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Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

A two-generation reproductive toxicity study is available on C12 AO. It was conducted according to Japan Ministry of Health and Welfare guideline and is comparable to the OECD 416 study design [Tesh JM (1983)]. The study was fully quality assured by the GLP certified testing facility where it was conducted.

Administration of the test substance continuously in the diet over two generations, at levels of up to 750 ppm, had no adverse effect on the general condition of male and female rats, but was associated with slight reductions in rate of bodyweight gain during maturation and gestation at levels of 375 ppm and above.

Mating performance, fertility, gestation and parturition were unaffected by treatment. Litter size at birth and viability of offspring up to weaning showed no consistent treatment-related effects.

At terminal necropsy of parents and offspring of both generations some variations in organ weights were found, but these were largely related to the deficits in bodyweight and no macroscopic or microscopic abnormalities were detected that could be related to ingestion of the substance.

The study report concludes no effects on fertility with a high dose NOAEL of 750 pmm in diet. Based on chemical intake data provided for the study, 750 ppm in diet translates to a dose range of ≥ 37 -128 mg/kg/day in male and female animals.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
37 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Reliability 1 for the 2-generation study.
Reliability 1 for the entire database.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In addition to the key study (i.e. the two-generation reproductive toxicity study) on C12 AO, there are combined repeat dose toxicity and reproductive developmental toxicity screening studies, conducted under GLP according to OECD TG 422, available on category members C12-14 AO and C12-18 AO.

In the C12-14 AO study the substance was administered via gavage to 10 animals/sex/group at 0, 40, 100 or 250 mg AO/kg bw/day [Ceccatelli et al (2008)]. Males were dosed for at least 14 days prior to mating and during 14 days pairing and female rats for 14 days prior to pairing, through the pairing and gestation period until the offspring reached Day 4 post-partum. No adverse effects on fertility were noted at any dose level. Litter size and mean number of pups at first litter check and sex ratio were not affected by treatment. No abnormal pups were noted at any dose level. However, a statistically significant increase in pup deaths was observed on postnatal days 0 -4 at the high dose of 250 mg AO/kg/day. Although the higher postnatal loss was within the range of historical control data, this resulted in a reduced number of pups and mean pup weight during development was reduced at this dose. The NOEL for reproductive/developmental toxicity in this study was 100 mg AO/kg bw/day.

In the C12-18 AO study the substance was administered via gavage to 10 rats/sex/group at 0, 40, 100 or 200 mg AO/kg bw/day [Hansen B (2010)]. Males were dosed for two weeks prior to mating, during mating and approximately two weeks after mating. Females were dosed for two weeks prior to mating and continuing up to and including day 3 post-partum or the day prior to sacrifice. No adverse effects on fertility were noted at any dose level. Litter size and mean number of pups at first litter check and sex ratio were not affected by treatment. No abnormal pups were noted at any dose level. However, treatment with 200 mg AO/kg bw/day resulted in a statistically significant increase in pre-implantation loss compared to controls. Post implantation loss was not influenced at any dose level. The NOAEL for reproductive/developmental toxicity in this study was 100 mg AO/kg bw/day.

Effects on developmental toxicity

Description of key information

A prenatal developmental toxicity study is available on category member C12-14 AO. The study was performed under GLP according to EPA OTS 798.4900 test guideline [York RG (1999)]. In the study presumed pregnant female rats (Crl:CDBR VAF/Plus; 25 animals/group) were dosed by oral gavage with 0, 25, 100 or 200 mg AO/kg bw/day on days 6 through 19 of presumed gestation. 

Two rats in the 200 mg AO/kg bw/day dosage group died; one of these deaths was attributed to the test substance, the other was the result of an intubation error. All other rats survived until scheduled sacrifice. Excessive salivation, rales, urine-stained abdominal fur, brown or red perioral substance, laboured breathing and gasping was occurred in 100 mg AO/kg bw/day dose groups. All necropsy observations were considered unrelated to treatment with the test substance.

Rats in the 100 mg AO/kg bw/day dose groups had significantly reduced body weight gains for the entire dosage period (calculated as days 6 to 20 of gestation) and body weight gains were significantly reduced in the 200 mg AO/kg bw/day dose group for the entire gestation period (DGs 0 to 20). The gravid uterine weight and the corrected DG 20 maternal body weight (DG 20 body weight minus the gravid uterine weight) were significantly reduced in the 200 mg AO/kg bw/day dosage group.

Absolute and relative feed consumption values were significantly reduced in the 200 mg AO/kg bw/day dose group. Relative feed consumption values were also significantly reduced in the 100 mg AO/kg bw/day dose group for the entire doseage period, while values for the gestation period were significantly reduced in all treatment groups.

Fetal body weights were significantly reduced in the 200 mg AO/kg bw/day dosage group. Live litter size was decreased, and the number of early resorptions was increased in the 200 mg AO/kg bw/day dosage group, but primarily as the result of one dam in this dose group that had a litter consisting of 16 early resorptions.

