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Key value for chemical safety assessment

Effects on fertility

Additional information

In the chosen key study,i.e. a reproduction/developmental toxicity screening study according to OECD 421 and GLP, 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate (C-1701 B_C_3) suspended in Polyethylene glycol 300 (PEG 300) was administered by oral gavage to 10 Wistar rats/sex/dose level at dose levels of 0 (vehicle control group), 100, 250 or 700 mg/kg bw/day for 14 days prior to cohabitation, throughout cohabitation and continuing through the day before necropsy for male rats or through day 4 of lactation for female rats that delivered a litter (Charles River Laboratories (2012), 20027631). A complete necropsy was performed on all P generation rats, and selected tissues were weighed, retained and processed for histopathological examination. All surviving F1 generation pups were euthanized on postnatal day 5, and examined for gross lesions. In this study, mortality (P and F1 generations), clinical signs (P and F1 generations), body weights (P and F1 generations), feed consumption, estrous cyclicity, mating and fertility parameters, natural delivery, litter observations, macroscopic findings (P and F1 generations), selected organ weights and microscopic findings (incl. sperm staging in males) were assessed.

Administration of the test item at dose levels of 100, 250 and 700 mg/kg bw/day once daily by oral gavage resulted in urine-stained abdominal fur in male and female rats. Mean body weight gains were slightly decreased (53 % of the control group mean value) during the first week of study (days 1-8) in P generation male rats at 700 mg/kg bw/day. In P generation female rats at the same dose level, mean body weight gains were slightly decreased (83 % of the control group mean value) throughout the overall gestation period (DGs 0-20). Mean food consumption values were slightly decreased (90 to 88 % of the control group mean value) during the first week in P generation male and female rats and the first week of pregnancy (DGs 0-7; 93 to 92 % of the control group mean value) in female rats at 700 mg/kg bw/day. There were no test item-related effects on estrous cycle, mating and fertility parameters, gestation and lactation. Reproductive organ weights were not altered by the administration of the test item.

Mean pup weights per litter on DLs 1 and 5 were slightly reduced (9 and 14 % reduction, relative to control group mean values, respectively) in the 700 mg/kg bw/day group, reflecting decreased body weight change in P generation females during gestation (17 % reduction, relative to the control group mean value) and also the slightly higher mean litter size (11.2 versus 10.4 in the control group). It is known from literature (Fleeman et al., 2005) that reductions in fetal body weights frequently occur concurrent with reduced maternal food consumption and maternal body weights, as seen in the current study results.

Histopathological examinations did not reveal any test item-related effects. There were no adverse clinical signs or gross lesions in the F1 generation pups attributed to administration of the test item to the P generation dams.

Under the conditions of this study, the no-observed-adverse-effect-level (NOAEL) for developmental toxicity for the test item was considered to be 250 mg/kg bw/day, based on the reductions in mean pup weights per litter at 700 mg/kg bw/day, which were probably related to maternal toxicity, as the reductions in pup weights were concurrent with decreased maternal body weights and a slightly higher litter size. Further, these reductions in mean pup weights per litter were not observed in the lower dose groups, where evidence of maternal toxicity was not apparent. Accordingly, the no-observed-adverse-effect level (NOAEL) for general toxicity was considered to be 250 mg/kg body weight/day; urine-stained abdominal fur was attributed to administration of the test substance and was observed at this dose level, but the observation was not concurrent with effects on other parameters.The no-observed-adverse-effect-level (NOAEL) for effects on fertility was found to be 700 mg/kg bw/day, i.e. the highest dose tested.

Short description of key information:
The toxicity to reproduction was investigated for 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate in a reproduction/developmental toxicity screening test in rats (OECD guideline 421, GLP). The NOAEL for developmental toxicity was determined to be 250 mg/kg bw/day. The NOAEL for general systemic toxicity was determined to be 250 mg/kg bw/day. The NOAEL for effects on fertility was determined to be 700 mg/kg bw/day, i.e. the highest dose tested.

