Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Oral (OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, rat): NOAEL (reproduction) = 1000 mg/kg bw/day

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
16 Nov 2017 - 24 Jan 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
recommended by guideline
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: The Lab Animals Breeding Center “Pushchino”, Branch of Institute of Bioorganic Chemistry RAS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approx. 11-13 weeks old at the initiation of dose administration (day 1)
- Weight at study initiation: males 336 ± 23 g, females 210 ± 14 g
- Housing: two animals in per sex in solid bottom polycarbonate cages (Type-4) with bedding, cages equipped with steel lids, steel separators for the food and steel label holders, all cages provided with environmental enrichment material. After mating, dams were housed alone to deliver and their litters were housed in these cages. Animals of satellite subgroup were housed 2-3 animals per cage in Type-4 cages.
- Diet: Laboratory Rodent Diet (SSNIFF V1534-300 autoclavable, Spezialdiaeten, GmbH), ad libitum
- Water: Filtered tap water, ad libitum
- Acclimation period: approx. 6 weeks

DETAILS OF FOOD AND WATER QUALITY:
Diet is analyzed by the manufacturer for nutritional components and environmental contaminants and routinely by BTL BIBC RAS for microbiological contaminants. Samples of water are analyzed routinely for microbiological contaminants.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 55
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: 16 Nov 2018 - 24 Jan 2018
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared as a suspension of the neat test item in vehicle (corn oil). The required amount of the test item was weighed and suspended it in a mortar with the addition of vehicle (corn oil) followed by stirring with the calculated volume of vehicle. Test item formulations were prepared every three days and stored at room temperature and checked visually for homogeneity prior to initiation of dosing. The test item formulations were stirred continuously during dosing.

VEHICLE
- Justification for use and choice of vehicle: delivery vehicle for fat-soluble compounds, recommended by guideline
- Amount of vehicle: 10 mL/kg bw
- Lot/batch no.: MKCC0462
- Quality Release Date: 05 Dec 2016
- Storage Conditions: Room temperature (20 ± 5 °C)
- Provider: Sigma-Aldrich CHEMIE GMBH, Germany
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: until evidence of copulation. If evidence of copulation was not detected after 8 days of pairing in presence of regular oestrus cycle, the female was housed with another male for which the mating was confirmed.
- Proof of pregnancy: presence of sperm following a vaginal lavage referred to as gestation day 0 (G0)
- After 8 days of unsuccessful pairing replacement of first male by another male with proven fertility.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability of the test item in the vehicle was confirmed following storage for 7 days at room temperature during a method validation study. Analysis of formulations for homogeneity and concentration was conducted in the test facility using a validated method. For homogeneity analysis, quadruplicate samples (3 mL of each) were collected from the top, middle and bottom strata of each dosing formulation prepared during the study, and collected from the top and bottom strata of each resuspended formulation after 2 days of storage, at the beginning of in-life phase. Samples collected from the middle stratum for homogeneity analysis were used for concentration assessment.
Duration of treatment / exposure:
Males were administrated 14 daily doses prior to mating and continuing throughout the mating period for a total of 28 doses.
Females received 14 daily doses prior to pairing and were dosed through lactation day 13 for a total of 50 - 60 doses.
Frequency of treatment:
7 days/week
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
Main subgroup (control, 100, 300, 1000 mg/kg bw/day): 12 males, 12 females
Satellite subgroup (control, 1000 mg/kg bw/day): 5 males, 5 females
Control animals:
yes, concurrent vehicle
Parental animals: Observations and examinations:
During the period of expected parturition (starting with gestation day 19), females were observed twice daily for initiation and completion of parturition and for signs of dystocia.

DETAILED CLINICAL OBSERVATIONS: Yes:

BODY WEIGHT: Yes

FOOD CONSUMPTION AND COMPOUND: Yes

for further details see Section 7.5.1
Oestrous cyclicity (parental animals):
Oestrous cycles was monitored before start of the treatment to select the females with regular cyclicity (4-5 day cycles). Vaginal smears were also monitored daily for the last week during the pre-mating period with continued monitoring into the mating period until there was evidence of mating. Vaginal smears were examined before necropsy to determine the stage of the oestrous cycle and allow correlation with histopathology of female reproductive organs.
Sperm parameters (parental animals):
Parameters examined in P male parental generations: Cowper’s glands, Epididymides, Glans penis, Levator ani & bulbocavernosus muscles (LABC) complex, Prostate, Seminal vesicles & coagulating glands, Testes
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 10 pups/litter (5/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: Pups delivered, Liverborn, Stillborn, Unknown vital status, Runts, Pups found dead, Pups cannibalized, Survival relative to number born, Body weight, Anogenital distance normalized, Sex
- Additional information: Each litter was examined daily for survival and abnormalities and all deaths were recorded.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All F0 male animals were sacrificed following completion of the mating period and 28 days of dose administration.
- Maternal animals: All surviving F0 female animals were sacrificed on lactation day 14 after litters have been euthanized on PND 13.

