Benzamide, N-[[4-[(cyclopropylamino)carbonyl]phenyl]sulfonyl]-2-methoxy-

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Two-generation reproductive toxicity (OECD 416), oral, rat:
NOAEL (reproductive) parental = 173 mg/kg bw/day (female); 1007 mg/kg bw/day (male)
NOAEL (systemic) parental = 39/55 mg/kg bw/day (male/female)
NOAEL (developmental) offspring = 173 mg/kg bw/day (male/female)

Link to relevant study records

Reference

Endpoint:

two-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Oct 2004 - 13 Jul 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)

Version / remarks:

adopted 22 Jan 2001

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministerium für Umwelt und Naturschutz, Landwirtschaft und Verbraucherschutz des Landes Nordrhein-Westfalen, Germany

Limit test:

no

Species:

rat

Strain:

other: Wistar Crl: (WI) WU BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River GmbH, Sulzfeld, Germany
- Age at study initiation: (P) 6-7 wks; (F1) 4 wks
- Weight at study initiation: (P) Males: 108-159 g; Females: 74-100 g, (F1) Males: 73-121 g; Females 60-109 g
- Fasting period before study: Not applicable, feeding study
- Housing: Singly except when co-housed for matings or with litters in Makrolon cages Type IIIh on low-dust soft-wood shavings or nesting material (day 20 p.c. to 14 p.p.).
- Diet: Kliba 3883.9.25 (Provimi Kliba SA, CH-4303 Kaiseraugst, Switzerland), ad libitum.
- Water: Tap water, ad libitum.
- Acclimation period: About 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): Minimum of 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): At least weekly.
- Mixing appropriate amounts with (Type of food): Dietary formulations were prepared by mixing appropriate amounts of test substance with diet (Kliba 3883.9.25) using a pre-mixture. A mixing granulator was used (manufactured by the company Loedige, Paderborn, Germany).
- Storage temperature of food: At room tempearture.

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: A maximum of 12 times during the three-week mating period; inseminated females were not further co-housed.
- Proof of pregnancy: Vaginal plug/sperm in vaginal smear referred to as Day 0 of pregnancy.
- After 14 days of unsuccessful pairing (lack of weight gain following insemination): Females were co-housed again over one week with the same male without checking insemination or measuring body weight and food intake during possible further pregnancy. According to the test laboratories experience, occurrence of sperms after the first co-housing day without a following pregnancy can happen, if an inexperienced male, co-housed with a female for the first time, inseminated the female outside the oestrus.
- Further matings after two unsuccessful attempts: Yes, Six (P) females of the 12000 ppm group, which did not get pregnant with the males appointed, were co-housed additionally over the whole day with another male of the same group, which had been shown to be fertile. These unscheduled co-housings were done to investigate the cause of lack of pregnancy of these females. On these females vaginal smears were taken for evaluation for positive sperm findings as done normally to prove a mating. Sperm positive (P) females were kept then individually until littering. Litters were killed when born, but no pup necropsies of these animals were done. The (P) females in question were necropsied when it became clear that they were not pregnant or after their littering. No other data than date of positive sperm finding and littering were recorded. Implantation sites of these females, if any, were not entered to the statistical analyses.
- After successful mating each pregnant female was caged singly in Makrolon cages Type IIIh on nesting material.
- Any other deviations from standard protocol: Generally, if a male had to be necropsied prior to co-housing the next male was co-housed with two females.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Checks on homogeneity and stability (low and high concentrations) and contents of active ingredient (all doses including control) were done at several times during the study. The dietary levels of the test substance were verified by HPLC-UV.
Homogeneity analysis: In samples taken from the top, middle and bottom of 100 and 15000 ppm mixtures the test substance was proven to be homogeneously distributed.
Stability analysis: The test substance concentration in 100 and 15000 ppm mixtures was within the range of ± 20% of their initially measured concentration after storage at animal room temperature for up to 15 days and after 15 days of freezer storage (95-104%).
Concentration analysis: The absence of the test substance in the control diet was confirmed. The test substance concentration (all concentrations including control) measured at four time points was in the range of ± 20% of the nominal concentrations (104-113%). The active ingredient at the low and high concentration was homogenously distributed.

Duration of treatment / exposure:

(P) Males: About 12 weeks before mating, 3 weeks during mating.
(P) Females: About 12 weeks before mating, 3 weeks during mating, about 22 days during gestation, 28 days during lactation until weaning.
(F1) Males: 28 days until weaning, during growth into adulthood (at least 10 weeks), mating and production of an F2 generation, until weaning (28 days) of the F2 generation.
(F1) Females: 28 days until weaning, during growth into adulthood (at least 10 weeks), mating and production of an F2 generation, until weaning (28 days) of the F2 generation.

Frequency of treatment:

Daily, 7 days/week

Details on study schedule:

- F1 parental animals not mated until 10 weeks after selected from the F1 litters.
- Selection of parents from F1 generation when pups were 28 days of age.
- Age at mating of the mated animals in the study: 18-19 weeks (P); 14 weeks (F1)

Dose / conc.:

480 ppm

Remarks:

corresponding to actual dose ingested during the pre-mating period:
39.2 mg/kg bw/day for P generation males (P0)
55.3 mg/kg bw/day for P generation females (P0)
50.3 mg/kg bw/day for F1 parental males (P1)
68.5 mg/kg bw/day for F1 parental females (P1)

corresponding to actual dose ingested during gestation and lactation:
37.2 mg/kg bw/day for P generation females (P0), Days 14 to 20 post coitum
58.5 mg/kg bw/day for P generation females (P0), Days 0 to 4 post partum
42.0 mg/kg bw/day for F1 parental females (P1), Days 14 to 20 post coitum
60.5 mg/kg bw/day for F1 parental females (P1), Days 0 to 4 post partum

Dose / conc.:

2 400 ppm

Remarks:

corresponding to actual dose ingested during the pre-mating period:
202.3 mg/kg bw/day for P generation males (P0)
260.3 mg/kg bw/day for P generation females (P0)
251.5 mg/kg bw/day for F1 parental males (P1)
323.7 mg/kg bw/day for F1 parental females (P1)

corresponding to actual dose ingested during gestation and lactation:
173.1 mg/kg bw/day for P generation females (P0), Days 14 to 20 post coitum
246.4 mg/kg bw/day for P generation females (P0), Days 0 to 4 post partum
196.2 mg/kg bw/day for F1 parental females (P1), Days 14 to 20 post coitum
272.6 mg/kg bw/day for F1 parental females (P1), Days 0 to 4 post partum

Dose / conc.:

12 000 ppm

Remarks:

corresponding to actual dose ingested during the pre-mating period:
1006.9 mg/kg bw/day for P generation males (P0)
1350.2 mg/kg bw/day for P generation females (P0)
1292.4 mg/kg bw/day for F1 parental males (P1)
1696.5 mg/kg bw/day for F1 parental females (P1)

corresponding to actual dose ingested during gestation and lactation:
840.5 mg/kg bw/day for P generation females (P0), Days 14 to 20 post coitum
1370.8 mg/kg bw/day for P generation females (P0), Days 0 to 4 post partum
1001.2 mg/kg bw/day for F1 parental females (P1), Days 14 to 20 post coitum
1449.1 mg/kg bw/day for F1 parental females (P1), Days 0 to 4 post partum

No. of animals per sex per dose:

25

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: Dose selection was based on results of a one-generation pilot study (Bayer Report AT02084, May 20, 2005), where rats received 1000, 4000 and 12000 ppm in the diet. In this study the NOEL was 1000 ppm in males and 4000 ppm in females. At higher concentrations the body weight gain was decreased (in 12000 ppm females during lactation). At necropsy three of ten 12000 ppm (P) females exhibited a discolored, pale liver. There were increased absolute and relative weights of the adrenals, liver and kidneys (females) as well as the (both sexes) at 12000 ppm. At 12000 ppm decreased pup weights were observed from day 7 post partum onwards resulting in reduced absolute weights of the uterus. Therefore, the 2-generation reproduction study was dosed at 480, 2400 and 12000 ppm.

Positive control:

None

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily and once daily on weekends and holidays.
- Cage side observations included: All signs of illness or clinical reactions to treatment (especially during littering) were individually recorded daily. All further clinical symptoms were also recorded. This investigation included the observation of the general state of health, behavior, condition of the fur, and the orifices as well as excretory products. Any further findings e.g. prolonged parturition, morbidity and mortality were recorded as well.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Prior to the first administration in the diet and then weekly as a rule and during the pregnancy and lactation periods as follows:
- Pregnancy on Day 0 (= day of a sperm positive smear or vaginal plug) 7, 14 and 20.
- Lactation on Day 0 (day of birth), 4, 1, 14, 21 and 28.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded directly prior to the first administration and thereafter weekly up to necropsy (males and females not pregnant) and during the pregnancy and lactation periods as follows: During pregnancy on Day p.c. 0, 7, 14 and 20; During lactation on Day p.p. 0, 4, 7, 14, 21 and 28; On the day of scheduled necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
The individual food consumption was measured (by weighing the quantity of food provided and back-weighing the amount, which remained unconsumed) as follows:
- Males: Weekly from Week 1 up to necropsy (except during mating period).
- Females: Weekly from Week 1 up to mating.
During pregnancy Day post coitum (p.c.) 0-7; 7-14; 14-20.
During lactation Day post partum (p.p.) 0-4, 4-7.
From these food intake data the intake of the test compound was calculated per animal and/or per kg body weight per day, week or for a given period.

OTHER:
Determination of insemination
During the mating periods vaginal smears were taken in the morning after rats had been co-housed over night to determine time of insemination and gestation length. The vaginal smears were obtained using a flame-sterilized platinum loop and plated out on slides. Smears were stained for about 1 minute with MAY GRUENWALD'S Eosine-methylene blue solution modified for microscopy. The date where sperms or a vaginal plug were found microscopically was taken as gestation Day 0 for calculating the gestation length. Females which exhibited marked weight gains although insemination had not been established were not further co-housed. No duration of pregnancy could be determined for these animals.

Oestrous cyclicity (parental animals):

Oestrus cycle length determination was done by evaluation of vaginal smears received daily over 19 consecutive days about 2 weeks prior to the mating period. The smears were examined microscopically for large serrated cells indicating that oestrus had occurred. This data was used to determine the oestrus cycle length and whether females were cycling properly.

Sperm parameters (parental animals):

Spermatological investigations were performed in all surviving (P) and (F1) males of the 0 and 12000 ppm group on the day of necropsy: Spermatozoa motilily, viability and morphology; Determination of spermatozoa in epididymis; Determination of homogenisation resistant spermatid heads in the testis

Litter observations:

STANDARDISATION OF LITTERS
- Performed on Day 4 postpartum: Yes
- Maximum of 8 pups/litter (4/sex/litter); excess pups (dead/alive) were examined for external effects.

PARAMETERS EXAMINED
The following parameters were examined in F1 / F2 offspring:
Number of live pups, pup weight, external alterations (=clinical and necropsy observation), number of dead pups, ano-genital measurements, sex of each pup, developmental milestones, including age and body weight when balano-preputial separation or vaginal opening occured.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals when no longer needed for matings.
- Maternal animals: All surviving animals at weaning of 28 days old pups or up to 2 days later.
All parental rats to be necropsied unscheduled were also killed by exsanguination under CO2 narcosis.