Effects on developmental toxicity

Description of key information
The toxicity to development was investigated for 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate in a prenatal developmental toxicity study according to OECD TG 414 and GLP. The NOAEL for developmental toxicity and maternal toxicity was determined to be 250 mg/kg bw/day.
Additional information

In the chosen key study, i.e. a developmental toxicity study according to OECD TG 414 and GLP, 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate (C-1701 B_C_3) suspended in Polyethylene glycol 300 was administered to 25 pregnant Crl:WI(Han) female rats/dose level by oral gavage (dose volume: 5 mL/kg bw) at dose levels of 0 (vehicle control), 100, 250 and 700 mg/kg bw/day on DGs 6-20 (Charles River Laboratories (2013), 20027630).All female rats were euthanized on DG 21 and examined for ovarian and uterine contents, and a gross necropsy of the thoracic, abdominal, and pelvic viscera was performed blind to dose group. The following parameters and end points were evaluated: viability, clinical signs, body weights, body weight changes, food consumption, mating performance, gross observations, ovarian and uterine contents, gravid uterine weights, and fetal sex, fetal body weights, and fetal gross external, soft tissue, and skeletal alterations, as well as ossification site averages.

Urine-stained abdominal fur, dehydration (based on skin turgor), and red perinasal substance occurred in the 700 mg/kg bw/day group. These clinical signs were attributed to administration of the test item. Additional clinical signs included but were not limited to excess salivation, thin body condition, urine-stained perivaginal area, all of which occurred in a single animal in the 700 mg/kg bw/day group; these clinical signs were also attributed to test item administration. Urine-stained abdominal fur also occurred in an increased number of animals at 250 mg/kg bw/day, and dehydration was noted in a single animal on a single occasion. No test item-related clinical signs were observed at 100 mg/kg bw/day.

Mean maternal body weights and body weight changes (absolute and corrected for gravid uterine weights) were reduced at 700 mg/kg bw/day, and mean absolute body weight gain between DGs 6 and 21 was reduced by 24 % when compared to the control group value. Likewise, mean absolute and relative food consumption values in this dose group were reduced by 14 % and 12 %, respectively, when compared to the control group values during this same interval. Mean body weight and body weight changes and food consumption values were not affected by the administration of the test substance in the other dose groups.

Slight reductions in fetal body weight averages (approximately 7 %) were noted at 700 mg/kg bw/day. Fetal morphology examinations revealed reduced numbers of ossified caudal vertebrae and hind limb tarsals, metatarsals, and phalanges at 700 mg/kg bw/day. No test item-related effects were observed at 100 and 250 mg/kg bw/day.

Taken together, daily test item administration at 700 mg/kg bw/day from DGs 6-20 caused maternal toxicity, as evidenced by clinical signs, significantly reduced food consumption and significantly reduced body weight and body weight changes. There were no compound-related effects regarding pregnancy or Caesarean-sectioning examination parameters. Mean fetal body weights were slightly reduced at 700 mg/kg bw/day. Fetal examinations revealed reductions in the mean number of ossification sites in the caudal vertebrae and hind limbs, but no test item-related effects regarding the incidence of malformations and other variations. The reductions in the mean number of ossification sites at the caudal vertebrae and hind limbs were morphological correlates of the reductions in fetal body weight averages, which occurred at a maternally toxic dose level. It is known from the literature (Fleeman et al., 2005) that reductions in fetal body weights and delays in ossification frequently occur concurrent with reduced maternal food consumption and maternal body weights, as seen in the current study results.

At 250 mg/kg bw/day, a higher incidence of urine-stained abdominal fur was present in the dams. Mild dehydration (based on skin turgor) occurred in a single rat on a single occasion. In the absence of any other changes, these findings were not considered as adverse. No embryo‑fetal effects were observed. Neither maternal nor embryo-fetal effects were observed at 100 mg/kg bw/day.

 

Under the conditions of this study with the test item, the no observed adverse effect-level (NOAEL) for maternal and embryo-fetal toxicity was established at 250 mg/kg bw/day.Reductions in fetal body weight averages and reductions in the mean number of ossification sites in the caudal vertebrae and hind limbs occurred at 700 mg/kg bw/day, which were considered related to maternal toxicity, as these effects were concurrent with decreased maternal food consumption and body weights. These reductions in fetal body weights and ossification sites were not observed at lower dose levels, including 250 mg/kg bw/day, where evidence of maternal toxicity was not apparent.

 

Overall based on the study results 2-ethoxyethyl (2Z)-2-cyano-2-[3-(3-methoxypropylamino)cyclohex-2-en-1-ylidene]acetate did neither display selective embryotoxicity nor teratogenicity.

 

Reference:

Fleeman TL, Cappon GD, Chapin RE, Hurtt ME (2005). The effects of feed restriction during organsogenesis on embryo-fetal development in the rat. Birth Defects Res B Repro Toxicol 74 (5):442-449.

Justification for classification or non-classification

The present data on reproductive toxicity do not fulfill the criteria laid down in 67/548/EEC and regulation (EU) 1272/2008, and therefore, a non-classification is warranted.