4.9	4.8	4.7
	S.D.	0.92	0.96	1.01	0.75
	N	21	24	18	23
FETAL WEIGHTS (g)
of all viable Fetuses	MEAN	38.4	39.0	37.0	34.9
	S.D.	3.79	6.46	6.21	3.82
	N	21	24	19	23
of Male Fetuses	MEAN	38.5	39.3	36.4	35.6
	S.D.	4.37	6.78	4.85	4.14
	N	21	24	18	22
of Female Fetuses	MEAN	38.1	38.5	37.1	34.2
	S.D.	4.42	6.54	6.34	4.26
	N	21	24	18	23

FETAL EXTERNAL EXAMINATIONS

Fetal external malformations

One external malformation was recorded for a control fetus (protruding tongue). There were no external malformations in any test substance-treated fetuses.

Tab. 9: Total external malformations

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Litters Fetuses	N N	21 190	24 209	19 174	23 219
Fetal Incidence	N (%)	1 (0.5)	0.0	0.0	0.0
Litter Incidence	N (%)	1 (4.8)	0.0	0.0	0.0
Affected fetuses/ litter	Mean (%)	0.5	0.0	0.0	0.0

Fetal ecxternal variations

No external variations were recorded.

Fetal external unclassified observations

Three unclassified external observations were recorded, i.e. ‘pale fetus’ in one control fetus, ‘head: bulge’ and ‘skin: lesion’ in one fetus, each, of the same low-dose litter (100 mg/kg bw/d). These findings are not considered to be related to treatment.

Tab. 10: Total external unclassified observations

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Litters Fetuses	N N	21 190	24 209	19 174	23 219
Fetal Incidence	N (%)	1 (0.5)	2 (1.0)	0.0	0.0
Litter Incidence	N (%)	1 (4.8)	1 (4.2)	0.0	0.0
Affected fetuses/ litter	Mean (%)	0.6	0.7	0.0	0.0

FETAL SOFT TISSUE EXAMINATIONS

Fetal soft tissue malformations

Soft tissue malformations occurred in single fetuses of test groups 0, 2 or 3 (0, 300 or 1000 mg/kg bw/d), as listed in Table 3. The distribution of the findings about the test groups does not indicate an association to the treatment and no statistically significant differences between the groups were noted. The total incidence of soft tissue malformations in treated animals did not differ significantly from the control group (Table 4) and was comparable to the historical control data.

Tab. 11: Individual fetal soft tissue malformations

Test group	Doe No.-fetus No. Sex	Finding
control	9-08 M	aortic arch atresia, heart: muscular ventricular septum defect
100 mg/kg bw	none
300 mg/kg bw	51-02 M 58-06 M	malpositioned kidney enlarged lens
1000 mg/kg bw	77-07 F 88-02 F 93-01 F	absent subclavian aortic arch atresia, heart: muscular ventricular septum defect absent gallbladder

Tab. 12: Total soft tissue malformations

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Litters Fetuses	N N	21 190	24 209	19 174	23 219
Fetal Incidence	N (%)	1 (0.5)	0.0	2 (1.1)	3 (1.4)
Litter Incidence	N (%)	1 (4.8)	0.0	2 (11)	3 (13)
Affected fetuses/ litter	Mean (%)	0.5	0.0	1.0	1.3

Fetal soft tissue variations

The examinations of the soft tissues revealed malpositioned carotid branches in all test groups including the control (0, 100, 300 and 1000 mg/kg bw/d). Absent lung lobe (Lobus inferior medialis) was noted in the test groups 0, 2 and 3. The incidences of malpositioned carotid branches in the low-dose group and of absent lung lobe in the mid-dose group, slightly exceed the historical control range. As there is not dose response and both are quite common findings in this rabbit strain showing high incidences in the historical control data of the test facility, no association with the treatment is assumed. Other variations, such as cystic dilatation of the brain and small gallbladder, occurred in individual fetuses of test group 2 (brain: cystic dilatation in 2 fetuses of different litters) or test group 3 (small gallbladder in one single fetus). No statistically significant or toxicologically relevant differences between the groups were noted, and the overall incidences were within the historical control range (Table 5).