GROSS NECROPSY
- Gross necropsy was done on all rats and consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera. Implantation sites were counted after staining with 10% aqueous ammonium sulfide. In cases where implausible discrepancies existed between the number of observed implantation sites and the number of pups delivered (more pups than implantation sites) the number of pups was used as number of implantation sites. In case of autolytic rats or rats killed pre-scheduled the number of pups was taken as the number of implantation sites.
Vaginal smears were performed and evaluated at scheduled necropsy as precaution but were not reported. Rats found dead or which had to be killed in moribund condition were necropsied as early as possible in the same way.

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues of adult (P) and (F1) rats were fixed in 10% formalin with the exception of total kidneys (P) or the left and two thirds of the right kidney and testes, which were fixed in Davidson's solution: Abnormalities, adrenals*, brain*, epididymides*, esophagus, kidneys*, larynx, liver*, ovaries and oviducts*, pituitary gland*, prostate*, physical identifier, seminal vesicles and coagulating glands*, skin in mammary region, spleen*, testes*, thyroid/parathyroids*, trachea, ureters, urinary bladder, uterus (incl. cervix)*, vagina, head with scull cap (* organ was weighed).
The organs mentioned above were microscopically examined at least in the control and high dose group. This included also all P/F1 rats, which died intercurrently (as far as possible) and those, which were killed moribund. Organs altered possibly by the treatment and any gross and/or microscopically changed lesions were examined in all rats. Staging of ovarian follicles was done in F1 females (control and high concentration) only. All reproductive organs of pairs without pups were investigated.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 28 days of age.

GROSS NECROPSY
All pups culled for standardisation of litter size, found dead or had to be killed moribund during lactation were necropsied and investigated macroscopically with particular attention on the organs of reproduction except for cases of autolysis or cannibalism. This included also visible skeletal abnormalities as far as possible. A lung flotation in water was performed during the necropsy of pups found dead on the day of the first litter inspection to determine whether pups had breathed at birth (life birth) or not.
All F1 weanlings not selected for further treatment were killed when they were at least 4 weeks old and examined macroscopically.
All F2 weanlings were killed after a 4 week lactation period and examined macroscopically.

HISTOPATHOLOGY / ORGAN WEIGTHS
Grossly abnormal tissues if any were fixed in all pups/weanlings. In F1 and F2 weanlings brain, spleen, thymus, uterus, urinary bladder (instillation fixed), ureters, urethra and kidneys of one male and one female out of the firstly necropsied 5 litters per group were fixed in 10% formalin.
The brain, spleen, thymus and uterus of one male and one female per F1/F2 litter were trimmed and weighed as soon as possible after dissection. The ratio of organ weights to body weights was calculated. Therefore, all these weanlings were weighed at day of necropsy.

Statistics:

Statistical evaluation was performed by a TASC-application (Toxicology Analysis Systems Customized) using the following methods:
Analysis of Variance (ANOVA) and in case of significant results Dunnett's test as post hoc test: Body weights/body weight gains of males/females, food consumption of the males and females, number of implantation sites/female, number of viable pups/female, organ weights at necropsy, time to insemination, life birth, viability and lactation index, oestrus data.
Chi-test and in case of significant differences Fisher's exact test with Bonferroni correction: Number of viable pups/group based on the number of implantations, insemination, fertility, gestation and rearing rate.
Kruskall-Wallis test and in case of significant differences Dunnett's test: number of prenatal loss per litter.

Sperm and spermatid count data were not evaluated statistically, because there were no meaningful differences between high dose and control groups.
Generally, differences between control group and treatment-groups were considered as statistically significant when p<0.05 (*) or p<0.01 (**).

Follicle counts were evaluated statistically using Wilcoxon Mann-Whitney U-test. Females without implantation sites and females and males, which died or which had to be sacrificed in moribund condition, were excluded from statistical evaluation of post mating parameters. This was true also for those (P) females, where additionally mated. Animals which spilled their food, if any, were excluded from calculation of means for food intake at the respective days.
Gestation length was not calculated for remated females with pups. Nutritional state of animals examined at their necropsy was reported only in cases where a deviation from normal nutritional state was noted. Mean pup weight of each individual litter was used as basis for calculating mean pup weight of the dose groups. Mean litter size calculation was based on number of female animals with living pups.

Reproductive indices:

The following reproductive indices were calculated from breeding and parturition records of animals in the study using the following formulae:
Insemination index (%): (No. of sperm positive females/No. of females co-housed with a male) x 100
Fertility index (%): (No. pregnant females/No. of sperm positive females) x 100
Gestation index (%): (No. of females completing delivery/No. of pregnant females) x 100
Rearing index (%): (No. of females rearing a litter up to day 21 p.p./No. of females that delivered a litter) x 100

Offspring viability indices:

The following viability indices were calculated from lactation records of litters in the study:
Live birth index (%): (No. of live pups at birth/total No. of pups born) x 100
Viability index (%): (No. of live pups on day 4 pre-culling/No. of live pups born) x 100
Lactation index (%): (No. of live pups after three weeks/No. of live pups after four days (after culling)) x 100 (moribund pups that died during the course of culling were not included)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- Increase in the number of P females showing adverse clinical signs, including piloerection, poor general condition, paleness and emaciation. Tabulated data is presented in Attachment 1.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, treatment-related

Description (incidence):

12000 ppm:
- During lactation five P females died or had to be sacrificed moribund. Tabulated data is presented in Attachment 1.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- During gestation and lactation P females gained less (p<0.01) body weight than controls. Tabulated data is presented in Attachment 2.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Compound intake:
- Based on food consumption and body weight data, the doses expressed as mean daily mg test substance/kg body weight during the premating, gestation and lactation periods are presented in Attachment 3. The values for the P and F1 generation are considered to be representative for the test substance intake for the entire study.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no histopathological lesions induced by the test substance at 480 ppm. Incidences of treatment-related organ changes found from 2400 ppm onwards are summarised in Attachment 7.

Intercurrent mortality:
- The increased intercurrent mortality (incidence P:0/0/0/5), seen in 12000 ppm P females is secondary to induced renal lesions caused by precipitation of foreign material in the kidneys and lower urinary tract.

Reproductive tract:
- There was no evidence of any induced finding in the testes, epididymides, prostate and seminal vesicles/coagulating glands up to and including 12000 ppm.
- In the ovaries, the amount and size of corpora lutea was similar throughout the groups up to and including 12000 ppm.
- At 12000 ppm there was an increased incidence of corpora lutea with a foamy cytoplasm.
- In the uterus of 12000 ppm P females with foamy corpus luteum cells more unregressed implantation sites were found.
- The vagina of the P females affected by the changes in the corpora lutea and uterine implantation sites showed a highly mucified vaginal epithelium more often.
- The numbers of animals without progeny did not show any dose-correlation. The histopathological investigation of these rats showed no evidence of a test substance induced effect.

Urinary tract:
- In the kidneys of 12000 ppm rats, precipitates of foreign (crystalline) material - presumably the test compound or metabolite - could be found more frequently in the collecting ducts and renal pelvis than at 0 ppm. As sequels of this condition the following changes were observed in the kidneys of male and female parental rats at 2400 ppm (partly) and 12000 ppm:
- Diffuse tubular basophllia, intratubular granulocytes, papillary necrosis, medullary fibrosis, dilation of the renal pelvis and transitional cell hyperplasia.
- A slight elevation of ureteric hypertrophy was encountered also in 12000 ppm P females.
- A slightly elevated incidence of diffuse transitional cell hyperplasia was observed in the urinary bladder of 12000 ppm P females.

Adrenals, liver and spleen:
- In the adrenal glands of 12000 ppm P females, an increased incidence of hypertrophy of the zona glomerulosa was seen. This was considered to be induced by the renal findings such as foreign material, basophilic tubules and renal pelvic dilation, therefore, it was regarded as treatment-related.
- In the liver an increased incidence of condensed cytoplasm, which is the morphological correlate of a decreased glycogen content, was found in P females at 12000 ppm more frequently than at 0 ppm.
- In the spleen a comprised congestion, increased haemopoiesis and high grade of iron pigment deposits were observed more frequently in males (from 2400 ppm onwards) and females (at 12000 ppm) of the P generation indicating an increased turnover of red blood cells.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- A reduced rearing index was calculated for P females as result of increased mortality of 12000 ppm dams (5 deaths) and some total litter losses of females. A summary of reproductive performance is presented in Attachment 4.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

480 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at this dose level.

Remarks on result:

other: corresponding to 39 and 55 mg/kg bw/day for males and females, respectively.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

2 400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 202 and 260 mg/kg bw/day for males and females, respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

2 400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at this dose level.

Remarks on result:

other: corresponding to 173 mg/kg bw/day.

Key result

Dose descriptor:

LOAEL

Remarks:

reproductive

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

reproductive performance

Remarks on result:

other: corresponding to 840 mg/kg bw/day.

Critical effects observed:

yes

Lowest effective dose / conc.:

2 400 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Critical effects observed:

yes

Lowest effective dose / conc.:

2 400 ppm

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- There was an increase in number of F1 male and female rats showing clinical signs such as poor general condition, piloerection, paleness, high-stepping gate, sunken flanks and/or, emaciation. Tabulated data is presented in Attachment 1.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, treatment-related

Description (incidence):

12000 ppm:
- Four F1 females were sacrificed pre-scheduled, two of them died and two had to be sacrificed moribund.

480 ppm:
- One F1 female had to be killed moribund.

Control:
- One F1 male died after mating.

Tabulated data is presented in Attachment 1.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- At termination of the premating period, F1 females exhibited 9% lower (p<0.01) body weights than controls. There was also a slight body weight depression (-4.5% compared to controls) in F1 males receiving 12000 ppm, which was not statistically significant.
- During gestation and lactation F1 females gained less (p<0.01) body weight than controls.

Tabulated data is presented in Attachment 2.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Compound intake:
- Based on food consumption and body weight data, the doses expressed as mean daily mg test substance/kg body weight during the premating, gestation and lactation periods are presented in Attachment 3. The values for the F0 and F1 generation are considered to be representative for the test substance intake for the entire study.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ measurements revealed a tendency to increased absolute and relative spleen weights in F1 males and females from 2400 ppm onwards, which were partly statistically significant. There was also a tendency to increased absolute and relative thyroid weights in F1 females from 2400 ppm onwards. Relative kidney weights were statistically significantly increased in F1 females at 12000 ppm. Other statistical significances or weight deviations were not toxicologically significant as a dose correlation was missing, the differences were minor or caused by variations in body weights. A summary of absolute and relative organ weights for F1 parental animals is presented in Attachment 6.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- 7/25 F1 females were found to be skinny.
- An increased incidence of gross changes in the kidneys was found F1 females (incidences with increasing dose: discoloration 1/0/0/5, enlargement 0/0/0/2, surface changes 1/0/0/5).

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Intercurrent mortality:
- The increased intercurrent mortality (incidence F1: 0/1/0/4) seen in 12000 ppm F1 females was secondary to induced renal lesions caused by precipitation of foreign material in the kidneys and lower urinary tract.
- Female No. 332 (480 ppm, F1 generation) exhibited an intrauterine fetus and fluid, which corresponded histopathologically to marked metritis and perimetritis.

