A mixture of: α-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyl-ω-hydroxypoly(oxyethylene); α-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyl-ω-3-(3-(2H-benzotriazol-2-yl)-5-tert-butyl-4-hydroxyphenyl)propionyloxypoly(oxyethylene)

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Species:

rat

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 a one-generation study conducted according to OECD guideline 415 (1983) doses of 2, 50 and 100 mg/kg bw/d were applied to rats via gavage (Ciba-Geigy Ltd 1991). The males were treated for 16 weeks including 70 days premating, 21 day mating; treatment was continued until sacrifice. Females were treated for 11 weeks including 14 day premating, 21 day mating and through gestation and lactation (day 21 post parturition).

Since no effects on insemination, fertility, duration required until mating and reproductive organs were seen, the NOAEL for reproduction toxicity for rats of both sexes is equal to or greater than 100 mg/kg bw/d.

Referring to systemic toxicity, changes in body weight and body weight gain, food consumption, as well as changes in liver and kidney with histopathological correlates were seen. Thus, the LOAEL for systemic toxicity was 50 mg/kg bw/d for both sexes; the NOAEL was 2 mg/kg bw/d.

Referring to offspring, increase in the number of stillborn pups and consequently decreased live birth index, and occurrence of dark abdominal skin partly associated with tail necrosis led to a LOAEL of 50 mg/kg bw/d; the NOAEL was 2 mg/kg bw/d. At the high dose group, 10% of the pups suffered from dark abdominal skin and the perinatal loss was 7% versus 0% in the control group. At the mid dose, perinatal loss was 3% and 0.6% of the pups showed dark abdominal skin.

Since there was no treatment related increase in postnatal pup mortality after day 4 and since body weight gain of the pups was only slightly affected during the lactation period at 100 mg/kg bw/d, it was concluded that the effects of the substance were mainly due to prenatal exposure and not due to exposure via lactation.

 

 

The preceeding range finding study was conducted with doses of 0, 20, 100 and 200 mg/kg bw/d of test substance (Ciba-Geigy Ltd 1990). Female SD-rats were treated one week before and throughout mating up to day 14 post partum (42 days) and males were treated one week before mating up to the end of the mating period of 12 days (19 days). No effects on the pregnancy rate, the days until successful mating and the number of pregnant females in any dose group were seen.

All pregnant females of the high dose group of 200 mg/kg bw which gave birth had solely stillborn pups. At 100 mg/kg bw/d the mean number of live born pups was significantly decreased while the mean number of pups dying or cannibalized between days 0 and 4 post partum was significantly increased. Since no effects on pup parameters were seen at 20 mg/kg bw/d this concentration was the NO(A)EL.

Referring to maternal toxicity, at 200 mg/kg bw/d reduced mean absolute body weight and mean body weight changes during gestation and lactation period were seen. Thus, the LOAEL was 200 mg/kg bw/d. The NOAEL was established at 100 mg/kg bw/d.

 

In biochemical and (ultra)morphological investigations of liver samples of males, dams and fetuses as well as whole fetuses the reduced live birth index and dark abdominal skin was correlated to hepatotoxic effects which are attributable to peroxisome proliferation (see IUCLID chapter 7.9.3: specific investigation). Since peroxisome proliferation is known to be a rodent specific phenomenon these effects were considered not to be relevant for classification.

No effects on insemination, fertility, duration until successful mating and on reproductive organs were seen in the one-generation study. Thus fertility was not affected by treatment with the test substance up to 200 mg/kg bw/d. Based on a reduced live birth index and dark abdominal skin the LOAEL for rat embryotoxicity was 50 mg/kg bw/d and the NOAEL was 2 mg/kg bw/d.

 

In biochemical and (ultra)morphological investigations of liver samples of males, dams and fetuses as well as whole fetuses the reduced live birth index and dark abdominal skin was correlated to hepatotoxic effects which are attributable to peroxisome proliferation (see IUCLID chapter 7.9.3: specific investigation).

Short description of key information:
In a one-generation study conducted according to OECD guideline 415 (1983) doses of 2, 50 and 100 mg/kg bw/d were applied to SD-rats via gavage (Ciba-Geigy 894538, 1991). The LOAEL for systemic toxicity and for embryotoxicity was 50 mg/kg bw/d for both sexes and the NOAEL was 2 mg/kg bw/d. No effects on fertility were seen upon treatment with the test substance up to the highest dose of 100 mg/kg bw/d. The substance causes peroxisome proliferation in parental and fetal liver as shown in specific investigations.

