Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Multiple studies were conducted. In the key two-generation rat study (2013/1347949) dose levels of 0, 100, 500 and 2000 ppm were tested.

The NOAEL for general, systemic toxicity is 500 ppm for the F0 and F1 parental rats, based on decreased food consumption and body weight/body weight gain observed at 2000 ppm in all F0 and F1 parental animals, as well as effects on hematology and clinical chemistry.

The NOAEL for fertility and reproductive performance for the parental rats is 500 ppm due to the reduction in implantation sites and pups delivered in the F1 parents of the 2000 ppm dose group.

The NOAEL for developmental toxicity in the F1 and F2 progeny is 100 ppm, due to the decrease in the pre-weaning pup body weights/pup weight gains observed at the 500 ppm dose. The NOAEL at 100 ppm was based on an effect only observed during the lactation portion of the study when the effective maternal dose is doubled. In addition, later pharmacokinetic studies in the rat indicated that doses exceeding 15 mg/kg bw/d follow non-linear kinetics, resulting in an even higher effective dose. In the context of risk assessment, the NOAEL of 100 ppm does not represent meaningful evidence of increased pup sensitivity.

Effect on fertility: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
51 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP and Guideline conform study.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Key study: Two-generation reproductive toxicity 2013/1347949

In this 2-generation reproduction study performed according to OECD 416, rats (24/sex/dose) were given 0, 100, 500 or 2000 ppm (equivalent to 0, 8, 39 and 155 mg/kg bw/d for males and 0, 12.0, 56 and 197 mg/kg bw/day for females, based on intake by F0 animals during pre-mating) starting 10 weeks before mating (F0) or at weaning (F1) until scheduled necropsy. No dose adjustment was performed during gestation and lactation. This lead to a significantly higher exposure during lactation equivalent to 18.7 (F0: 12.2 / F1: 23.0), 88.5 (F0: 61.0 / F1: 107.2) and 300.1 (F0: 206.5 / 356.8) mg/kg bw/day at 100, 500, and 2000 ppm, respectively.

A clear effect on the number of pups cannibalized and pup mortality was visible in both F1 and to a greater extent the F2 generation at 2000 ppm. Investigation of the affected pups revealed clear indications of reduced maternal care and insufficient nursing, including empty stomachs. The study concluded that significant pup effects observed in the high dose animals are secondary to lactation and thus maternal toxicity.

The NOAEL (no observed adverse effect level) for general, systemic toxicity is 500 ppm (51 mg/kg bw/d) for the F0 and F1 parental rats, based on decreased food consumption and body weight/body weight gain observed at 2000 ppm (186 mg/kg bw/d) in all F0 and F1 parental animals, as well as effects on hematology and clinical chemistry. The NOAEL for fertility and reproductive performance for the parental rats is 500 ppm (51 mg/kg bw/d) due to the reduction in implantation sites and pups delivered in the F1 parents of the 2000 ppm (186 mg/kg bw/d) dose group. The NOAEL for developmental toxicity in the F1 and F2 progeny is 100 ppm (11 mg/kg bw/d), due to the decrease in the pre-weaning pup body weights/pup weight gains observed at the 500 ppm (51 mg/kg bw/d) dose.

There are two final comments regarding the developmental NOAEL of 100 ppm (11 mg/kg bw/d) from the definitive rat 2-generation study using the LOAEL based on reduced pup body weight at 500 ppm (51 mg/kg bw/d).

1. The effects that set this NOAEL were only observed during the lactation portion of the study when the effective maternal dose is doubled. Therefore, the relevance of using this endpoint as a NOAEL to define an independent adverse developmental finding is questionable.

2. Later pharmacokinetic studies in the rat indicated that doses exceeding 15 mg/kg bw/d follow non-linear kinetics - as oral doses increase, there is a disproportionate increase in plasma concentration of the test substance (also refer to IUCLID chapter 7.1). All of the pup effects that were seen in the 2-generation reproduction studies (and the later discussed developmental toxicity studies) were at doses that exceed a kinetically derived maximum tolerated dose (MTD or KMD). The effective internal exposure at the LOAEL was measured in the PK study to be more than 25X the exposure expected with linear kinetics.