The percentages of fetuses and litters with alterations in the 200 mg AO/kg bw/day dosage group were significantly increased and reflected delays in skeletal ossification related to the significantly reduced fetal body weights in this group. Similar but lower incidence and less severe delays in ossification also occurred in the 100 mg AO/kg bw/day dose group. The NOAELs for both maternal toxicity and developmental toxicity in this study are 25 mg AO/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
25 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Reliability 1 for the rat and rabbit developmental toxicity studies.
Reliability 1 for the database..
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In addition to the key study, there are several supporting studies to establish developmental toxicity potential.

In a combined repeat dose toxicity study with the reproduction/developmental toxicity screening test conducted with C12-14 AO under GLP according to OECD TG 422 the substance was administered via gavage to 10 animals/sex/group at 0, 40, 100 or 250 mg AO/kg bw/day [Ceccatelli et al (2008)]. Males were dosed for at least 14 days prior to mating and during 14 days pairing and female rats for 14 days prior to pairing, through the pairing and gestation period until the offspring reached Day 4 post partum. At doses of ≥100 mg AO/kg bw/day, reduced activity, body weight gain and food consumption were noted in males and/or females. Doses of 250 mg AO/kg bw/day also resulted in increased liver weights and microscopic changes in liver and kidney. Forestomach lesions were observed at both 100 and 250 mg AO/kg bw/day. The parental toxicity NOAEL was 40 mg AO/kg bw/day. Evaluation of reproductive and developmental parameters demonstrated that litter size and mean number of pups at first litter check were not affected by treatment. The sex ratio was also not affected by treatment. No abnormal pups were noted at any dose level. However, at 250 mg AO/kg bw/day a statistically significant increase in pup death was observed on postnatal days 0-4. Although the higher postnatal loss was in the range of historical control data, this resulted in a reduced number of pups. Mean pup weight development was reduced at 250 mg AO/kg bw/day. At necropsy, there were no findings in pups. The NOEL for reproduction/developmental toxicity was 100 mg AO/kg bw/day.

In a rat two-generation reproductive toxicity study with a 30% aqueous solution of C12 AO conducted according to Japan Ministry of Health and Welfare guidelines (comparable to the OECD 416 study design) and fully quality assured by the GLP certified testing facility, the test substance was administered continuously in the diet over two generations at levels of up to 750 ppm [Tesh JM (1993); see Table 25]. The test substance had no adverse effect on the general condition of male and female rats but was associated with slight reductions in rate of bodyweight gain during maturation and gestation at levels of 375 ppm and above. Litter size at birth and viability of offspring up to weaning showed no consistent treatment-related effects. On Day 25 post-partum only, there was a dosage-related reduction in the rate of bodyweight gain of F1 and F2 offspring at 375 and 750 ppm. Thereafter there were no demonstrable effects on body weights. At terminal necropsy of parents and offspring of both generations some variations in organ weights were found, but these were largely related to the deficits in bodyweight and no macroscopic or microscopic abnormalities were detected that could be related to ingestion of the substance. The study report concludes no effects on development at up to the high dose NOAEL of 750 ppm in diet. Based on chemical intake data provided for the study, this translates to a dose range of ≥ 37 -128 mg AO/kg/day in males and 47 – 119 mg AO/kg bw/day in females.

In a prenatal developmental toxicity study with a 30% aqueous solution of C12 AO conducted in a GLP-certified laboratory according to Japan Ministry of Health and Welfare guidelines (similar to OECD TG 414), doses of 50, 100 or 200 mg/kg bw/day(equivalent to 15, 30 or 60 mg AO/kg bw/day)were administered by gavage to pregnant Sprague-Dawley rats on days 7 to 17 of gestation[Tesh JM (1980)]. Each group consisted of 32 pregnant animals: 21 per group were sacrificed on gestation day 20 and their fetuses were examined for morphological development. The remaining 11 females per group were allowed to give birth and their offspring were evaluated for viability, growth, and the attainment of a number of developmental milestones as well as neurobehavioral effects and fertility. Treatment of the F1 dams was continued through weaning, but the F1 animals were not subsequently dosed. Maternal toxicity in the form of decreased body weight and food water consumption was observed in the 200 mg/kg bw/day group. This group also had an increase in water consumption. There were no effects observed at the two lower dose levels. Developmental effects consisting of decreased fetal weight and delayed ossification of some skeletal elements were observed in the 200 mg/kg bw/day group. There were no effects on growth, viability, behavior, or fertility in the F1 litters in any dose group. In summary, the 200 mg/kg bw/day group produced maternal toxicity and associated developmental effects; 100 mg/kg bw/day was a clear no observed adverse effect level. Therefore, the LOAEL for maternal and developmental effects was 200 mg/kg bw/day (equivalent to 60 mg AO/kg bw/day) and the NOAEL was 100 mg/kg bw/day (equivalent to 30 mg AO/kg bw/day)