Effects on developmental toxicity

Description of key information

In a GLP-compliant prenatal developmental toxicity study conducted according to OECD guideline 414 (1981) with rats a minor developmental delay in ossification was seen at the highest tested dose of 150 mg/kg bw/d (Ciba-Geigy 924079, 1993). In the range-finder study, at this there was one fetus with dark skin of the abdomen and the hind limbs at this dose-level. Treatment of dams was done during organogenesis as required by the OECD testing guideline at that time. The delay in ossification was considered to be a consequence of maternal toxicity. Thus, the NOAEL was 30 and the LOAEL for developmental toxicity was 150 mg/kg bw/d. The substance caused peroxisome proliferation and related adverse effects on rat pups in special investigations that included gestation days 16 -20. 
A GLP-compliant prenatal developmental toxicity study in rabbits conducted according to OECD guideline 414 included the final days of gestation. No embryotoxicity and no teratogenicity was observed. A slight reduction in pup and litter weight occurred as a result of the marked maternal toxicity at the highest dose group of 100 mg/kg bw (Covance 2013b).

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Species:

rabbit

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

Experimental data on teratogenicity is available for rats and rabbits. For rats, several studies including mechanistic investigations are available because of the effects on offspring observed in the one-generation study and because the main teratogenicity study covered only organogenesis. For rabbits, the prenatal developmental toxicity study covers all stages until the end of gestation.

 

In the prenatal developmental toxicity study with rats conducted according to OECD guideline 414 (1981) doses of 1, 30 and 150 mg/kg bw/d were applied to pregnant SD-rats via gavage during gestation days 6 to 15. On day 21 of gestation, females were sacrificed and fetuses were examined for visceral and skeletal abnormalities. The treatment schedule covered only the phase of organogenesis as required in that version of the OECD guideline.

Referring to maternal toxicity, at 30 mg/kg bw/d a slightly reduced food consumption of females was seen. At 150 mg/kg bw/d, reduction in food consumption, body weight and body weight gain, as well as reduced mean carcass weight and net body weight gain from day 6 to 21 were seen. Since the reduced food consumption seen at 30 mg/kg bw/d did not represent an adverse effect, the NOAEL was set at this dose level. The LOAEL for maternal toxicity was 150 mg/kg bw/d.

 

Referring to effects on the offspring, a minor developmental delay in ossification was seen at 150 mg/kg bw/d only. However, this effect was considered to be a consequence of maternal toxicity. The NOAEL was 30 and the LOAEL was 150 mg/kg bw/d. No signs of teratogenicity were evident. Thus the NOAEL for teratogenicity in rats was 150 mg/kg bw/d.

 

The teratogenicity study in rats does not cover the exposure during the final gestation days (day 16 -20 for rats). During the one-generation study, adverse effects on the offspring were observed. As shown in special investigations, these effects are likely due to the exposure of dams during the final days of gestation.

Pregnant rats were treated during gestation days 6 to 14, 17 or 20 with a dose of 150 mg/kg bw (Ciba-Geigy Ltd 1994) and sacrificed at the end of treatment. It was shown that the test substance induces peroxisome proliferation in rat fetal liver and that this is concommittant with depletion of glycogen storage and oxidative stress in the fetus. Abdominal discoloration of fetuses wasobserved in single animals, and this is consistent with abdominal discoloration in 10% of the high dose group pups in the one-generation study.

Major treatment related findings in dams consisted of a moderate to striking hepatic peroxisome proliferation as evidenced by electron microscopy and biochemically confirmed by the moderate to strong induction of a number of diagnostic enzyme activities such as peroxisomal fatty acid beta-oxidation, lauric acid 11- and 12-hydroxylase, and catalase. These alterations occurred without any obvious consequences of toxicological significance for the hepatic glutathione status and the liver content of the lipid peroxidation product malondialdehyde. The activities of Se-dependent and Se-independent glutathione peroxidases remained unchanged. Inversely, fetal livers from treated dams experienced a striking peroxisome proliferation as early as day 15 of gestation. This alteration was identified by electron microscopy and confirmed by substantially to strongly induced activities of peroxisomal fatty acidb-oxidation and lauric acid 11- and 12-hydroxylation. In contrast to the respective dams, low constitutive expression and poor or even lacking inducibility of the protective fetal enzyme systems of catalase and glutathione peroxidases were regarded in charge of the observed decrease in total hepatic glutathione content and doubling of the liver malondialdehyde contents at day 21 of gestation. Histologically detectable subcutaneous and skeletal muscular haemorrhages in fetuses from treated dams after 18 and 21 days of gestation are presumed to be the result of oxidative endothelial cell lesions. Oxidative stress is suspected to be also the reason for the lack of glycogen accumulation in fetuses up to day 21 of gestation.