This unproportioned dose-multiplying effect is particularly relevant during the lactational phase of the study, where the maternal rats are already consuming two fold higher levels of the test substance, plus the additional systemic exposure due to saturated elimination kinetics.

Therefore, though the study NOAEL of 100 ppm (11 mg/kg bw/d) is accurate, in the context of risk assessment it does not represent meaningful evidence of increased pup sensitivity but rather an effect that is limited to a high-dose restricted mode(s) of action that is not relevent to humans.

 

 

Supporting study: Two-generation reproduction toxicity 2013/8001785

The test substance was administered to BrlHan:WIST@Jcl(GALAS) rats (24 males and 24 females per group) via the diet at dose levels of 0, 100, 300, and 1000 ppm over two successive generations to evaluate the potential effects on reproductive performance of parental animals and on growth and development of their offspring.

 

No changes in general appearance or deaths related to the test substance administration were observed in either sex of F0 and F1 parental animals in any of the treated groups.

Body weights, body weight gains and food consumption were not affected by the test substance treatment even in males of the 1000 ppm group, as well as in both sexes of parental animals in the 100 and 300 ppm groups. However, food consumption of both F0 and F1 females decreased significantly in the 1000 ppm group during the lactation period.

Postmortem examinations of parental animals also revealed no effects of the test substance administration in the 100 and 300 ppm groups. In the 1000 ppm group, however, the absolute and/or relative liver weights of males and absolute and relative adrenal weights of females increased significantly in both generations. Weight changes were not accompanied by histopathological abnormalities.

 

Reproductive performance of parental animals was not affected in any test substance-treated groups. Although mean days of age at completion of preputial separation in F1 males of the 1000 ppm group significantly exceeded the control value, this was within the historical control range and considered not to be an indication of delay in sexual maturation but merely a suggestion of growth retardation because body weights of F1 males on the day of completion did not differ significantly from the control value even in the 1000 ppm group. The number of primordial follicles in F1 females of the 1000 ppm group was comparable to that of the control group.

 

The test substance treatment did not affect offspring in such parameters as litter sizes, sex ratios, general appearances, viability indices, lactation indices, anogenital distances, body weights, reflex responses, necropsy findings and organ weights in the 100 and 300 ppm groups. In the 1000 ppm group, body weights of male and female pups were significantly lower than their respective controls on postnatal day 21 in both F1 and F2 generations.

 

These results lead to the conclusion that the NOAEL is 300 ppm (corresponding to 24.1 mg/kg bw/d) for systemic toxicity on parental animals and for growth and development of their offspring. It is also concluded that the test substance does not disturb parental reproductive performance up to the highest dose level of 1000 ppm (corresponding to 79.0 mg/kg bw/d) under the present study conditions.

 

 

Supporting study: Dose-range finding study for two-generation reproduction toxicity 2009/7010953

In the scope of a range finding study for the 2-generation study (2013/8001785) the test substance was administered to groups of 8 male and 8 female rats at doses of 0, 150, 1500, 3000, and 6000 ppm. Rats were pre-dosed for 2 weeks, and dosed through mating, gestation and lactation.

Body weight gain was significantly decreased in males of the 3000 and 6000 ppm dose group starting from week 2 or 1 of treatment until week 5, being below control levels until termination. Female rats of the high dose group (6000 ppm) exhibited body weight loss in week 1 and significantly reduced body weight until their termination. Females of the 3000 ppm dose group had significantly decreased body weight starting from day 20 of gestation until termination.

Food consumption was significantly reduced at several time points in males starting from the 3000 ppm dose group. Females showed significantly lower food consumption at several time points starting from 3000 ppm in the premating interval. Significantly lower food consumption was also observed in the female animals during the whole gestation period and the entire lactation period (≥ 1500 ppm). A number of organs showed significant reductions in absolute weight (brain, pituitary, epididymides, seminal vesicles, prostate) and alterations in relative weight (liver, spleen, adrenals, seminal vesicle, prostate) in male rats. Females exhibited a significant increase in absolute and relative liver weights as well as significantly increased relative weights of the thymus and adrenals.