GROSS NECROPSY
- Gross necropsy consisted of: refer to details under 7.5.1
The number of former implantation sites and corpora lutea (in total, corpora lutea (CL) of pregnancy and newly formed CL after the postpartum ovulation) was counted and recorded.

HISTOPATHOLOGY / ORGAN WEIGHTS
Refer to details under 7.5.1
Postmortem examinations (offspring):
SACRIFICE
- Pups were sacrificed on post-natal day (PND) 13 post-partum.
- Dead pups and pups killed on PND 13 were carefully examined externally for gross abnormalities. Particular attention was paid to the external reproductive genitals which were examined for signs of altered development. Blood samples from the day 13 pups were assessed for serum levels of thyroid hormone (T4).

GROSS NECROPSY
- Gross necropsy consisted of external examinations.
Statistics:
See Section 7.5.1
Reproductive indices:
The following parameters of reproductive performance were calculated for males and females:
- Pre-coital interval (days): Rats paired over a 12-hour dark cycle were considered to have been paired for 1 day
- Male Mating Index (%): (No. of males with evidence of mating / Total No. of males used for mating) X 100
- Female Mating Index (%): (No. of females with evidence of mating / Total No. of females used for mating) X 100
- Male Fertility Index (%): (No. of males siring a litter / Total No. of males used for mating) X 100
- Male Copulation Index (%): (No. of males siring a litter / No. of males with evidence of mating or females with confirmed pregnancy) X 100
- Female Fertility Index (%): (No. of females with confirmed pregnancy / Total No. of females used for mating) X 100
- Female Conception Index (%): (No. of females with confirmed pregnancy / No. of females with evidence of mating) X 100

The following parameters were evaluated for dams of each group:
- Duration of gestation (days): The time elapsed between confirmed mating and the day of first pup was delivered
- Gestation index (%): Number (N) of rats with live offspring / Number of pregnant rats
- Pre-natal loss of offspring: Implantations minus live births
- Post-natal loss of offspring: Live births minus alive at PND 13
Offspring viability indices:
The following parameters were evaluated for each litter:
- Dead pups (N, %): Including pups found dead, euthanazied in extremis and cannibalized. Missing pups were presumed cannibalized (PND 0-1, 1-4, 1-7, 1-13)
- Postnatal survival (N, % per litter): Number of live pups, % per litter (PND 0-1, PND 1-4, PND 1-7, PND 1-13)
- Viability index (N/N, %): Number of live pups / Number of liveborn pups on PND 0, PND 4, 7, 13
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Red urine was observed for three days in one female (No.113) from group 4 during daily handling, which was associated with the severe renal tubular necrosis and supposed to be test substance-related.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female in the 300 mg/kg bw/day group was euthanized on lactation day 8 due to poor clinical condition. The cause was determined to be trauma of esophagus and, therefore, was not attributed to test item administration. All other males and females at all dose levels survived until the scheduled necropsy.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Decrease in food consumption was observed in all groups compared to the pre-treatment period (study days 0-1). Since there were no corresponding effects on mean body weight gain and food consumption have increased by the end of the recovery period in satellite subgroup, the decrease in mean food consumption were attributed to the high caloric content of the vehicle.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
There were no test item-related effects on hematology parameters in males. Effects observed in females of different dosing groups and the control were of small magnitude and were not considered toxicologically important.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The following alterations in serum chemistry parameters were considered to be related to test item administration in the 1000 mg/kg bw/day group males: mean serum ALT (↑85.7%) and mean serum ALP (↑66.2%); these differences were significant (p<0.001 and p<0.01) when compared to the vehicle control group. The increase in ALP was slightly dose-related; the mean changes observed in the 100 and 300 mg/kg body weight/day group were higher (not significantly) of that in control group (↑20.4%, ↑17.2%, respectively). Additionally, mean serum globulins were significantly increased, and A/G ratio was reduced (p<0.05) in the 300 mg/kg bw/day male group (↑12.4, ↓9.5%, respectively).
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Necrosis of renal tubules and infiltration of pelvis observed in females at a dose of 300 mg/kg bw/day and in severe grade at a dose 1000 mg/kg bw/day is considered adverse.
Histopathological findings: neoplastic:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
no effects observed
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No test item-related effects on reproductive performance were observed at any dosage level. One male from 100 mg/kg bw/day group and one male from 1000 mg/kg bw/day group have no evidence of mating and did not sire a litter. The precoital interval was approximately the same in all groups. All females in groups were with evidence of mating. One gravid female from 300 mg/kg bw/day group did not deliver.