Reproductive tract:
- There was no evidence of any induced finding in the testes, epididymides, prostate and seminal vesicles/coagulating glands up to and including 12000 ppm.
- In the ovaries, the amount and size of corpora lutea was similar throughout the groups up to and including 12000 ppm.
- At 12000 ppm there was an increased incidence of corpora lutea with a foamy cytoplasm.
- Counting of primary follicles and corpora lutea done in controls and 12000 ppm F1 females revealed no treatment-related changes.
- The numbers of animals without progeny did not show any dose-correlation.
- The histopathological investigation of these rats showed no evidence of a test substance induced effect.

Urinary tract:
- In the kidneys of 12000 ppm rats precipitates of foreign (crystalline) material - presumably the test compound or metabolite - could be found more frequently in the collecting ducts and renal pelvis than at 0 ppm. As sequels of this condition the following changes were observed in the kidneys of male and female parental rats at 2400 ppm (partly) and 12000 ppm: Diffuse tubular basophllia, intratubular granulocytes, papillary necrosis, medullary fibrosis, dilation of the renal pelvis and transitional cell hyperplasia. In 12000 ppm females in addition cortical tubules showed small vacuoles or inclusions, which were more frequent in the F1 generation.
- In 12000 ppm F1 females the ureters showed an increased incidence of hypertrophy. A slight elevation of ureteric hypertrophy was also seen at 2400 ppm.
- A slightly elevated incidence of diffuse transitional cell hyperplasia was observed in the urinary bladder of 12000 ppm F1 females.

Adrenals, liver and spleen:
- In the adrenal glands of 12000 ppm F1 females and one F1 male, an increased incidence of hypertrophy of the zona glomerulosa was seen. This was considered to be induced by the renal findings such as foreign material, basophilic tubules and renal pelvic dilation, therefore, it was regarded as treatment-related.
- In the liver an increased incidence of condensed cytoplasm, which is the morphological correlate of a decreased glycogen content, was found in F1 females at 12000 ppm more frequently than at 0 ppm.
- In the spleen a comprised congestion, increased haemopoiesis and high grade of iron pigment deposits were observed more frequently in males (partly from 2400 ppm onwards) and females (at 12000 ppm) of the F1 generation indicating an increased turnover of red blood cells.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

480 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at this dose level.

Remarks on result:

other: corresponding to 50 and 69 mg/kg bw/day for males and females, respectively.

Key result

Dose descriptor:

LOAEL

Remarks:

Systemic

Effect level:

2 400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Remarks on result:

other: corresponding to 252 and 324 mg/kg bw/day for males and females, respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at this dose level.

Remarks on result:

other: corresponding to 1292 and 1001 mg/kg bw/day for males and females, respectively.

Critical effects observed:

yes

Lowest effective dose / conc.:

2 400 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Critical effects observed:

yes

Lowest effective dose / conc.:

2 400 ppm

System:

haematopoietic

Organ:

spleen

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- Incidences of pups exhibiting the clinical findings of no milk spot, paleness thin and/or cold to touch were increased.

A summary of clinical findings and litter parameters of F1 and F2 generations are presented in attachment 8.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

12000 ppm:
- F1 pups exhibited a reduced (p>0.05) viability index (litters of dams, which died during lactation were excluded) as a result of an increase in pup deaths (including total litter loss in 2 females), which must be interpreted as a treatment-related effect though a comparable low viability index in control F2 pups was noted and no effect was seen on viability of 12000 ppm F2 pups.
- The lactation indices were slightly reduced in both generations. Though the lactation indices at 12000 ppm were within historical control range a test substance-related effect on this parameter could not be excluded, if the toxicity observed in the dams is considered.

The fact that there were higher mean litter sizes of F2 pups in group 2400 and 12000 ppm than in F1 pups of corresponding dose groups is based on the result that some F1 litters had died completely.

A summary of clinical findings and litter parameters of F1 and F2 generations are presented in attachment 8.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- Treatment-related depressed pup (males and females) and litter weights were evident (pups: partly statistically significant from Day 7 p.p. onwards); litters: about 11-16% from Day 0 p.p. onwards).

A summary of mean litter and pup weights is presented in Attachment 9.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Compound intake:
- Based on food consumption and body weight data, the doses expressed as mean daily mg test substance/kg body weight during the premating, gestation and lactation periods are presented in Attachment 3. The values for the P and F1 generation are considered to be representative for the test substance intake for the entire study.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Sexual maturation:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- Female F1 post weanlings exhibited a slight delay in time of vaginal opening (p<0.01). This finding was interpreted to be due to body weight retardation. Additionally, an estrogenic or androgen effect was excluded as ano-genital measurements in F2 pups revealed no differences between controls and treated animals up to 12000 ppm.

Summary data of sexual maturation milestones for F1 post-weanlings is presented in Attachment 10.

Anogenital distance (AGD):

not examined

Description (incidence and severity):

Not applicable.

Nipple retention in male pups:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- For some absolute or relative organ weights statistically significantly higher or lower means were obtained compared to means of the control group. In all cases, these changes were attributed to differences in body weight as follows:
- Reduced absolute thymus weights in female F1 weanlings were not correlated with statistically significantly changed relative weights.
- Elevated relative brain weights of F1 females corresponded with lower absolute brain weights.

A summary of organ weight data for F1 weanlings is presented in Attachment 11.

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Developmental immunotoxicity:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

2 400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at this dose level.

Remarks on result:

other: corresponding to 173 mg/kg bw/day.

Key result

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

body weight and weight gain

Remarks on result:

other: corresponding to 841 mg/kg bw/day.

Critical effects observed:

no

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- The incidences of pups exhibiting the clinical findings of no milk spot, paleness thin and/or cold to touch were increased by the intake of the test substance.

A summary of clinical findings and litter parameters of F1 and F2 generations are presented in attachment 8.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality / viability:

no mortality observed

Description (incidence and severity):

The lactation indices were not changed remarkably up to 2400 ppm and slightly reduced at 12000 ppm in both generations. Though the lactation indices at 12000 ppm were within historical control range a test substance-related effect on this parameter can not be excluded, if the toxicity observed in the dams is considered.

A summary of clinical findings and litter parameters of F1 and F2 generations are presented in attachment 8.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- Treatment-related depressed pup (males and females) and litter weights were evident (pups: partly statistically significant from Day 7 p.p. onwards); litters: about 11-16% from Day 0 p.p. onwards).

A summary of mean litter and pup weights is presented in Attachment 9.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Compound intake:
- Based on food consumption and body weight data, the doses expressed as mean daily mg test substance/kg body weight during the premating, gestation and lactation periods are presented in Attachment 3. The values for the F1 and F2 generation are considered to be representative for the test substance intake for the entire study.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

12000 ppm:
- For some absolute or relative organ weights statistically significantly higher or lower means were obtained compared to means of the control group. In all cases, these changes were attributed to differences in body weight as follows:
- Reduced absolute thymus weights in F2 weanlings were not correlated with statistically significantly changed relative weights.
- Reduced absolute spleen weights of male F2 weanlings were not associated with statistically significant changed relative weights.
- Elevated relative brain weights of F2 weanlings corresponded with lower absolute brain weights.

A summary of organ weight data for F2 weanlings is presented in Attachment 11.

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Developmental immunotoxicity:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOAEL

Generation:

F2

Effect level:

2 400 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at this dose level.

Remarks on result:

other: corresponding to 196 mg/kg bw/day.

Key result

Dose descriptor:

LOAEL

Generation:

F2

Effect level:

12 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

clinical signs

body weight and weight gain

Remarks on result:

other: corresponding to 1001 mg/kg bw/day.

Critical effects observed:

no

Key result

Reproductive effects observed:

yes

Lowest effective dose / conc.:

12 000 ppm

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The study was performed in accordance to OECD TG 416 under GLP conditions and is considered reliable. Under the conditions described, the parental systemic LOAEL in rats is 2400 ppm (corresponding to 202 mg/kg bw/day in males and 260 mg/kg bw/day in females) and the parental systemic NOAEL is 480 ppm (39 mg/kg bw/day in males and 55 mg/kg bw/day in females), based on histopathological changes in spleen and urinary tract accompanied with organ weight changes. The NOAEL for parental reproductive parameters in the P animals was 2400 ppm (173 mg/kg bw/day) due to a reduced rearing index at 12000 ppm. Parental animals of the F1 generation did not show any adverse effect on reproductive behaviour or function, and thus, a NOAEL of 12000 ppm was derived for the F1 parental animals. The offspring NOAEL is 2400 ppm (173 mg/kg bw/day in F1 offspring and 196 mg/kg bw/day in F2 offspring) due to pup weight decrease at 12000 ppm.

Effect on fertility: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

173 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

The toxicity to reproduction was assessed in a 2-generation study conducted with male and female rats according to OECD TG 416 (2001), under GLP conditions. The study is considered of reliable quality and validity (RL1), fulfills the criteria of a key study and thus, is suitable for assessment of the present endpoint.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A two-generation reproduction toxicity study (M-283510-01-1) was conducted in Wistar rats. The study was performed under GLP condition and in accordance with OECD TG 416 (adopted 2001).

In this study, the parental animals (25/sex/dose) received doses of 480, 2400 and 12000 ppm in their diet. During the premating period, the test substance intake was: (P) 39, 202 and 1007 mg/kg bw/day (males); 55, 260 and 1350 mg/kg bw/day (females) and (F1): 50, 252 and 1292 mg/kg bw/day (males); 69, 324 and 1697 mg/kg bw/day (females). Post coitum (Day 14 to 20), the dietary dose levels of (P) females were 37, 173 and 841 mg/kg bw/day and 42, 196, 1001 mg/kg bw/day for (F1) females. Postpartum (Day 0 to 4), dietary dose levels were 59, 246, 1371 for (P) females and 61, 273, 1449 mg/kg bw/day for (F1) females.

Parental animals received the test substance for a period of about 12 weeks and were then allowed to mate over a period of up to three weeks. F1 offspring were nursed up to an age of four weeks. Some of the F1 offspring were selected for further treatment and for breeding a F2 generation. Generally, the test substance was administered to each animal up to its necropsy.

At 12000 ppm, mortality was increased in P and F1 females due to clinical findings of poor general condition and severe renal alterations. Additionally at this dose, an increase in water intake and urination was found in F1 rats. At 12000 ppm, body weight gain was decreased in F1 males (slightly) and females during the premating phase and in P and F1 females during gestation and lactation. At necropsy, skinny female rats were found more often at 12000 ppm than in the controls. Additionally at this dose, kidneys of F1 females were found to be discoloured, enlarged and/or changed at their surface.

The ovaries of P and/or F1 females receiving 12000 ppm showed increased incidence of foamy corpus luteum cells, the uterus exhibited more unregressed implantation sites and the vagina showed a mucified epithelium more often. The number of primary follicles and corpora lutea was unchanged.