Tab. 13: Total soft tissue variations

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Litters Fetuses	N N	21 190	24 209	19 174	23 219
Fetal Incidence	N (%)	4 (2.1)	8 (3.8)	7 (4.0)	8 (3.7)
Litter Incidence	N (%)	4 (19)	6 (25)	5 (26)	4 (17)
Affected fetuses/ litter	Mean (%)	2.0	4.8	3.5	3.7

Fetal soft tissue unclassified observations

Two unclassified soft tissue observations were recorded: an altered texture of kidney was seen in one control fetus, furthermore, a blood coagulum around urinary bladder was recorded in four control, two low-dose, three mid-dose and three high-dose fetuses. These findings were not considered biologically relevant.

Tab. 14: Total soft tissue unclassified observations

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Litters Fetuses	N N	21 190	24 209	19 174	23 219
Fetal Incidence	N (%)	5 (2.6)	2 (1.0)	3 (1.7)	3 (1.4)
Litter Incidence	N (%)	2 (9.5)	2 (8.3)	2 (11)	3 (13)
Affected fetuses/ litter	Mean (%)	2.7	1.1	1.5	1.3

FETAL SKELETAL EXAMINATIONS

Fetal skeletal malformations

Skeletal malformations were detected in single fetuses of test groups 0 and 2 (0 and 300 mg/kg bw/d), as shown in Tab. 7. No statistically significant differences between the test substance-treated groups and the control were noted (Tab. 8). The overall incidences were well within the historical control range of the test facility.

Tab. 15: Individual fetal skeletal malformations

Test group	Doe No.-fetus No. Sex	Finding
control	1-03 M 9-14 F 20-10 M	malpositioned and bipartite sternebra branched rib misshapen thoracic vertebra
100 mg/kg bw	none
300 mg/kg bw	74-07 M	absent lumbar vertebra, branched rib
1000 mg/kg bw	none

Tab. 16: Total skeletal malformations

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Litters Fetuses	N N	21 190	24 209	19 174	23 219
Fetal Incidence	N (%)	3 (1.6)	0.0	1 (0.6)	0.0
Litter Incidence	N (%)	3 (14)	0.0	1 (5.3)	0.0
Affected fetuses/ litter	Mean (%)	1.3	0.0	0.7	0.0

Fetal skeletal variations

For all test groups, skeletal variations of different bone structures were observed, with or without effects on corresponding cartilages. The observed skeletal variations were related to several parts of fetal skeletons and were neither statistically significantly nor dose-dependently changed in comparison to the control group (Tab. 9). The overall incidences of skeletal variations were comparable to the historical control data.

Tab. 17: Total fetal skeletal variations

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Litters Fetuses	N N	21 190	24 209	19 174	23 219
Fetal Incidence	N (%)	171 (90)	180 (86)	156 (90)	187 (85)
Litter Incidence	N (%)	21 (100)	24 (100)	19 (100)	23 (100)
Affected fetuses/ litter	Mean (%)	89.8	84.7	89.2	84.2

Fetal skeletal unclassified cartilage observations

Some isolated cartilage findings without impact on the respective bone structures, which were designated as unclassified cartilage observations, occurred in all test groups (Tab. 10). The observed unclassified cartilage findings were related to the sternum and the ribs and did not show any relation to dosing. Therefore, they were assessed as not treatment-related.

Tab. 18: Total unclassified cartilage observations

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Litters Fetuses	N N	21 190	24 209	19 174	23 219
Fetal Incidence	N (%)	25 (13)	32 (15)	16 (9.2)	15 (6.8)
Litter Incidence	N (%)	10 (48)	11 (46)	8 (42)	12 (52)
Affected fetuses/ litter	Mean (%)	14.1	13.7	8.4	7.4

Assessment of all fetal external, soft tissue and skeletal observations

There were noted external, soft tissue and skeletal malformations in the test groups 0, 2 and 3 (0, 300 or 1000 mg/kg bw/d). The distribution of total malformations about the groups was not related to dose.