Estrous cyclicity was significantly increased in high dose dams. Dams of the 6000 ppm dose group showed increased length of gestation, reduced numbers of implantations and number of pups delivered and all pups died between lactation day 0 and 4. The number of implantations was also reduced in dams of the 3000 ppm dose group. Sex ratio and viability index at birth were not affected. The number of pups lost or found dead increased in a dose-dependent manner.

Both male and female pups had significantly decreased body weight at doses 1500 ppm, although body weight at birth was comparable. At necropsy male pups showed decrease absolute (3000 ppm) and relative (≥ 1500 ppm) weight of the brain of absolute thymus weight (≥ 1500 ppm) and absolute and relative weight of the spleen (≥ 1500 ppm). Female pups showed decreased absolute (3000 ppm) and relative (≥ 1500 ppm) weight of the brain of absolute thymus weight (≥ 3000 ppm) and absolute and relative weight of the spleen (≥ 1500 ppm) as well as a decrease of absolute uterus weight (3000 ppm).

Based on these findings doses of 100, 300, and 1000 ppm were chosen for the two-generation reproductive toxicity study (2013/8001785).

Supporting: One-generation reproductive toxicity 2012/1090369

The objective of this study was to compare the possible adverse effects of two batches (standard and high-purity batch) of the test substance on reproduction. The test substance was administered to groups of 25 male and 25 female healthy young Wistar rats (F0 parental generation) as a constant homogeneous addition to the food at concentrations of 0 and 1500 ppm. At least 75 days after the beginning of treatment, F0 animals were mated to produce a litter (F1 generation). Mating pairs were from the same dose group. The study was terminated with the terminal sacrifice of the F0 parental animals. Test diets containing containing the test substance were offered continuously throughout the study.

The overall mean dose administered to the male and female Wistar rats during the entire study period was approx. 154 mg/kg bw/d (standard batch) and approx. 156 mg/kg bw/d (high purity batch).

 

Under the conditions of the present one-generation study no substantial difference was observed between the two batches of the test substance when tested for effects on general systemic toxicity, fertility and reproductive performance, as well as developmental toxicity in pre-weaning F1 offspring. The tested dose of 1500 ppm caused systemic toxicity in the F0 parents (reduced food consumption, clinical-pathological changes) and a corresponding slight inhibition of preweaning pup development.

 

 

Supporting: Cross Fostering 2012/1016029

The test substance was administered to groups of 20 male and 20 female healthy young Wistar rats (F0 parental generation) during different study phases as a constant homogeneous addition to the food in different concentrations (0 and 1500 ppm). At least 76 days after the beginning of treatment, F0 animals were mated to produce a litter (F1 generation). Mating pairs were from the same dose group. As information was sought on whether exposure of the offspring during in utero and/or lactational windows resulted in neonatal morbidity or mortality, litters of unexposed dams were cross-fostered with litters of dams which were exposed to the test substance during these specific periods of reproduction. The diets containing the test substance were offered continuously throughout the different study phases.

The overall mean dose administered to the male and female Wistar rats during the entire study period was approx. 156 mg/kg bw/d in the 1500 ppm dose groups. A supplementary control group was added to this study to clarify the decreased viability index in cross-fostered pups after cesarean section. For this purpose, 50 time-mated animals of the same rat strain and source were delivered on gestation day 0 (GD 0) aged 10-12 weeks. The animals remained untreated. The study was terminated with the terminal sacrifice of control weanlings and control parental females.

 

Under the conditions of the present cross-fostering reproduction toxicity study the NOAEL for general, systemic toxicity is below 1500 ppm for the F0 parental rats, based on decreased food consumption observed at 1500 ppm, particularly during lactation in the F0 parental female animals.

 

The NOAEL for fertility and reproductive performance for the F0 parental rats is at least 1500 ppm, the highest dose tested. Postnatal survival and viability was unaffected by treatment.

 

The NOAEL for developmental toxicity in the F1 progeny is below 1500 ppm, due to the reduced pup body weight parameters in animals exposed during lactation. Importantly, no developmental toxicity occurred in the offspring fostered, which were exposed in utero, but not during lactation. In addition, no difference was detected between the developmental effects in the pups fostered in groups exposed postnatal via the milk and postnatal via milk after dams were pre-treated prenatally. Taken together, the study demonstrates that there are no direct or indirect consequences to the offspring of dosing during the premating or gestation periods and that the effect on pups is clearly a post-natal effect that occurs during the lactation period.