In the kidneys of 12000 ppm rats, precipitates of foreign material were found more frequently in the collecting ducts and renal pelvis. As sequels of these precipitations, transitional cell hyperplasia, tubular basophilia, intratubular granulocytes, papillary necrosis, medullary fibrosis, renal pelvic dilation and/or vaculation/inclusions in the proximal tubules were observed in the kidneys of 2400 ppm and 12000 ppm parental rats. In females at 12000 ppm, these findings correlated with increased relative kidney weights. From 2400 ppm onwards, hypertrophy was recorded in the ureters and/or hyperplasia of the transitional epithelium of the urinary bladder was present more frequently in females.

In the adrenal cortex, the incidence of hypertrophy of the zona glomerulosa was increased in females at 12000 ppm, this was considered secondary to the marked renal lesions at this dose level. In the liver, hepatocellular glycogen content was reduced in 12000 ppm P and F1 females. In the spleen, increased ferrous pigment deposits, congestion and increased haemopoiesis were noted at 2400 ppm and above in males and/or females (P and F1). From 2400 ppm onwards, absolute and relative spleen weights increased in P and F1 rats. From 2400 ppm onwards, thyroid weights were increased in F1 females, however, without any morphological correlate.

Sperm analysis and examination of oestrus cycling revealed no substance-related effects up to 12000 ppm. The parameters of the reproductive performance such as insemination, fertility and gestation indices as well as gestation length and the number of litters born were not influenced by the test substance up to 12000 ppm. At 12000 ppm, P females exhibited a reduced rearing index (78.6% compared to 95.8% in high-dose females and controls, respectively). As the effect on rearing might be correlated to systemic toxicity indicated by reduced body weight gain during gestation/lactation and maternal death of 20% during lactation (5/25 P females) in the P generation, the reduced rearing index is considered as secondary non-specific consequence of systemic toxicity in maternal animals. This is further supported by the absence of any effect on reproductive function and/or performance in the following F1 parental generation.  

The litter data, such as mean number of implantations, prenatal loss, live birth indices, sex distribution, number of pups born and litter sizes were not affected by the test substance at levels of up to 12000 ppm. At 12000 ppm, a reduced viability index (F1 pups) including two total litter losses were noted and a reduced lactation (F1 and F2 pups) index was evident. At this dose level, more pups showing clinical findings (paleness, cold to touch, missing milk spot, thin) were evident.

The pup/litter weights were depressed at 12000 ppm. Due to the depressed body weight at this dose level, vaginal opening in F1 females was delayed. However, anogenital distance measurements in F2 pups did not indicate any treatment-related effect.

The effects seen in the F1 and F2 pups at 12000 ppm were considered to be secondary effects due to the maternal toxicity seen in the P and F1 animals, notably, increased mortality, decreased body weight gain and severe renal alterations.

At pup or weanling necropsies, no remarkable macroscopic findings were noted. Changes of brain, thymus and/or spleen weights were noted in pups, but were interpreted to be secondary to depressed body weights.

Under the conditions of the study, the parental systemic LOAEL in rats was 2400 ppm (corresponding to 202 mg/kg bw/day in males and 260 mg/kg bw/day in females) based on organ changes in the spleen and urinary tract. The parental systemic NOAEL was 480 ppm (39 mg/kg bw/day in males, 55 mg/kg bw/day in females). For a discussion on the relevance to humans for the findings in the urinary tract, please refer to the endpoint summary for carcinogenicity.

The NOAEL for parental reproductive parameters in the P animals was 2400 ppm (173 mg/kg bw/day) due to a reduced rearing index at 12000 ppm. Parental animals of the F1 generation did not show any adverse effect on reproductive behaviour or function, and thus, a NOAEL of 12000 ppm was derived for the F1 parental animals. The offspring NOAEL is 2400 ppm (173 mg/kg bw/day in F1 offspring and 196 mg/kg bw/day in F2 offspring) due to pup weight decrease at 12000 ppm.

Effects on developmental toxicity

Description of key information

Prenatal developmental toxicity (OECD 414), oral, rat: 

NOEL maternal toxicity = 250 mg/kg bw/day

NOEL developmental toxicity = 1000 mg/kg bw/day

 

Prenatal developmental toxicity (OECD 414), oral, rabbit: 

NOAEL maternal toxicity = 125 mg/kg bw/day

NOEL developmental toxicity = 125 mg/kg bw/day

 

Prenatal developmental toxicity (OECD 414), oral, rabbit:

LOAEL maternal toxicity = 250 mg/kg bw/day

NOAEL developmental toxicity = 250 mg/kg bw/day

 

Two-generation reproductive toxicity (OECD 416), oral, rat:

NOAEL (systemic) parental = 39/55 mg/kg bw/day (male/female)

NOAEL (developmental) offspring = 173 mg/kg bw/day (male/female)

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 Jan 2004 - 10 Oct 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 Jan 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 27 Jun 2018

Deviations:

yes

Remarks:

See Principles of method if other than guideline.

Principles of method if other than guideline:

Deviations to OECD guidleine 414 (2018): Diet not analysed (phytoestrogens); no investigations on the thyroid (weight, histopathology, no T4, T3/TSH levels measured); anogenital distance of pups not measured; reproductive tract of fetuses or cryptorchidism not examined; no comparison between external vs. internal (gonadal) sex morphology.

GLP compliance:

yes (incl. QA statement)

Remarks:

INTERMINISTERIAL GROUP FOR CHEMICAL PRODUCTS, Paris, France

Limit test:

no

Species:

rat

Strain:

other: Sprague-Dawley Crl:CD(SD)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River laboratories, St Germain-sur-l'Arbresle, France.
- Age at study initiation: Adult virgin, not further specified.
- Weight at study initiation: 243-318 g (females selected for study).
- Fasting period before study: No data.
- Housing: Pregnant females were housed individually in suspended, stainless steel, wire mesh cages.
- Diet: Certified rodent pelleted and irradiated diet A04C-10 from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France), ad libitum. Feed was stored in an identified room, controlled for temperature and humidity. Diet was used only until the date of expiry.
- Water: Water from the municipal supply was provided with an automatic watering system, ad libitum.
- Acclimation period: At least 12 days prior to mating.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: aqueous solution of methylcellulose 400 (Fluka, Mulhouse, France) at 0.5%

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test substance was suspended (w/v) in an aqueous solution of methylcellulose 400 (Fluka, Mulhouse, France) at 0.5% and stored at approximately 5 °C (± 3 °C). Formulations were prepared twice (F1 and F2) during the study. The suspensions were mixed continuously before and during treatment with an electromagnetic stirrer.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% aqueous methylcellulose 400; methylcellulose is commonly used as thickener or emulsifier in various food and cosmetic applications and toxicological studies to generate appropriate consistency.
- Concentration in vehicle: 6, 25 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Quantification was performed by High Performance Liquid Chromatography using UV detection. Homogeneity of the suspensions was checked on the first formulation (F1) for the lowest and the highest concentrations (6 and 100 g/L). In addition, the intermediate concentration of the first formulation (F1) and all concentrations of the second formulation (F2) were checked. Stability of the test substance in suspension in the vehicle at concentrations of 0.1 and 250 g/L was determined in a previous study (SA03017), and was found to be stable for 28 days under similar conditions to those of the current study.

Homogeneity and concentration checks of dosing suspensions of the test substance were between 94 and 99% of nominal values, which was within the in-house target range of 90 to 110% of the nominal concentration.

Details on mating procedure:

- Impregnation procedure: Cohoused
- M/F ratio per cage: 1/1
- Length of cohabitation: Overnight
- Further matings after two unsuccessful attempts: Not referred to.
- Verification of same strain and source of both sexes: Yes, stock males of same strain from same supplier.
- Proof of pregnancy: Vaginal plug or sperm in vaginal smear referred to as Day 0 of pregnancy.

Duration of treatment / exposure:

Gestation Day (GD) 6 to 20

Frequency of treatment:

Once daily, 7 days/week

Duration of test:

Until GD 21

Dose / conc.:

60 mg/kg bw/day

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

1 000 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The range of doses was selected in agreement based on results obtained in a range-finding study (SA 03017), where pregnant rats received up to 1000 mg/kg bw/day of the test substance. The high dose of 1000 mg/kg bw/day was a limit dose which was expected to cause possible slight maternal toxicity. The mid dose of 250 mg/kg bw/day provided an approximate four-fold factor between the dose levels. The low dose of 60 mg/kg bw/day was expected to be a clear No Observed Effect Level (NOEL) in both dams and fetuses.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Clinical signs daily from GD 0 through GD 21; mortality twice daily (in the morning and in the afternoon, except at weekends and public holidays when check was carried out once daily).
- Cage side observations included: All clinical signs, mortality (dead or moribund animals)

BODY WEIGHT: Yes
- Time schedule for examinations: On GD 0, 6, 8, 10, 12, 14, 16, and 18

FOOD CONSUMPTION: Yes
Full feeder weights were measured on GD: 1, 6, 8, 10, 12, 14, 16 and 18; Empty feeder weights were measured on GD: 6, 8, 10, 12, 14, 16, 18 and 21.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on GD 21, by inhalation of carbon dioxide
- Organs examined:
Examination of uterine content of all females was performed. Each female was first subjected to macroscopic examination of the visceral organs including examination of the kidney and urinary bladder for the presence of gritty material. Kidneys and urinary bladder were taken at necropsy from all dams, tissues and carcasses were then discarded. Histopathological examinations were performed on the kidney at all dose levels and on the urinary bladder at 1000 mg/kg/day and in the control group only. The reproductive tract was weighed (gravid uterine weight), dissected out and the caesarean parameters were recorded without knowledge of treatment group.

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
- Other: Sex of live fetuses, individual weight of live fetuses; if possible, sex and weight of dead fetuses were recorded and included in the study file, as well.
Uterine horn(s) without visible implantations were immersed in a 10% solution of ammonium sulfide according to the SALEWSKI method (1964) in order to visualize any sites which were not apparent.
Intra-uterine death was classified according to GLEICH and FROHBERG (1977) as:
- Early resorptions: macroscopic discrimination between fetal residues and placental material not possible.
- Late resorptions: distinct macroscopic discrimination between fetal and placental remains possible.

Dead fetuses: Defined as dead conceptuses showing distinct digits on fore- and hind-paws.
Runt fetuses: Defined as live fetuses weighing less than 4 g at cesarean section of the dam.

Fetal examinations:

- External examinations: Yes: All the live fetuses were killed by subcutaneous injection (0.02 mL/fetus) of Dolethal (18.22 g/100 mL, sodium pentobarbital) and subjected to an external examination.
- Soft tissue examinations: Yes: Approximately half of the live fetuses from each litter were stained in Bouin's fluid for subsequent internal examination following free-hand sectioning.
- Skeletal examinations: Yes: The remaining half of the live fetuses from each litter were skinned, eviscerated and stained with alizarin red S and alcian blue for skeletal examination of bone and cartilage.
Structural deviations were classified as follows:
Malformations: Very rare or obviously lethal changes.
Minor anomalies: Slight, relatively rare structural changes not obviously detrimental.
Common variants: Structural changes occurring in more than approximately 5% of the control population.
- Head examinations: No

Statistics:

Mean and standard deviation for all maternal, litter and fetal parameters were calculated for each group. Statistical analyses were performed separately for all pregnant females and for all pregnant females with live fetuses, for body weight change and food consumption parameters. As the data were identical, only tables and appendices for all pregnant females are presented.