In a combined repeat dose toxicity study with the reproduction/developmental toxicity screening test conducted with C12-18 AO under GLP according to OECD TG 422 the substance was administered via gavage to 10 rats/sex/group at 0, 40, 100 or 200 mg AO/kg bw/day [Hansen B (2010)]. Males were dosed for two weeks prior to mating, during mating and approximately two weeks after mating. Females were dosed for two weeks prior to mating and continuing up to and including day 3 post-partum or the day prior to sacrifice. A satellite group of non-mated animals was treated in the same manner as the main study animals and kept without treatment at least 14 days after the first scheduled sacrifice of main study females. No test item related mortality or influence on body weight was noted in any group. No signs of systemic toxicity were noted for the male and female animals treated with 40 mg AO/kg bw/day. Effects noted were as follows: At ≥100 mg AO/kg bw/day, Increased salivation; dose dependent increase of macrophages with vacuolisation in the mesenteric lymph nodes. This effect was still noted at the end of the recovery period. At 200 mg/kg bw/day, periodic reduction in food intake (males & females); periodic reduction in food intake; laboured breathing (one male) piloerection (females); changes in haematological parameters; effects in the forestomach - squamous cell hyperplasia with submucosal inflammatory reaction and hyperkeratosis/parakeratosis in the stratum corneum (males and females). These lesions were reversible within the 16-day recovery period. The NOEL for parental toxicity from this study was 40 mg AO/kg bw/day.

No test item related influence was noted on the female fertility index or pre-coital time at any of the tested dose levels. There were no test item related differences in the number of corpora lutea, implantation sites, in the number and sex of pups, runts or malformed pups. No test item related influence was noted in the values calculated for the gestation length, the gestation index, the birth index and the live birth index between the control group and animals in any test group. No test item related increase in pre-implantation loss was noted in the dams after treatment with 40 or 100 mg AO/kg bw/day throughout the study up to day 3 post-partum. Treatment with 200 mg AO/kg bw/day resulted in a statistically significant increase in pre-implantation loss compared to controls. Post implantation loss was not influenced at any dose level. In pups no test item related effects were noted for mortality, abnormal behaviour, mean and total litter weight or external abnormalities at any of the doses tested. Due to the pre-implantation loss noted at the high dose of 200 mg AO/kg bw/day the NOAEL for reproduction/developmental toxicity was 100 mg AO/kg bw/day. 

Toxicity to reproduction: other studies

Additional information

No other studies concerning toxicity to reproduction are currently available.

It should be noted that in repeated dose studies with category member C12 -14 AO treatment-related ocular changes were noted.  In the two-generation reproductive toxicity study with C12 AO, there were no treatment-related observations of ocular problems noted in any of the three generations (no effects noted clinically or in histopathology). One observation of a cataract was noted in a low-dose adult animal in the F1 generation by histopathology.  This single instance was not dose-related and therefore not treatment-related.  Given the high spontaneous rate of cataracts in albino rats (Durand et al. 2001), it is not surprising to see a single observation of a cataract in a study in which a large number of animals were evaluated.  It is also worth noting that the observation of a cataract provides evidence of the laboratory’s ability to detect cataract by histopathology.

1) Durand, G et al. Spontaneous polar anterior subcapsular lenticular opacity in Sprague-Dawley rats.  Comp Med. 2001 Apr;51(2):176-179.

Justification for classification or non-classification

In a two-generation reproduction study in the rat with the category member C12 AO administered in the diet, P0 parental toxicity was evident (moderately impaired body weight performance) at 750 ppm (70 mg/kg/ bw/day). There were no adverse effects of treatment on oestrous cyclicity, mating performance, fertility, gestation, parturition or development of the offspring through two successive generations. In addition, in a combined repeat dose and reproductive and developmental toxicity screening study in the rat with C12 -14 AO administered by gavage, parental toxicity was evident at doses 100 mg/kg/day. No adverse effects of treatment were noted on mating performance, fertility, gestation or parturition. Postnatal effects related to pup mortality and mean body weight at the highest dose of 250 mg/kg/day were associated with frank parental toxicity and were within historical control range, thus not indicative of direct reproductive toxicity. Based on these two reproductive toxicity studies, no classification for reproductive toxicity is warranted.

In a prenatal developmental toxicity study in rats and in a combined repeat dose and reproduction/developmental toxicity screening study in rats with C12-14 AO, maternal toxicity was observed at doses ≥ 100 mg/kg/day. Developmental effects secondary to maternal toxicity, including delayed ossification and reduced pup weights, were also observed at these doses. In another prenatal developmental toxicity study with the category member C12 AO, maternal toxicity and similar secondary developmental effects were observed at the high dose of 60 mg/kg/day active. However, in a prenatal developmental toxicity study in rabbits with C12 AO at doses up to 48 mg/kg/day, no maternal or developmental effects were observed. And in a two-generation reproductive toxicity study in rats at doses of up to 37 -128 mg/kg/day, no treatment-related effects on litter size or viability of offspring were noted in the presence of slight body weight reductions in the parental animals at this dose. Based on the results in the prenatal developmental toxicity studies on C12 AO and C12 -14 AO, no classification for developmental toxicity is warranted.

Additional information