GESTATION LENGTH AND PARTURITION OBSERVATION
Mean gestation lengths were similar amongst all groups when the 100, 300, and 1000 mg/kg bw/day groups were compared to the vehicle control group. One female (No.93) from 300 mg/kg bw/day dose group did not deliver and had signs of post-implantation loss. Prolonged labor was observed in three females (No.139, No.128 and No.115 from the control group, 100 and 300 mg/kg bw/day groups, respectively) that was not attributed to the test item administration.

LOSS OF OFFSPRING
No statistically significant differences were noted between the vehicle control and test item-treated groups. One female (No.93) from 300 mg/kg bw/day dose group did not deliver in the presence of one implantation site. The other females from all dose groups delivered approximately the same number of live pups. The numbers of stillborn pups as well the values of pre-natal and post-natal loss of offspring were approximately the same between groups. Three dams from high dose group had abnormal pups identified as runts. However, there was no statistical difference from the control group, and this finding was considered to be substance unrelated. Also, one dam from the control group had pups with a visual abnormality of hindlimbs layout and brain hemorrhages in one of them. These pups have been identified as abnormals.

For further details refer to section 7.5.1
Key result
Dose descriptor:
NOAEL
Remarks:
reproduction
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse reproductive effects observed
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Microscopic examination of brain tissue collected from pup 139-8m (control group), and pup 115-8f (300 mg/kg body weight/day dose group) with signs of hemorrhage revealed moderate focal subarachnoid hemorrhage. The findings identified cannot be attributed to F0 parental test item administration.
Histopathological findings:
no effects observed
Behaviour (functional findings):
not examined
Developmental immunotoxicity:
not examined
LITTER VIABILITY ON PND0
There were no test item-related changes in the mean number of pups born, live litter size and survival between birth and PND 0 (relative to number born). Three pups from 1000 mg/kg bw/day group were identified as runts. However, there was no statistical difference from the control group in the percentage number, and this finding was considered to be test item unrelated.

SEX, BODY WEIGHT AND ANOGENITAL DISTANCE AT BIRTH
The mean number of females and males born, the percentage of males, body weights at birth, and absolute and relative anogenital distance in substance-treated groups did not differ significantly from the values in the control group. An increase in the mean number of females (↑24.1%) at birth in the 1000 mg/kg bw/day group were not significantly different compared to the vehicle control group and the percentage of males per litter was approximately similar to control value.

POST-NATAL SURVIVAL
The mean number of liveborn pups, live litter size and post-natal survival in the all dose-treated group did not significantly differ from the values in control group. The percentage of dead pups during postnatal period PND 1-13 was not increased in test item treated groups, and the viability index was similar to the control group value.

CLINICAL OBSERVATIONS
The general physical condition of all F1 pups in the 100, 300 and 1000 mg/kg bw/day groups were unaffected by test item administration. On the PND 0-1, several liveborn pups were found dead in groups 1, 2 and 3 (1, 1 and 2 pups respectively). Four, 1, 1 and 4 pups in the groups 1, 2, 3 and 4 were missing and presumed to have been cannibalized. One pup from control group 1 was euthanized in extremis on PND 0 in moribund condition. Litter 86 (group 3) and litter 131 (group 4) were euthanized in extremis due to starvation, which was associated with clinical condition of dams. The poor clinical condition of the female 86 was caused by a trauma of esophagus. The cause of emaciation of litter 131 was determined to be agalactia.

BODY WEIGHTS, BODY WEIGHT GAIN AND PERCENTAGE OF MALES
Mean body weights and body weight gains in male and female pups were unaffected by test item administration. No statistically significant differences from the vehicle control group were noted. The percentage of males in the litters during PND 0-13 was also approximately the same in all groups.