For details on statistics performed please refer to "Any other information on materials and methods incl. tables".

Indices:

Maternal body weight (BW) changes for interval periods; Corrected body weight change (CBWC); Average food consumption (FC) during intervals in g/day; Pre-implantation loss per litter; Post-implantation loss per litter; Number of live fetuses per litter; Number of dead fetuses per litter; Percentage of dead fetuses per litter; Percentage of male fetuses per litter; Mean fetal body weight per litter; Mean fetal body weight per litter and per sex; Mean fetal body weight per group; Mean fetal body weight per sex per group; Percentage of fetuses affected per litter; Mean percentage of fetuses affected per group; Percentage of litters affected per group.

Historical control data:

Historical control data from studies conducted in-house were provided and referred to in order to allow comparison with concurrent controls.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- Two females at 1000 mg/kg bw/day and one female at 250 mg/kg bw/day regurgitated the dose suspension on one occasion. As these incidences occurred in isolation and in so few animals, this finding was considered to be incidental.
- One female at 1000 mg/kg bw/day had reduced motor activity, vaginal discharge and red traces on the cage tray on GD 21. These clinical signs were considered to be due to the commencement of parturition and not related to treatment.
- Another female at 1000 mg/kg bw/day had vaginal discharge on GD 14, however, this finding is common in this strain of rat at this stage of gestation and was therefore considered to be a normal occurrence.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Body weight change was reduced by 32% between GD 6 to 8 at 1000 mg/kg bw/day, compared to the controls, though the effect was not statistically significant. Thereafter, body weight change was comparable to the controls at this dosage.
- Maternal corrected body weight change was reduced by 11% at 1000 mg/kg bw/day, compared to the controls.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, one female had yellow sediment in both kidneys and prominent lobulation of the liver, one female was found to have an enlarged liver with white foci on the left lobe and one female had a white area on the left lobe.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, one female was found to have a moderate bilateral papillary necrosis in the kidney, associated with mixed cell infiltrate. This change correlated with the macroscopic finding of yellow bilateral sediment in the kidney of this female at autopsy.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Not applicable.

Changes in number of pregnant:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOEL

Remarks:

systemic

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No effects at this dose level.

Key result

Dose descriptor:

LOEL

Remarks:

systemic

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

gross pathology

histopathology: non-neoplastic

Key result

Dose descriptor:

NOEL

Remarks:

developmental

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No effects at this dose level.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Changes in postnatal survival:

no effects observed

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were three fetuses observed with malformations, two occurred at 250 mg/kg bw/day and one at 60 mg/kg bw/day. At 250 mg/kg bw/day, one fetus had an umbilical hernia and one fetus had anasarca, whilst at 60 mg/kg/day one fetus had a cleft palate. In isolation and in the absence of a dose response, these malformations were considered to be incidental.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Two fetuses with malformations were noted, one occurred at 1000 mg/kg bw/day and comprised of hemivertebra in the atlas vertebra and malpositioned axis vertebra. The second malformation occurred at 60 mg/kg bw/day, where one fetus had a small and split palatine (the same fetus which had a cleft palate at the external observation). In isolation, these malformations are considered to be incidental.

The following two variants were found at a higher incidence at 1000 mg/kg bw/day; incomplete parietal ossification and incomplete interparietal ossification. In addition, the incidence of 13th costal cartilage absent (unilateral/bilateral), classified as a variant, was marginally higher at 1000 mg/kg bw/day, compared with the controls. However, as in all three cases the incidence was found not to be statistically significantly different from the control group at both the fetal and litter level, these findings were considered to be fortuitous.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

At 1000 mg/kg bw/day, one fetus had the malformation retinal fold associated with a small lens, whilst a second fetus had the malformation right-sided aortic arch and pulmonary trunk. As these two malformations occurred in isolation, they were considered to be incidental. In addition, the incidence of the malformation absent renal papilla (unilateral) was marginally higher in terms of both mean percentage of fetuses affected and percentage of litters affected, compared with the control group. However, as the incidence was not statistically significant for either parameter, when compared to the control group, and the incidence in terms of litters (the litter being regarded as the experimental unit), was within the in-house historical control range, this finding was considered to be fortuitous.

Summary data for the incidence of absent renal papilla (unilateral), including historical control range are presented in Attachment 1.

There were no other treatment-related anomalies or variants noted in any treatment group as the findings did not occur in a dose-related manner, were within the in-house historical control range or occurred in isolation.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOEL

Effect level:

1 000 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects seen at this dose level.

Key result

Abnormalities:

effects observed, non-treatment-related

Key result

Developmental effects observed:

no

Conclusions:

The study was performed in accordance to OECD TG 414 under GLP conditions and is considered reliable. Body weight change was reduced in the dams treated at 1000 mg/kg bw/day between GD 6 to 8. At microscopic examination, one female was found to have a moderate bilateral papillary necrosis in the kidney, associated with mixed cell infiltrate. The change correlated with the macroscopic finding of yellow bilateral sediment in the kidney of this female. No litter parameters were affected. At external, visceral and skeletal fetal examination, no treatment-related findings were observed. The NOEL for maternal toxicity was 250 mg/kg bw/day, while 1000 mg/kg bw/day was a NOEL in terms of fetal development.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Mar 2004 - 07 Jan 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The highest dose level chosen induced maternal toxicity and death

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 Jan 2001

Deviations:

yes

Remarks:

The highest dose level exceeded the maximum tolerated dose. Animals were housed with a16-hour light period.

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 25 Jun 2018

Deviations:

yes

Remarks:

See Principles of method if other than guideline

Principles of method if other than guideline:

Deviations to OECD guidleine 414 (adopted 2018): Diet not analysed (phytoestrogens); missing investigations (thyroid weight and histopathology; reproductive tract of fetuses or cryptorchidism; comparison between external vs. internal (gonadal) sex morphology); animals were housed with a 16-hour light period. The highest dose level exceeded the maximum tolerated dose.

GLP compliance:

yes (incl. QA statement)

Remarks:

INTERMINISTERIAL GROUP FOR CHEMICAL PRODUCTS, Paris, France

Limit test:

no

Species:

rabbit

Strain:

other: New Zealand White Crl:KBL(NZW)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Chatillon-sur-Chalaronne, France.
- Age at study initiation: approx. 18 weeks.
- Weight at study initiation: 3.46-3.52 kg at gestation day 3 (time-mated females were used)
- Fasting period before study: No data
- Housing: Individual housing of pregnant females in polycarbonate cages on a perforated cage floor.
- Diet: A known weighed amount of 110C-10 pelleted animal diet from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France) was distributed every one or two days.
- Water: Water from the municipal supply was provided with an automatic watering system, ad libitum. Filters servicing the watering system were changed regularly and sterilization of the system was periodically performed.
- Acclimation period: Approx. 4 days (arrival on GD 1 or 2, GD 6 = first day of dosing)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21
- Humidity (%): 40-70
- Air changes (per hr): Approx. 15
- Photoperiod (hrs dark / hrs light): 8/16

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous methylcellulose 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test substance was periodically (six times formulations at 12.5 and 31.25 g/L and four times formulations at 125 g/L) suspended (w/v) in an aqueous solution of methylcellulose 400 (Fluka, Mulhouse, France) at 0.5% and stored at approximately 5 °C (± 3 °C).

VEHICLE
- Justification for use and choice of vehicle: 0.5% aqueous methylcellulose; methylcellulose is commonly used as thickener or emulsifier in various food and cosmetic applications and toxicological studies to generate appropriate consistency.
- Concentration in vehicle: 0, 12.5, 31.25, 125 mg/mL
- Amount of vehicle: 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Quantification was performed by High Performance Liquid Chromatography using UV detection. Stability of the test substance in 0.5% aqueous methylcellulose was demonstrated in study SA 03017 at concentrations of 0.1 and 250 g/L for up to 28 days, under similar conditions to those of the current study.
Homogeneity (surface, middle and bottom) of the suspensions was checked on the first formulation (F1) for the lowest and the highest concentrations (12.5 and 125 g/L). The mean values obtained from the homogeneity check were used as measured concentrations. In addition, the intermediate concentration of the first formulation (F1) and all concentrations of the remaining formulations were checked.

Homogeneity and concentration results ranged from 94 to 108% and were, therefore, within the in-house target range (90 to 110% of nominal concentration).

Details on mating procedure:

- Impregnation procedure: Purchased timed pregnant
- Verification of same strain and source of both sexes: Yes, males of same strain from same supplier.
- Proof of pregnancy: Day of insemination referred to as Day 0 of pregnancy.

Duration of treatment / exposure:

Gestation day (GD) 6 to 28

Frequency of treatment:

Once daily, 7 days/week

Duration of test:

Until GD 29

Dose / conc.:

50 mg/kg bw/day

Dose / conc.:

125 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The range of dosages was selected based on the results obtained in a previous range finding study in pregnant rabbits (SA 03081), where animals received 0, 125, 250, 500, 750 or 1000 mg/kg bw/day of the test substance from GD 6 to 28. The two highest dose levels caused maternal deaths and clearly exceeded a Maximum Tolerated Dose (MTD) for the dams.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily from GD2 through GD29 for clinical signs; cages checked twice daily for dead or moribund animals or signs of abortion (once daily at weekends and public holidays)
- Cage side observations included: Clinical signs, mortality, signs of abortion
Any animal showing signs of abortion or morbidity, particularly if death appeared imminent, was killed by intravenous injection of Dolethal to prevent loss of tissues through autolysis. All females which aborted, were killed for humane reasons or found dead in the cage were necropsied.
At 500 mg/kg bw/day, following the deaths of 7/25 females at this dosage, the decision was taken to terminate the group without further evaluation.

BODY WEIGHT: Yes
- Time schedule for examinations: On GD 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 29

FOOD CONSUMPTION: Yes
Full feeder weights were measured on GD: 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28
Empty feeder weights were measured on GD: 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 29
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on GD 29.
- Organs examined:
Animals killed in extremis by intravenous injection of Dolethal (Sanofi, Libourne, France) (including females which aborted) or found dead, were autopsied. The number and type of implantations and corpora lutea were noted when present. In the case of no visible uterine implants, but with corpora lutea, uterine horn(s) were immersed in a 20% solution of ammonium sulfide according to the Salewski method (1964). The urinary bladder and kidney were taken and preserved in 10% neutral buffered formalin. Both organs were subsequently examined microscopically at 125 mg/kg/day and from the control females. Tissues and carcasses were then discarded.
On GD 29, surviving females were killed by intravenous injection of Dolethal for examination of their uterine content. Each female was first subjected to macroscopic examination of the visceral organs and the number of ribs was recorded. In the case of no visible uterine implants, but with corpora lutea, uterine horn(s) were immersed in a 20% solution of ammonium sulfide according to the Salewski method. The urinary bladder and kidney were taken and preserved in 10% neutral buffered formalin. Both organs were subsequently examined microscopically at 125 mg/kg/day and from the control females. Tissues and carcasses were then discarded. The reproductive tract was weighed (gravid uterine weight), dissected out and the caesarean parameters were recorded without knowledge of treatment group.