Three fetuses had more than one malformation. Respectively, for male control fetus No. 9-08 and female high-dose fetus No. 88-02 (1000 mg/kg bw/d) an aortic arch atresia and a ventricular septum defect were recorded. Male mid-dose fetus No. 74-07 (300 mg/kg bw/d) had a branched rib in combination with an absent lumbar vertebra. No ontogenetic pattern is recognizable for the individual malformations nor was there any cluster of any of these individual malformations seen in the other offspring of these test groups. Further malformations, such as protruding tongue, absent subclavian, enlarged lens, absent

gallbladder, malpositioned kidney, misshapen thoracic vertebra, as well as malpositioned and bipartite sternebra were scattered observations in individual fetuses of test groups 0, 2 or 3. They were not dose-related and most of them can be found in the historical control data at comparable or higher frequency, thus, a relation to the treatment is not assumed. The total incidences of malformations are summarized in Tab. 11.

Tab. 19: Total fetal malformations

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Litters Fetuses	N N	21 190	24 209	19 174	23 219
Fetal Incidence	N (%)	5 (2.6)	0.0	3 (1.7)	3 (1.4)
Litter Incidence	N (%)	4 (19)	0.0	3 (16)	3 (13)
Affected fetuses/ litter	Mean (%)	2.3	0.0	1.6	1.3

External variations did not occur in any fetus in this study. Four soft tissue variations and a range of skeletal variations were noted in all test groups including controls. If all different types of variations are summarized, none of the incidences showed a relation to dosing (Tab. 12) and can be found in the historical control data at a comparable frequency.

Tab. 20: Total fetal variations

		Control	100 mg/kg bw	300 mg/kg bw	1000 mg/kg bw
Litters Fetuses	N N	21 190	24 209	19 174	23 219
Fetal Incidence	N (%)	171 (90)	181 (87)	156 (90)	190 (87)
Litter Incidence	N (%)	21 (100)	24 (100)	19 (100)	23 (100)
Affected fetuses/ litter	Mean (%)	89.8	85.2	89.2	85.5

A spontaneous origin is assumed for the unclassified external unclassified soft tissue and unclassified skeletal cartilage observations which were observed in several fetuses of test groups 0, 1, 2 and 3. The

distribution and type of these findings do not suggest any relation to treatment. Finally, fetal examinations revealed that there is no adverse effect of the compound on the respective morphological structures up to the highest dose tested (1000 mg/kg bw/d).

Conclusions:

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test item to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) at doses as high as 1000 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 1000 mg/kg bw/d.

Executive summary:

The test item was tested for its prenatal developmental toxicity in New Zealand White rabbits. The test substance was administered as an aqueous suspension to groups of 25 inseminated female New Zealand White rabbits orally by gavage in doses of 100, 300 and 1000 mg/kg body weight/day (mg/kg bw/d) on gestation days (GD) 6 through 28. The vehicle control group, consisting of 25 females, was dosed with the vehicle (0.5% Sodium carboxymethyl cellulose [CMC] suspension in deionized water (with 10 mg/100 mL Cremophor EL) in parallel. A standard dose volume of 10 mL/kg body weight was used for each test group. At terminal sacrifice on GD 29, 19-24 females per group had implantation sites. Food consumption and body weight of the animals were recorded regularly throughout the study period. The state of health of the animals was checked each day. On GD 29, all females were sacrificed and assessed by gross pathology (including weight determinations of the unopened uterus and placentas). For each doe, corpora lutea were counted and number and distribution of implantation sites (differentiated between resorptions, live and dead fetuses) were determined. The fetuses were removed from the uterus, sexed, weighed and further investigated for any external, soft tissue and skeletal (inclusive cartilage) findings.

The stability of the test substance in 0.5% CMC suspension in deionized water (with 10 mg/100 mL Cremophor EL) over a maximum of 7 days at room temperature was demonstrated. The homogeneous distribution of the test substance in the vehicle was shown. The correctness of the prepared concentrations was shown. Clinical observations revealed no toxicologically relevant difference between the animals

receiving 100, 300 or 1000 mg/kg bw/d of the test item and the controls. Neither food consumption nor gross or net body weight gain were adversely influenced by the treatment. No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age. Similarly, no influence of the test substance on uterine weight, placental weight, fetal weight and sex distribution of the fetuses was noted at any dose. Fetal examinations revealed no toxicologically relevant adverse effects of the test substance on embryofetal development.