CLINICAL PATHOLOGY
There were no test item-related effects on a thyroxin level in serum of F1 offspring on postnatal day 13. Mean thyroxin concentrations in the 100, 300 and 1000 mg/kg bw/day dose groups did not significantly differ from the values in the vehicle control group.

NUMBER OF NIPPLES/AREOLAE
The test item did not increase nipples/areolae retention in males at PND 13. Areolae were observed in the following males, single in groups: 138-4m (Group 1, 6 areolae), 130-2m (Group 2, 4 areolae) and 125-2m (Group 3, 4 areolae), that was not treatment related. Female offspring exposed to 0, 100, 300 and 1000 mg/kg bw/day of the test item did not display decreased nipples at PND 13.

WEIGHTS OF THYROID GLANDS
There were no changes in the absolute and relative weights of the thyroid glands in F1 offspring males and females in all test item treatment groups compared with the control group.

MACROSCOPIC OBSERVATIONS
The number of pups necropsied on PND 0 as found dead or euthanized in extremis, was 13, 14, 11 and 6, respectively, in the control group, 100, 300 and 1000 mg/kg bw/day dose groups. No internal findings that could be attributed to F0 parental test item administration were noted at the necropsies of these pups. In the control group, pups no. 139-7 and no. 139-8 were suspected of an anomaly of the hind limbs, which was not confirmed. In addition, brain hemorrhage was revealed in the pup no. 139-8 as well as in the pup no. 115-8 from 300 mg/kg bw/day dose group. Aside from the presence or absence of milk in the stomach, no other internal findings were noted. Litter 86 (group 3) and litter 131 (group 4) were euthanized in extremis on PND 8 and PND 10, respectively, due to pups emaciation, which was associated with clinical condition of dams (trauma of esophagus in female No.86 and agalactia in female No. 131). There were no significant observations in all pups in the test item-treated groups and in the vehicle control group during scheduled necropsy on PND 13. No signs of demasculinization were found in any male in all groups.

MICROSCOPIC OBSERVATIONS
Microscopic examination of brain tissue collected from pup 139-8m (control group), and pup 115-8f (300 mg/kg bw/day dose group) with signs of hemorrhage revealed the following findings: moderate focal subarachnoid hemorrhage was observed in the posterior parietal cortex and in the thalamus of pup 139-8m, and marked focal hemorrhage was observed in the frontal and temporal cortex of pup 115-8f. The findings identified cannot be attributed to F0 parental test item administration. The processing of skeletons by Dawson's technique did not reveal any anomalies of ossification in pups 139-7f and 139-8m.
Key result
Dose descriptor:
NOAEL
Remarks:
development
Generation:
F1
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxicologically relevant effects observed
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Tables summarising individual animal data are attached as pdf documents to the technical dossier.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7.1, of Regulation (EC) No. 1907/2006.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

The reproductive toxicity after oral exposure of 4-(octadecylamino)-4-oxoisocrotonic acid (CAS No. 3077-27-8) has been investigated in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD TG 422 and under GLP conditions (BTL, 2018). The test item in the vehicle (corn oil) was administered orally by gavage once daily (7 days/week) to three groups of Sprague-Dawley rats at the doses 100, 300 and 1000 mg/kg bw/day. A concurrent control group received the vehicle (corn oil) on a comparable regime and in the same volume of 10 mL/kg body weight. Each group of the main subgroup consisted of 12 males and 12 females used for dosing and mating. Males were administrated 14 daily doses prior to mating and continuing throughout the mating period for a total of 28 doses. Females received 14 daily doses prior to pairing and were dosed through lactation day 13 for a total of 50-60 doses. Satellite animals (five males and five females from control group and high dose group) were not mated. These satellite animals were dosed for a total of 28 and 55 consecutive days for males and females, respectively, and were sacrificed after a two weeks recovery period. All animals were observed twice daily for mortality and morbidity. Detailed clinical observations, body weights, and food consumption were recorded weekly. All F0 females were allowed to deliver and rear their pups until lactation day 13. Clinical pathology evaluations (hematology, coagulation, and serum chemistry) were performed in half of F0 males and half of F0 females as well as in all satellite animals at the end of the in-life phase. Two F0 females in poor clinical condition and with emaciation of pups were euthanized in extremis. Complete necropsies were conducted on all F0 animals and satellite animals, and selected organs were weighed. Selected tissues were examined microscopically from half of F0 animals in the vehicle control and high-dose groups. Lower dose level groups and satellite subgroups were evaluated for macroscopic observations and for presumptive target organs.