Ovaries and uterine content:

The ovaries and uterine content were 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
- Other: Number of live and dead fetuses, individual weight of fetuses

Intra-uterine resorption death was classified according to GLEICH and FROHBERG (1977) as:
- Early resorptions: macroscopic discrimination between fetal residues and placental material not possible.
- Late resorptions: distinct macroscopic discrimination between fetal and placental remains possible.

Dead fetuses: Defined as dead conceptuses showing distinct digits on fore and hind-paws.
Runt fetuses: Defined as live fetuses weighing less than 28 g at cesarean section of the dam.

Fetal examinations:

- External examinations: Yes: All live fetuses were killed by subcutaneous injection of Dolethal and subjected to an external examination.
- Soft tissue examinations: Yes: The bodies of all fetuses were dissected for soft tissue anormalities and sexed.
- Skeletal examinations: Yes: After soft tissue examination all fetuses were fixed in absolute ethanol before staining (a modification of the Staples and Schnell staining technique was performed).
- Head examinations: Yes: After internal examination of the neck, the head of fetuses from approximately half of each litter was immersed in Bouin's fluid and the internal structures examined after fixation.
Structural deviations were classified as follows:
Malformations: Very rare or obviously lethal changes.
Minor anomalies: Slight, relatively rare structural changes not obviously detrimental.
Common variants: Structural changes occurring in more than approximately 5% of the control population.

Statistics:

Mean and standard deviation for all maternal, litter and fetal parameters were calculated for each group. Only for body weight descriptive statistics were done as the only exception.

For details on statistics performed please refer to "Any other information on materials and methods incl. tables".

Indices:

Maternal body weight (BW) changes for interval periods; Corrected body weight change (CBWC); Average food consumption (FC) during intervals; Pre-implantation loss per litter; Post-implantation loss per litter; Number of live fetuses; Number of dead fetuses; Percentage of dead fetuses per litter; Percentage of male fetuses per litter; Mean fetal body weight per litter; Mean fetal body weight per litter and per sex; Mean fetal body weight per group; Mean fetal body weight per sex per group; Percentage of fetuses affected per litter; Mean percentage of fetuses affected per group; Percentage of litters affected per group.

Historical control data:

Historical control data from studies conducted in-house were provided and referred to in order to allow comparison with concurrent controls.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg bw/day, the principal clinical signs observed in decedent animals were yellow sediment in the urine in all animals, associated with red traces on the cage tray in five cases. All animals had few/no feces on one or more occasion, and one female had reduced motor activity, tremors and was warm to touch on the day of death. As this dose level had clearly exceeded a Maximum Tolerated Dose (MTD), treatment at 500 mg/kg bw/day was terminated. Surviving females were killed prior to scheduled autopsy with no macroscopic examination performed and no tissues taken.

Of the surviving females at 500 mg/kg bw/day, treatment-related clinical signs consisted of yellow sediment in the urine of 3/18 dams and few faeces for 4/18 dams, prior to termination of this dose group.

At 125 mg/kg bw/day, treatment-related clinical signs consisted of yellow sediment in the urine of 2/25 dams.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 500 mg/kg bw/day, 7/25 dams died between GD 9 and 20. Four dams were killed for humane reasons and three were found dead.

At 125 mg/kg bw/day, one female was found dead on GD 19, shortly after dosing, with no prior adverse findings. At autopsy, red contents were found in the trachea, all lobes of the lung were discolored and a marbled pattern was noted on the kidney. This animal was considered to have died due to gavage error.

At 50 mg/kg bw/day, one female was found dead on GD 15; this animal had reduced activity and slow respiration on GD 15. At autopsy, macroscopic findings consisted of red contents in the trachea, and hemorrhaging and discoloration in the lungs. The cause of death was judged to have been gavage error.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg bw/day, all decedent animals showed a marked body weight loss, associated with very poor food consumption prior to death.

In surviving females at 500 mg/kg bw/day, 2/18 showed a significant body weight loss of between 0.21 and 0.29 kg prior to termination of this dose level.

No effect on mean body weight change or mean corrected body weight change (maternal body weight change independent of the uterine weight) was noted at 125 or 50 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg bw/day, all decedent animals showed a marked body weight loss, associated with very poor food consumption prior to death.

Poor food consumption, which was associated with significant body weight loss was observed in 2/18 surviving females at 500 mg/kg bw/day, prior to termination of the group.

Food consumption was similar to the control group at 125 and 50 mg/kg bw/day.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At 500 mg/kg bw/day, the following macroscopic findings were noted in decedent animals:
Kidney: Yellow sediments (4/7); Marbled pattern (2/7)
Urinary bladder: Yellow sediments (2/7); Red contents (1/7)
Liver: Prominent lobulation (5/7)
Uterus: Red contents (2/7)

At autopsy, yellow sediment in the kidney was noted in 1/25 females at 125 mg/kg bw/day, whilst the decedent female at 125 mg/kg bw/day and 1/25 females at 50 mg/kg bw/day had a kidney with a marbled pattern appearance. In the absence of microscopic findings in this target organ at 125 mg/kg bw/day, these findings were considered to be treatment-related but not toxicologically relevant.

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

At the microscopic examination of the urinary bladder and kidney at 125 mg/kg bw/day, no treatment-related findings were noted.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Not applicable.

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The pregnancy rate was 100% at 125 mg/kg bw/day, 96% at 50 mg/kg bw/day and 92% in the control group. The pregnancy rate was not established at 500 mg/kg bw/day as the majority of females were culled with no macroscopic examination.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

125 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No adverse effects at this dose level.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

500 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

gross pathology

mortality

Key result

Dose descriptor:

NOEL

Remarks:

developmental

Effect level:

125 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: No effects at this dose level.

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

At 50 mg/kg bw/day, mean fetal weights for the combined sexes and for females only were slightly reduced compared to the controls. However, in the absence of an effect at the higher dose level of 125 mg/kg bw/day, this finding was considered to be incidental.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Description (incidence and severity):

Not applicable.

Changes in postnatal survival:

not examined

Description (incidence and severity):

Not applicable.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were three fetuses with external malformations, two fetuses had umbilical hernias; one at 125 mg/kg bw/day and one at 50 mg/kg bw/day, and one fetus at 50 mg/kg bw/day had forepaw brachydactyly. In isolation and in the absence of a dose-related response, these findings were considered to be incidental.

The few anomalies observed occurred in a non dose-related manner, in isolation, or in the case of the finding short tail, where the incidence at 125 mg/kg bw/day was slightly higher than in the control group, both in terms of mean percentage of fetuses affected (1.1%) and percentage of litters affected (4.2%), the incidence was within the in-house historical control range (0.0-3.1% and 0.0-4.3%, respectively) and was therefore considered to have occurred by chance.

No variant findings were noted.

The incidence of runt fetuses was similar at 125 and 50 mg/kg bw/day, and only slightly higher than the control group. Since these incidences were within the in-house historical control range, did not occur in a dose-related manner and there was no obvious effect on mean fetal weights, this was considered to be fortuitous.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

Malformations observed at the skeletal examination consisted of one fetus at 125 mg/kg bw/day and one control fetus with hemivertebrae in at least one thoracic vertebrae. As the incidence was similar in the control and treated group this finding was considered to be fortuitous. In addition, two fetuses from separate litters at 125 mg/kg bw/day had hemivertebrae in at least one lumbar vertebrae. The mean percentage of fetuses affected (0.9%) and percentage of litters affected (8.3%) was slightly outside the historical control range of 0.0-0.5% and 0.0-4.2%, respectively. However, in the complementary study (M-273153-01-1, 2005) where dams were dosed at 250 mg/kg bw/day, there were no fetuses with hemivertebrae in either the thoracic or lumbar region. Therefore, this finding was considered to be incidental. There were two incidences at 50 mg/kg bw/day and one incidence in the control group of the finding: at least one thoracic centrum and thoracic hemicentrum fused. At such a low frequency and in the absence of this finding at 125 mg/kg bw/day, this observation was considered to be a chance finding. At 50 mg/kg bw/day, one fetus had a malformation which included unossified radius, bent ulna and absence of the 1st metacarpal and 1st proximal phalanx. In isolation, this observation was considered to be a chance finding.

The following anomaly was observed: at least two caudal vertebrae fused, occurred at a higher frequency at 125 mg/kg bw/day than in the controls in terms of both fetal and litter parameters. This condition was associated with the absence of some caudal vertebrae in 3/4 cases. It should be noted however, that all four fetuses were from the same litter and the incidence was only slightly outside the in-house historical control range in terms of mean percentage of fetuses affected with a figure of 1.5% compared to a range of 0.0-1.0% for the historical data, but was within the historical control range for percentage of litters affected, where the figure was 4.2% compared to the historical control range of 0.0-4.2%. In addition, in the complementary study (M-273153-01-1, 2005) where pregnant rabbits were dosed at 250 mg/kg bw/day, there were no fetuses with this finding. This finding was therefore considered to be fortuitous.

The following variant findings occurred at a higher frequency in the treated groups or at 125 mg/kg bw/day alone, compared to the controls, in terms of both fetal and litter parameters: 13th thoracic rib(s) (unilateral/bilateral) short, 13th thoracic rib(s) (unilateral/bilateral) (including short) detached, hyoid centrum incomplete ossification or unossified, hindpaws 5th middle or 5th distal phalanges unossified, less than 15 caudal vertebrae. However, as all findings were within the historical control range for both fetal and litter parameters at the higher dose level of 125 mg/kg bw/day, these findings were considered to be fortuitous.

A summary of skeletal findings is presented in Attachment 1.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The only malformation observed at the visceral examination was ventricular septum defect in the cranial region in one fetus at 50 mg/kg bw/day. In isolation and at the low dose only, this finding was considered to be incidental.

The incidence of the anomaly retroesophageal subclavian artery, was slightly higher at 125 mg/kg bw/day compared to the controls, in terms of mean percentage of fetuses affected (1.9%) and percentage of litters affected (12.5%), however, the incidence was within the in-house historical control range of 0.0-4.4% and 0.0-12.5% for the corresponding figures, and was therefore considered to be a chance finding.

The only variant finding which was slightly higher in the treated groups compared with the controls for both mean percentage of fetuses affected (3.3% at 125 mg/kg bw/day) and percentage of litters affected (12.5% at 125 mg/kg bw/day) was short innominate artery, but as the increase was not dose-related and was within the in-house historical control range of 1.0-3.9% for the fetal parameter and 9.1-13.0% for the litter parameter, at 125 mg/kg bw/day, this finding was considered to be incidental.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOEL

Effect level:

125 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No effects at this dose level.

Remarks on result:

other: The high dose level of 500 mg/kg bw/day could not be evaluated for fetal toxicity due to severe maternal toxicity.

Key result

Abnormalities:

effects observed, non-treatment-related

Key result

Developmental effects observed:

no

Conclusions:

The study was performed in accordance to OECD TG 414 under GLP conditions and is considered reliable. The high dose level of 500 mg/kg bw/day was terminated prior to commencement of scheduled autopsy, due to the high mortality rate observed at this dosage. At 125 mg/kg bw/day, treatment-related clinical signs consisted of yellow sediment in urine in 2/25 dams. Yellow sediment in the kidney was also noted at autopsy in 1/25 females. No treatment-related changes were detected at the microscopic examination of the kidney or the urinary bladder. No adverse effects were observed at 50 mg/kg bw/day. No treatment-related effects on litter parameters were observed up to the dose level of 125 mg/kg bw/day. There were no external, visceral or skeletal findings at the fetal evaluation which were considered to be treatment-related up to the dose level of 125 mg/kg bw/day.