Effects on developmental toxicity

Description of key information

Multiple studies were conducted. In the key rabbit developmental study (2011/8000161) there were no adverse effects from the test substance in maternal animals at any of the doses tested (0, 8, 16, 32 mg/kg bw/d). Additionally, there were no treatment-related adverse effects on fetuses at any of the doses tested. Under the conditions of this study, both the maternal and fetal NOAEL was determined to be 32 mg/kg bw/d. In the key rat developmental study (2014/8000288) the test substance was administered orally, via gavage, at a dosage of 0, 50, 100, or 200 mg/kg bw/d. The dose level of 200 mg/kg bw/d produced overt maternal toxicity, as evidenced by body weight loss and one death. The dose level of 100 mg/kg bw/d also produced a slight but statistically significant reduction in food consumption during gestation days 6 -9. For fetal parameters, treatment-related effects were limited to a statistically significant increase in the litter incidences of several skeletal variations as supernumerary rib and zygomatic bone fused with maxilla in the 200 mg/kg bw/d group. These findings were only observed at a maternally toxic dose level that exceeds a kinetically derived maximum tolerated dose. The NOAEL for maternal and fetal (developmental) toxicity was 50 and 100 mg/kg bw/d, respectively.

On the basis of the result from the availabe developmental toxicity studis it is concluded that the test substance did not cause malformations in the rabbit or the rat, and therefore is not considered teratogenic.

A separate whitepaper summarizing some of the particular findings in the rat and rabbit developmental toxicity studies is available and attached in IUCLID Chapter 13.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
100 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
GLP and guideline compliant
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

Key study (rat): Developmental toxicity 2014/8000288

A teratogenicity study in rats was conducted to evaluate the potential maternal toxicity and prenatal developmental toxicity of the test substance. The test substance was administered orally, via gavage, to groups of 22-24 pregnant female Wistar Hannover (BrlHan:WIST@Jcl[GALAS]) rats once per day from days 6 to 19 of gestation at a dosage of 0, 50, 100, or 200 mg/kg/day.

The dose level of 200 mg/kg/day produced overt maternal toxicity, as evidenced by body weight loss in some individual females during gestation days 6-9, a statistically significant decrease in mean body weights on gestation day 9, and mild to moderate reductions in food consumption during gestation days 6 through 15. One treatment-related death occurred in the 200 mg/kg group. The necropsy of the dead animal revealed red-colored ileum as well as lung congestion. The dose level of 100 mg/kg/day also produced a slight but statistically significant reduction in food consumption during gestation days 6-9. There was no treatment-related change in maternal parameters examined in the 50 mg/kg group.

Examination of ovary and uterus revealed no treatment-related effects on gravid uterine weights, the numbers of corpora lutea and implantation sites, or percent pre- and post-implantation losses in any treated group.

For fetal parameters, treatment-related effects were limited to a statistically significant increase in the litter incidences of such skeletal variations as supernumerary rib and zygomatic bone fused with maxilla in the 200 mg/kg group. No other statistically significant change was found in any fetal parameters examined in the 200 mg/kg group or other treated groups. No evidence of an increased incidence of particular malformations was detected in any treated groups through external, visceral or skeletal examination. Only sporadic malformations were observed.

Based on the results of present study, it is concluded that a dose level of 50 mg/kg bw/day of the test substance is the no-observed-adverse-effect-level (NOAEL) for maternal toxicity, based on reduced food consumption and/or body weight gain at 100 mg/kg bw/day and above. The NOAEL for developmental toxicity is 100 mg/kg bw/day of the test substance, based on a slightly increased incidence of skeletal variations at 200 mg/kg bw/d. No fetal findings were evident at any dose level that did not cause maternal toxicity. The test substance is not teratogenic under the conditions of this study.

Key study (rabbit): Developmental toxicity 2011/8000161

Female rabbits were artificially inseminated and dosed with 0, 8, 16 or 32 mg/kg bw/d of the test substance from day 6-27 of gestation. Rabbits were necropsied on gestation day 28 and subject to macroscopic examination and assessment of fetal external, visceral and skeletal abnormalities.