F0 male and female mating and fertility, male copulation and female conception indices, pre-coital interval, gestation length, and the process of parturition were unaffected by test item administration at all dosage levels. One male from 100 mg/kg bw/day group and one male from 1000 mg/kg bw/day group had no evidence of mating and did not sire a litter. All females in all groups were with evidence of mating. One gravid female with one implantation site from 300 mg/kg bw/day group did not deliver. The other females from all dose groups delivered approximately the same number of live pups. The numbers of stillborn pups as well as the values of pre-natal and post-natal loss of offspring were approximately the same between groups. Three dams from high dose group had abnormal pups identified as runts. However, there was no statistical difference from the control group, and this finding was considered to be not test item-related.

No test item-related effects on F0 reproductive performance, gestation length, parturition, or reproductive organs were noted at any dosage level. There were no changes in thyroid weights, and thyroxin serum level in parental animals. Based on these results, the NOAEL for F0 reproductive toxicity was considered to be 1000 mg/kg body weight/day.

Effects on developmental toxicity

Description of key information

Oral (OECD 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, rat): NOAEL (development) = 1000 mg/kg bw/day

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 1), and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.7.1, of Regulation (EC) No. 1907/2006.
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

The reproductive toxicity after oral exposure of 4-(octadecylamino)-4-oxoisocrotonic acid (CAS No. 3077-27-8) has been investigated in a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test according to OECD TG 422 and under GLP conditions (BTL, 2018). The study has been discussed in detail above and in IUCLID section 7.5.1. Here, only findings related to developmental toxicity are summarised.

All F0 females were allowed to deliver and rear their pups until lactation day 13. Live pups were counted and sexed and litters weighed on day 0-1 (after the completion of parturition), day 4, day 7 and day 13 post-partum. The anogenital distance (AGD) of each delivered pup was measured at the end of delivery (on day 0-1 post-partum). Any abnormalities of the offspring were recorded. The number of nipples/areolae in male and female pups was counted on post-natal day 13 (PND 13). F1 pups were euthanized on PND 13 and examined for gross abnormalities with particular attention to the external reproductive genitals and with weighing of thyroid glands. Thyroids were examined microscopically from F1 offspring in the vehicle control and high-dose groups. Thyroxin level was assayed in PND 13 pups and adult males.

There were no test item-related changes in the mean number of pups born, live litter size, postnatal survival and index of viability. The mean body weights at birth, the percentage of males, and absolute and relative anogenital distance in test item-treated groups did not differ significantly from the values in the control group. The percentage of males in the litters during PND 0-13 was also approximately the same in all groups. The general physical condition of all F1 pups in the 100, 300 and 1000 mg/kg bw/day groups, mean body weights, and body weight gains in male and female pups, were unaffected by the test item administration. The test item did not increase nipples/areolae retention in males at PND 13. There were no test item-related effects on a serum thyroxin level of F1 offspring on postnatal day 13. Mean thyroxin concentrations in the 100, 300 and 1000 mg/kg bw/day dose groups did not significantly differ from the values in the vehicle control group. There were no changes in the absolute and relative weights of the thyroid glands in F1 offspring males and females in all test item treatment groups compared with the control group. The number of pups necropsied on PND 0 as found dead or euthanized, did not significantly differ among groups. No internal findings that could be attributed to F0 parental test item administration were noted at the necropsies of these pups. One litter in 300 mg/kg bw/day dose group and one litter in 1000 mg/kg bw/dose group were euthanized on PND 8 and PND 10, respectively, due to pups emaciation, which was associated with the clinical condition of dams. There were no significant observations in all pups in the test item-treated groups and in the vehicle control group during scheduled necropsy on PND 13.

Based on these results, the No-Observed-Adverse-Effect-Level (NOAEL) for F1 developmental toxicity was considered to be 1000 mg/kg bw/day.

Justification for classification or non-classification

The available data on toxicity to reproduction following exposure via the oral route of the registered substance do not meet the classification criteria according to Regulation (EC) No. 1272/2008 (CLP) and are, therefore, conclusive but not sufficient for classification.