In conclusion, a dose level of 500 mg/kg bw/day clearly exceeded a Maximum Tolerated Dose (MTD) in the pregnant rabbit. A dose level of 125 mg/kg bw/day was a No Observed Adverse Effect Level (NOAEL) for general toxicity in the dams due to the findings observed in the kidney, whilst being a No Observed Effect Level (NOEL) for developmental toxicity, in the New Zealand White Rabbit.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 Nov 2004 - 08 Jun 2005

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Only one dose level was chosen, but the study was complementary performed to study no. SA03349 (M-261627-01-1).

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 22 Jan 2001

Deviations:

yes

Remarks:

Animals were housed with a 16-hour light period. Only one dose level was tested, but the study was complementary performed to study no. SA03349 (M-261627-01-1).

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

adopted 2018

Deviations:

yes

Remarks:

See Principles of method if other than guideline

Principles of method if other than guideline:

Deviations to OECD guidleine 414 (2018): Diet not analysed (phytoestrogens); no investigations on thyroid weight and histopathology; reproductive tract of fetuses or cryptorchidism not examined; no comparison between external vs. internal (gonadal) sex morphology; animals were housed with a 16-hour light period. Only one dose level was tested, but the study was complementary performed to study no. SA03349 (M-261627-01-1).

GLP compliance:

yes (incl. QA statement)

Remarks:

INTERMINISTERIAL GROUP FOR CHEMICAL PRODUCTS, Paris, France

Limit test:

no

Species:

rabbit

Strain:

other: New Zealand White Crl:KBL(NZW)

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Chatillon-sur-Chalaronne, France.
- Age at study initiation: Approx. 18 weeks
- Weight at study initiation: 3.47-3.48 kg at gestation day 3 (time-mated females were used)
- Fasting period before study: No data
- Housing: Individual housing of pregnant females in polycarbonate cages on a perforated cage floor.
- Diet: A known weighed amount of 110C-10 pelleted animal diet from S.A.F.E. (Scientific Animal Food and Engineering, Augy, France) was distributed every one or two days.
- Water: Water from the municipal supply was provided with an automatic watering system, ad libitum. Filters servicing the watering system were changed regularly and sterilization of the system was periodically performed.
- Acclimation period: Approx. 4 days (arrival on GD 1 or 2, GD 6 = first day of dosing)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21
- Humidity (%): 40-70
- Air changes (per hr): Approx. 15
- Photoperiod (hrs dark / hrs light): 8/16

Route of administration:

oral: gavage

Vehicle:

other: 0.5% aqueous methylcellulose 400

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The appropriate amount of test substance was periodically (six times formulations at 62.5 g/L) suspended (w/v) in an aqueous solution of 0.5% methylcellulose 400 (Fluka, Mulhouse, France) and stored at approximately 5 °C (± 3 °C).

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% aqueous methylcellulose; methylcellulose is commonly used as thickener or emulsifier in various food and cosmetic applications and toxicological studies to generate appropriate consistency.
- Concentration in vehicle: 62.5 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Quantification was performed by High Performance Liquid Chromatography using UV detection.
Stability of the test substance in 0.5% aqueous methylcellulose was demonstrated in study SA 03017 at concentrations of 0.1 and 250 g/L for up to 28 days, under similar conditions to those of the current study.
Homogeneity (surface, middle and bottom) of the suspensions was checked on the first formulation (F1) for the lowest and the highest concentrations (62.5 g/L).
The mean values obtained from the homogeneity check were used as measured concentrations. For all other formulations at 62.5 g/L, three samples were taken from the surface (Sa, Sb and Sc). Sample Sa was analysed, Sb and Sc being stored frozen for possible verification purposes.

Homogeneity and concentration results ranged from 92 to 107% and were, therefore, within the in-house target range (90 to 110% of nominal concentration).

Details on mating procedure:

- Impregnation procedure: Purchased timed pregnant
- Verification of same strain and source of both sexes: Yes, males of same strain from same supplier.
- Proof of pregnancy: Day of insemination referred to as Day 0 of pregnancy.

Duration of treatment / exposure:

GD 6 to 28

Frequency of treatment:

Once daily, 7 days/week

Duration of test:

Until GD 29

Dose / conc.:

250 mg/kg bw/day

No. of animals per sex per dose:

25

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dosage was selected in agreement based on the results obtained in a previous developmental toxicity study in the pregnant rabbit (SA03349) where animals received 50, 125 or 500 mg/kg/day of the test substance.

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Once daily from GD2 through GD29 for clinical signs; cages checked twice daily for dead or moribund animals or signs of abortion (once daily at weekends and public holidays)
- Cage side observations included: Clinical signs, mortality, signs of abortion
Any animal showing signs of abortion or morbidity, particularly if death appeared imminent, was killed by intravenous injection of Dolethal to prevent loss of tissues through autolysis. All females which aborted, were killed for humane reasons or found dead in the cage were necropsied.

BODY WEIGHT: Yes
- Time schedule for examinations: on GD 3, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 29

FOOD CONSUMPTION: Yes
Full feeder weights were measured on GD: 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26 and 28
Empty feeder weights were measured on GD: 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 29
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on GD 29
- Organs examined:
Animals killed for humane reasons by intravenous injection of Dolethal (Sanofi, Libourne, France), including females which aborted or found dead, were autopsied. The kidney and urinary bladder were retained from all females and preserved in 10% neutral buffered formalin. The number and type of implantations and corpora lutea were noted when present. In the case of no visible uterine implants, but with corpora lutea, uterine horn(s) were immersed in a 20% solution of ammonium sulfide according to the Salewski method (1964). Tissues and carcasses were then discarded.
On GD 29, surviving females were killed by intravenous injection of Dolethal for examination of their uterine content. Each female was first subjected to macroscopic examination of the visceral organs and the number of ribs was recorded. The kidney and urinary bladder were retained from all females and preserved in 10% neutral buffered formalin. In the case of no visible uterine implants, but with corpora lutea, uterine horn(s) were immersed in a 20% solution of ammonium sulfide according to the Salewski method. Tissues and carcasses were then discarded. The reproductive tract was weighed (gravid uterine weight), dissected out and the caesarean parameters were recorded without knowledge of treatment group.

Ovaries and uterine content:

The ovaries and uterine content were 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
- Other: Number of live and dead fetuses, individual weight of fetuses

Intra-uterine resorption death was classified according to GLEICH and FROHBERG (1977) as:
- Early resorptions: macroscopic discrimination between fetal residues and placental material not possible.
- Late resorptions: distinct macroscopic discrimination between fetal and placental remains possible.

Dead fetuses: Defined as dead conceptuses showing distinct digits on fore and hind-paws.
Runt fetuses: Defined as live fetuses weighing less than 28 g at cesarean section of the dam.

Fetal examinations:

- External examinations: Yes: All live fetuses were killed by subcutaneous injection of Dolethal and subjected to an external examination.
- Soft tissue examinations: Yes: The bodies of all fetuses were dissected for soft tissue anormalities and sexed.
- Skeletal examinations: Yes: After soft tissue examination all fetuses were fixed in absolute ethanol before staining (a modification of the Staples and Schnell staining technique was performed).
- Head examinations: Yes: After internal examination of the neck, the head of fetuses from approximately half of each litter was immersed in Bouin's fluid and the internal structures examined after fixation.
Structural deviations were classified as follows:
Malformations: Very rare or obviously lethal changes.
Minor anomalies: Slight, relatively rare structural changes not obviously detrimental.
Common variants: Structural changes occurring in more than approximately 5% of the control population.

Statistics:

Mean and standard deviation for all maternal, litter and fetal parameters were calculated for each group. Statistical analyses were performed separately for all pregnant females and for all pregnant females with live fetuses. However, data was presented in the report for all pregnant females only, as both sets of data were very similar.

For details on statistics performed please refer to "Any other information on materials and methods incl. tables".

Indices:

Maternal body weight (BW) changes for interval periods; Corrected body weight change (CBWC); Average food consumption (FC) during intervals; Pre-implantation loss per litter; Post-implantation loss per litter; Number of live fetuses; Number of dead fetuses; Percentage of dead fetuses per litter; Percentage of male fetuses per litter; Mean fetal body weight per litter; Mean fetal body weight per litter and per sex; Mean fetal body weight per group; Mean fetal body weight per sex per group; Percentage of fetuses affected per litter; Mean percentage of fetuses affected per group; Percentage of litters affected per group.

Historical control data:

Historical control data from studies conducted in-house were provided and referred to in order to allow comparison with concurrent controls.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

For the decedent animals at 250 mg/kg bw/day, clinical signs for female OR2F0801 consisted of few faeces on several occasions, mucoid faeces on one occasion, yellow sediments in the urine on GD 24-26 and 28 and anogenital soiling, red traces on tray and placental tissue on tray on GD 28. Clinical signs for female OR2F0804 included no/few faeces on several occasions, yellow sediment in the urine on GD 23-28, red traces on tray on GD 26-28 and placental/fetoplacental tissue on tray on GD 28.

In surviving dams at 250 mg/kg bw/day, 5/23 females had yellow sediment in the urine on one or more occasions. In addition, one female had red traces on tray on GD 28. Other clinical signs occurred at a similar frequency in the control group or occurred in isolation and were therefore considered to be incidental findings.

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

Not applicable.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 250 mg/kg bw/day, 2/25 dams were killed or found dead in the cage on GD28 after aborting (no further information on mortality available).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

For the decedent animals at 250 mg/kg bw/day, the following body weight findings were recorded:
Between GD 10 and 28 (terminal body weight) female O2F0801 lost 0.49 kg in body weight, whilst between GD 20 and 28 (terminal body weight) female OR2F0804 lost 0.57 kg in body weight. In both cases, the body weight loss was associated with a marked reduction in food consumption.

For the surviving animals, no effect on mean maternal body weight change was observed at 250 mg/kg bw/day. Mean maternal corrected body weight change (maternal body weight change independent of the uterine weight) was slightly more pronounced at 250 mg/kg bw/day (-0.30 kg) compared with the controls (-0.24 kg), though the effect was not statistically significant. However, in the absence of an effect on mean maternal body weight change or mean fetal weight, this finding was considered to be incidental.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

For the decedent animals at 250 mg/kg bw/day, there was a marked reduction in food consumption.

For the surviving animals, mean maternal food consumption was comparable to the controls at 250 mg/kg bw/day.

Food efficiency:

not examined

Description (incidence and severity):

Not applicable.

Water consumption and compound intake (if drinking water study):

not examined

Description (incidence and severity):

Not applicable.

Ophthalmological findings:

not examined

Description (incidence and severity):

Not applicable.

Haematological findings:

not examined

Description (incidence and severity):

Not applicable.

Clinical biochemistry findings:

not examined

Description (incidence and severity):

Not applicable.

Endocrine findings:

not examined

Description (incidence and severity):

Not applicable.