In follow up biochemical and (ultra)morphological investigations of liver samples of males, dams and fetuses as well as whole fetuses the embryotoxicity was correlated to hepatotoxic effects which are attributable to peroxisome proliferation (see IUCLID chapter 7.9.3: specific investigation). Since peroxisome proliferation is known as a rodent specific phenomenon these effects were not considered to relevant for classification and labeling when determining the legal classification in Annex I of 67/548/EEC.

In the prenatal developmental toxicity study with rabbits conducted according to OECD guideline 414 (2001) doses of 10, 30 and 100 mg/kg bw/d were applied to pregnant New Zealand White rabbits via gavage during gestation days 6 to 28 inclusive (Covance 2013). On day 29 of gestation, females were sacrificed and fetuses were examined for visceral and skeletal abnormalities. The doses were chosen based on the results of a dose-range-finding study with 5 pregnant rabbits per dose group (Covance 2014). The highest dose group caused marked toxicity (reduced food consumption and body weight gain) that was higher than expected from the results of the dose-range-finding study with 50, 150 and 300 mg/kg bw. Mean body weight gain at 100 mg/kg bw was reduced by 70% compared to the control group and animals lost weight during the first week of treatment. As a result eight rabbits of this dose group aborted their litters. Abortion is considered secondary to strongly reduced food consumption and body weight gain and is often observed with rabbits at excessive toxicity. Effects on food consumption and body weight gain were also seen at the mid dose and the low dose of 10 mg/kg bw is the NOEL for maternal toxicity. In surviving litters mean uterine/implantation data were within the expected range in all groups and yielded no evidence of a compound-related effect.

No indication of teratogenic or embryotoxic effects were seen at any dose group. Mean foetal weight and therefore mean litter weight were reduced compared to controls at 100 mg/kg/day (17% and 25%, respectively, both p <0.001) and this was considered to be as a result of the marked maternal toxicity seen at that dose level. Sex ratio and mean placental weight were unaffected by treatment. Abdominal discoloration was seen in one rabbit fetus of the high dose group and this single case is considered to be incidental.

Justification for selection of Effect on developmental toxicity: via oral route:
The study in rabbit is considered to be the most appropriate study since the substance is a potent peroxisome proliferator in rats.

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available one-generation study is considered reliable and suitable for classification purposes under 67/548/EEC.

Based on the available studies the substance would have been classified for reproduction toxicity according to Directive 67/548/EEC and Directive EC/1272/2008. However, in biochemical and (ultra)morphological investigations of liver samples of males, dams and fetuses as well as whole fetuses the embryotoxicity was correlated to hepatotoxic effects which are attributable to peroxisome proliferation (see IUCLID chapter 7.9.3: specific investigation).

As a result no classified for fertility was included in the legal classification (Index no. 607-176-00-3) under Directive 67/548/EEC, as amended for the 28th time in Directive 2001/59/EC.

The developmental toxicity studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for developmental toxicity under Directive 67/548/EEC, as amended for the 28th time in Directive 2001/59/EC.

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available one-generation study is reliable and suitable for classification purposes under Regulation 1272/2008.

Based on the available studies the substance would have been classified for reproduction toxicity according to Directive 67/548/EEC and Directive EC/1272/2008. However, in biochemical and (ultra)morphological investigations of liver samples of males, dams and fetuses as well as whole fetuses the embryotoxicity was correlated to hepatotoxic effects which are attributable to peroxisome proliferation (see IUCLID chapter 7.9.3: specific investigation). As a result no classification for reproductive toxicity was included in the legal classification (Index no. 607-176-00-3) of

Regulation (EC) No. 1272/2008, as amended for the second time in Directive EC 286/2011.

The available developmental studies is reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for developmental toxicity under Regulation (EC) No. 1272/2008, as amended for the third time in Directive (EC 2 618/2012).

 

In the one generation study, no effects via lactation were observed.