Under the conditions of this prenatal developmental toxicity study, the oral administration of the test item to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) at doses as high as 1000 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity. In conclusion, the no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 1000 mg/kg bw/d.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a developmental toxicity study conducted according to the OECD Guideline 414 (Prenatal Developmental Toxicity Study, version of 1981), groups of 25 sexually mature and mated female rats received the test substance by oral gavage (arachis oil) at dosages of 50, 150, or 300 mg/kg daily for ten consecutive days from day 6 to day 15 post-coitum (inclusive). A further group of 25 rats of the same strain which received the vehicle (arachis oil) over the same period served as the control group. Doses were determined based on the results of a screening study.

The animals were sacrificed on day 20 post-coitum and the results of the study can be summarized as follows:

1) There were no clinical observations and no necropsy findings which might be considered to be related to the treatment with the test substance.

2) At a dose level of 300 mg/kg, maternal body weight gain during gestation was reduced by 16%, indicating that the dose selection was adequate for hazard assessment.

3) There was no effect of treatment on pregnancy incidence, implantations, or post-implantation loss

4) The mean number of foetuses per dam was slightly reduced in group 4 (300 mg/kg). This finding is considered to be related with the slightly reduced mean number of implantations per female and therefore not to be a compound-related effect.

5) Sex distribution was not affected by the treatment, but mean foetal weight of group 4 (300 mg/kg) was slightly increased. This finding is considered to be related with the slightly reduced mean number of foetuses per female in this group and not to indicate any effect of treatment.

6) Nature and incidence of malformations and variations did not reveal any compound-related effect.

In conclusion, administration of the test substance during organogenesis by oral gavage at a dose level of 300 mg/kg elicited slight maternal toxicity (slightly reduced body weight gain and - possibly - slightly reduced food consumption), but no embryotoxicity, and no teratogenicity. Administration of 50 and 150 mg/kg did not elicit any maternal toxicity, embryotoxicity, or teratogenicity. Effects on body weight gain was not observed in the subchronic toxicity study in rats, but this may be related to the use of arachis oil instead of an aqueous suspension as vehicle.

In a prenatal developmental toxicity study following OECD guideline 414 and under GLP, the test item was administered to pregnant New Zealand White rabbits daily by stomach tube from implantation to one day prior to the expected day of parturition (GD 6-28). Analyses confirmed the correctness of the prepared concentrations, their homogeneous distribution and the stability of the test substance in the vehicle. Clinical observations revealed no toxicologically relevant difference between the animals receiving 100, 300 or 1000 mg/kg bw/d 4,6-Bis(octylthiomethyl)-o-cresol and the controls. Neither food consumption nor gross or net body weight gain were adversely influenced by the treatment. No differences of toxicological relevance between the control and the treated groups (100, 300 or 1000 mg/kg bw/d) were determined for any reproductive parameters, such as conception rate, mean number of corpora lutea, mean number of implantations, as well as pre- and postimplantation loss. All differences observed are considered to reflect the normal range of fluctuations for animals of this strain and age. Similarly, no influence of the test substance on uterine weight, placental weight, fetal weight and sex distribution of the fetuses was noted at any dose. Fetal examinations revealed no toxicologically relevant adverse effects of the test substance on embryofetal development. In conclusion, under the conditions of this prenatal developmental toxicity study, the oral administration of the test article to pregnant New Zealand White rabbits from implantation to one day prior to the expected day of parturition (GD 6-28) at doses as high as 1000 mg/kg bw/d caused neither evidence of maternal nor developmental toxicity. The no observed adverse effect level (NOAEL) for maternal and prenatal developmental toxicity is 1000 mg/kg bw/d.

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for the purpose of classification under Regulation 1272/2008.Based on the data, classification for repeated dose toxicity is not warranted under Regulation (EC) No.1272/2008.