There were no adverse effects from the test substance in maternal animals at any of the doses tested. Additionally, there were no treatment-related adverse effects on fetuses at any of the doses tested.

Under the conditions of this study, both the maternal and fetal NOAEL was determined to be 32 mg/kg/day.

Supporting study (rat): Developmental toxicity 2013/8001786

The purpose of this study was to provide information for the selection of dosages to be used in a later study of developmental toxicity in the rat (embryo-fetal toxicity and teratogenic potential).

The test substance was administered orally via gavage to BrlHan:WIST@Jcl(GALAS) rats. There were 8 mated females per group which were dosed from Days 6 through 19 of gestation at dose levels of 0, 20, 100, 500 and 1000 mg/kg/day.

On the basis of these results, oral administration of the test substance at doses of 500 mg/kg/day and more was not considered suitable for the main study because approximately half of the maternal rats died or were euthanized due to moribundity during the dosing period, bodyweight and food consumption were reduced and there were histological changes in the heart and liver.

Absolute and relative weights of the liver and absolute weight of the heart, both of which are target organs in general toxicity studies, were significantly increased in the maternal rats at a dose of 100 mg/kg/day, although there were no corresponding histopathological changes.

Consequently, the test substance, administered at doses of approximately 100 mg/kg/day, was suggested to be suitable as the highest dose level in the main study.

Supporting study (rat): Developmental toxicity 2013/8001787

Female rats were mated (1:1) and dosed with 0, 10, 30 or 100 mg/kg bw/d from day 6-19 of gestation. Rats were necropsied on gestation day 20 and subject to macroscopic examination and assessment of fetal visceral and skeletal abnormalities.

The maternal NOAEL is set at 30 mg/kg bw/d based on transiently lowered bodyweight gains and food consumption and increased adrenal weight. The NOAEL for developmental effects is set conservatively at 30 mg/kg bw/d based on an increased incidence of lumbar ribs (a skeletal variation). Since no irrversible stuctural effects occurred the dose of 30 mg/kg bw/d repesents rather a NOEL than a NOAEL and the true NOAEL for developmental effects of the study can be considered to be 100 mg/kg bw/d.

Supporting study (rabbit): Developmental toxicity 2011/8000281

This study was conducted to select dose levels of the test substance prior to the initiation of a teratogenicity study in rabbits. The test substance was administered orally by gavage to pregnant female Japanese White (Kbl:JW) rabbits, 8 per group, once a day on days 6 through 27 of gestation at a dosage of 0, 10, 30, 100, or 300 mg/kg/day to evaluate the potential effects on maternal animals and their fetuses.

In the 10 mg/kg dose group, no effects on maternal animals or on fetal abnormalities or alterations were reported. In the 30 mg/kg dose group, body weights of maternal animals were lower and there were significantly lower values reported for body weight gain and mean food consumption. At 30 mg/kg, there were also slight increases reported for mean percent resorptions and fetal death. Fetal weight of females and placental weights were slightly lower at 30mg/kg when compared to controls.

At doses of 100 mg/kg and higher, there was a decrease in body weight and food consumption. There was also maternal death, abortion and premature delivery at the higher doses, which resulted in an insufficient number of live fetuses for evaluation following cesarean section.

Based on these results, a dose level of 30-40 mg/kg/day is recommended for the high-dose level in a definitive teratogenicity study in rabbits, which is expected to get enough live fetuses for teratological evaluation without resulting in critical effects on maternal pregnancy.

NOAELs were not established by this study.

Toxicity to reproduction: other studies

Description of key information

No study available

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008.

No test substance specific adverse effects were seen on fertility, reproductive performance and developmental toxicty which would justify a classification according to Regulation (EC) 1272/2008. The effects observed on pup development in the generational studies were post-natal effects that occurred during lactation at doses exceeding the kinetic MTD. They are considered non-relevant for classification.

In summary, the substance is not considered to be classified for toxicity to reproduction under Regulation (EC) No 1272/2008, as amended for the eighth time in Regulation (EU) No 2016/918.

Additional information