Urinalysis findings:

not examined

Description (incidence and severity):

Not applicable.

Behaviour (functional findings):

not examined

Description (incidence and severity):

Not applicable.

Immunological findings:

not examined

Description (incidence and severity):

Not applicable.

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

Not applicable.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Number of abortions:

effects observed, treatment-related

Description (incidence and severity):

At 250 mg/kg bw/day, 2/25 dams aborted. Both females showed yellow sediments in urine on several occasions and body weight loss associated with reduced food consumption. No treatment-related findings were observed in either female at the macroscopic examination.

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

not examined

Description (incidence and severity):

Not applicable.

Changes in number of pregnant:

no effects observed

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

LOAEL

Remarks:

systemic

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

body weight and weight gain

clinical signs

food consumption and compound intake

mortality

Key result

Dose descriptor:

LOAEL

Remarks:

developmental

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

number of abortions

Key result

Abnormalities:

effects observed, treatment-related

Localisation:

uterus

Description (incidence and severity):

2/25 dams aborted also showing severe signs of systemic toxicity

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

effects observed, non-treatment-related

Description (incidence and severity):

The percentage of male fetuses was higher at 250 mg/kg bw/day compared to the controls, the effect being statistically significant, but this finding was considered incidental as the percentage of male fetuses was closer to the norm of approximately 50% (46.3%) at 250 mg/kg bw/day than was the case for the controls (45.2%).

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

not examined

Description (incidence and severity):

Not applicable.

Changes in postnatal survival:

not examined

Description (incidence and severity):

Not applicable.

External malformations:

effects observed, non-treatment-related

Description (incidence and severity):

At 250 mg/kg bw/day one fetus had multiple malformations consisting of macroglossia, small naris, head misshapen, malpositioned or elongated digit, and fleshy hindpaw, tail and neck. In the control group four fetuses from separate litters had different malformations consisting of cleft palate, absent eye bulge, malpositioned umbilical cord and ectrodactyly of forepaws. As these malformations occurred as isolated incidences, they were considered to be fortuitous.

The only anomalies observed in the treated group were short tail in one fetus and swollen abdomen in two fetuses from separate litters. At such low incidences these findings were considered to have occurred by chance.

There were no variant findings at the external fetal examination.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

At 250 mg/kg bw/day, two fetuses from separate litters had malformations, one fetus had fused thoracic centrum whilst the second fetus had fused caudal vertebrae which were malpositioned and bipartite. In the control group there were four malformed fetuses from four separate litters. Two fetuses had total sternoschisis with absent 1st digit on forepaws in one case. The two other fetuses had fused centrum in the thoracic or lumbar region, plus absent radius and 1st digit of forepaws in one case. As the malformations occurred as isolated incidences at 250 mg/kg bw/day, they were considered to be fortuitous.

The occurrence of anomalies was very similar between the treated and control groups, occurred at a higher frequency in the control group, or as isolated findings, and were therefore regarded as chance findings.

The variant findings of 5th distal phalanges of hindpaws unossified, and less than 15 caudal vertebrae, occurred at a higher incidence in the treated group compared to the controls. However, as the incidence for both mean percentage of fetuses affected and percentage of litters affected for each parameter was within the in-house historical control range, these findings were considered to have occurred by chance.

The following variant findings occurred at a higher incidence in the treated group compared to the controls; 13th thoracic ribs detached, pubis unossified, and insertion point(s) of pelvic girdle unossified. Since the incidence of these findings was slightly outside the in-house historical control range at the litter level or at both the fetal and litter level, statistical analysis was conducted on these parameters at both the fetal and litter level. Since none of the parameters analysed were found to be statistically significant at either the fetal or litter level, these findings were considered to be fortuitous.

A summary of skeletal findings is presented in Attachment 1.

Visceral malformations:

effects observed, non-treatment-related

Description (incidence and severity):

At 250 mg/kg bw/day, one fetus had the following malformations: ventricular septum defect in the cranial region, left and right ventricles of the heart enlarged plus dilated pulmonary trunk. In the control group there were four fetuses from separate litters with malformations. One fetus had dilated cerebral lateral ventricles, retinal fold and microphthalmia (unilateral) plus a small thalamus, a second fetus had a ventricular septum defect in the median region of the heart and a diaphragmatic hernia, diaphragmatic hernia was also observed in a third fetus whilst retroesophageal aortic arch was noted in a fourth fetus. In isolation, the malformations observed at 250 mg/kg bw/day are considered to be fortuitous.

Two fetuses from separate litters at 250 mg/kg bw/day had the anomaly misshapen kidney(s), compared with no incidences in the control group. The mean percentage of fetuses affected was within the historical control range, whilst the percentage of litters affected was marginally outside the historical control range.

The incidence of the variant findings misshapen thymus and small gall bladder, were higher at 250 mg/kg bw/day compared with the controls. However, as the mean percentage of fetuses with misshapen thymus and percentage of litters affected in the treated group was well within the in-house historical control range, this finding was considered to be fortuitous. The mean percentage of fetuses with a small gall bladder and percentage of litters affected at 250 mg/kg bw/day, was very marginally outside the in-house historical control range.

To further evaluate the relevance of the slightly higher incidence of misshapen kidney(s), and small gall bladder at 250 mg/kg bw/day, statistical analysis was conducted on these parameters at both the fetal and litter level. As neither parameter was statistically significantly increased at the fetal or litter level, these findings were considered to have occurred by chance.

A summary of visceral findings is presented in Attachment 1.

Other effects:

not examined

Description (incidence and severity):

Not applicable.

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects at this dose level.

Key result

Abnormalities:

effects observed, non-treatment-related

Key result

Developmental effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day

Treatment related:

yes

Relation to maternal toxicity:

developmental effects as a secondary non-specific consequence of maternal toxicity effects

Conclusions:

The study was performed in accordance to OECD TG 414 under GLP conditions and is considered reliable. In the treatment group at 250 mg/kg bw/day, two dams were sent to necropsy on GD 28 after aborting. Clinical signs for the females consisted of no/few/mucoid faeces on several occasions, yellow sediments in the urine on, anogenital soiling, red traces on tray and placental tissue on tray on GD 28. Between GD 10-28 and GD 20-28, respectively the first female lost 0.49 kg and the second female 0.57 kg in body weight. In both cases, the body weight loss was associated with a marked reduction in food consumption. No treatment-related findings were observed in either female at the macroscopic examination.
In surviving females, 5/23 animals had yellow sediment in the urine on one or more occasions. In addition, one female had red traces on tray on GD 28. No effect on mean maternal body weight change, mean maternal corrected body weight change or mean maternal food consumption was observed at this dosage.
No treatment-related necropsy findings were observed in the dams at the macroscopic examination.
At cesarean section, no effect on litter parameters was observed. No treatment-related findings were observed at the external, visceral or skeletal fetal examination.
In conclusion, a dose level of 250 mg/kg bw/day was considered to cause clear maternal toxicity but no developmental toxicity. Therefore, the LOAEL for maternal toxicity and NOAEL for developmental toxicity were both 250 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

173 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

Developmental toxicity was assessed in rat (OECD TG 414 and 416) and rabbit (OECD TG 414), under GLP conditions. The studies are considered of reliable quality and validity (RL1 or RL2), fulfilling the criteria of a key study. Thus, both are suitable for assessment of the present endpoint.

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

Three studies are available addressing prenatal developmental toxicity of the test substance. The studies were conducted in rats or rabbits in accordance with OECD 414 under GLP conditions.

In the first study (M-261462-01-1), female Sprague-Dawley rats received the test substance by oral gavage from gestation day (GD) 6 to 20 at doses of 60, 250 and 1000 mg/kg bw/day.

There were no treatment-related mortalities in any dose group. At 1000 mg/kg bw/day, body weight change was reduced by 32% between GD 6 to 8 in dams, thereafter; body weight change was comparable to the controls. At autopsy: one female was noted with yellow sediment in both kidneys and prominent lobulation of the liver, one female had an enlarged liver with white foci on the left lobe and one female had a white area on the left lobe. At the microscopic examination, one female was found to have a moderate bilateral papillary necrosis in the kidney, associated with mixed cell infiltrate. The change correlated with the macroscopic finding of yellow bilateral sediment in the kidney in this female. At 250 and 60 mg/kg bw/day, there were no treatment-related maternal findings.

For all dose groups, no litter parameters were affected, including the number of live fetuses, early and late resorptions, fetal death status, percentage of male fetuses and mean fetal body weights. At external, visceral and skeletal fetal examination, no treatment-related findings were observed.

In conclusion, for developmental toxicity, the NOEL was determined to be 1000 mg/kg bw/day and a dose level of 250 mg/kg bw/day was the NOEL for maternal toxicity.

In the second developmental toxicity study (M-261627-01-1), female New Zealand White rabbits were exposed to the test substance by gavage from gestation days 6 -28. The doses given were 50, 125 and 500 mg/kg bw/day.

The high dose level of 500 mg/kg bw/day was terminated prior to commencement of scheduled autopsy, due to the high mortality rate observed at this dosage. At 125 mg/kg/day, treatment-related clinical signs consisted of yellow sediment in urine in 2/25 dams. Yellow sediment in the kidney was also noted at autopsy in 1/25 females. No treatment-related changes were detected at the microscopic examination of the kidney or the urinary bladder. No treatment-related adverse effects were observed at 50 mg/kg bw/day.

At a dosage of 125 and 50 mg/kg bw/day, no litter parameters were affected. There were no external, visceral or skeletal findings at the fetal evaluation that were considered to be treatment-related at either dosage.

In conclusion, a dose level of 500 mg/kg bw/day clearly exceeded a Maximum Tolerated Dose (MTD) in the pregnant rabbit and was taken as the LOAEL. The dose level of 125 mg/kg bw/day was the NOAEL in the dam due to the findings observed in the kidney.

As only two treated groups were fully evaluated in the previous study, a complementary third study was performed (M-273153-01-1). Female New Zealand White rabbits were exposed to the test substance at a single dose level of 250 mg/kg bw/day, by gavage from gestation days (GD) 6 to 28. A concurrent control group was included.

Two dams were necropsied on GD 28 after aborting. Both females showed yellow sediments in urine on several occasions and lost weight. No treatment-related findings were observed in either female at the macroscopic examination.

In the surviving females, 5/23 animals had yellow sediment in the urine on one or more occasions. No effects on mean maternal body weight change were observed at this dosage. All surviving females showed no macroscopic findings at necropsy.

At caesarean section, no effects on litter parameters were observed at the dose level of 250 mg/kg bw/day. No treatment-related findings were found at the external, visceral or skeletal foetal examination.

In conclusion, a dose level of 250 mg/kg bw/day was considered to cause clear maternal toxicity evident by severe systemic toxicity correlated with abortions in two females. No fetal developmental toxicity was observed. Therefore, the NOAEL for developmental toxicity was set to be 250 mg/kg bw/day. Under the test conditions, the LOAEL for maternal toxicity was 250 mg/kg bw/day.

Based on the two studies in rabbits, the overall NOAEL for maternal toxicity was 125 mg/kg bw/day and the NOAEL for developmental toxicity was 250 mg/kg bw/day.

Justification for classification or non-classification

The available data on toxicity to reproduction of the test substance do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.