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Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
1 000 mg/kg bw/day
Additional information

Information is available from two oral reproductive toxicity studies performed using treatment levels up to 1000 mg/kg bw/day. A good level of agreement was obtained for the parental and reproductive NOAELs obtained from these investigations.

A two-generation reproductive toxicity study by oral route in Sprague-Dawley rats, performed according to OECD Guideline 416 and under GLP (Centre International de Toxicologie, 2004a), did not show any effect on mating, fertility, gestation, fecundity or delivery or sperm parameters. No effects were observed on the progeny from delivery until weaning. The NOAEL for adult toxicity was 250 mg/kg bw/day based on a statistically significant reduction in body weight gain in F0 males, an increased absolute and relative kidney weight in F0 and F1 males and increased absolute and relative liver weights in F1 males at 500 mg/kg bw/day.

In a GLP compliant one generation study (OECD Guideline 415), increased parental adrenal and liver weights, but no alteration in reproductive or litter parameters, were apparent in male and female rats given ETBE by oral gavage at doses up 1000 mg/kg bw/day for 16 weeks (Safety Research Institute for Chemical Compounds, 2008; Fujii et al., 2010). There were no effects on fertility, however pup viability was slightly reduced during early lactation in the high dose group, with total litter loss noted in 2 moribund dams that were sacrificed on PND 2 and PND 4 while a third dam exhibiting no clinical signs had lost all pups by PND 5. This resulted in statistically non-significant reductions in pup viability on PND 4 and PND 21. When discussing these findings, the investigators comment that the loss of these litters occurred because the dams failed to take proper care of their pups (Safety Research Institute for Chemical Compounds, 2008), a position reiterated by Fujii et al. (2010) who "suggest that ETBE secondarily affects pup viability when overt systemic toxicities appear on (sic) sensitive dams at the dose level of 1000 mg/kg-d". The authors considered the findings to be biologically relevant since they were outside the historic range for total litter loss for this strain of rat in the laboratory conducting the investigation. However, as noted by de Peyster (2010), the large standard deviations associated with these results suggest that findings from this handful of animals had influenced the group mean, with compromised maternal condition in a small number of dams (rather than a more generalised, direct effect of ETBE on F1 pup viability) considered the likely cause. Overall, therefore, the NOAEL of 300 mg/kg bw/day for effects on the F1 generation assigned to this study appears conservative.

Information on the reproductive toxicity of ETBE summarised in this dossier has been the subject of a published expert review (de Peyster, 2010) which concluded that there was no evidence of adverse effects on male or female gamete production and transport, female oestrous cycle, sexual behaviour, fertility, gestation length, or parturition when ETBE was evaluated specifically for effects on mating and fertility in male and female rats at dose levels up to 1000 mg/kg bw/day. While one sub-chronic toxicity investigation (Chemical Industry Institute of Toxicology, 1996a) reported some evidence of spermatocyte degeneration in F344 rats exposed to 1750 or 5000 ppm ETBE by inhalation for 13 weeks, the testes were preserved using neutral buffered formalin (a procedure that can lead to structural changes during fixation) and not Bouin’s fixative (the preferred option which readily penetrates the tunica albuginea that encapsulates the testis). Such technical differences may explain, at least in part, why findings of increased percentages of seminiferous tubules exhibiting slight spermatocyte degeneration by have not been replicated elsewhere.

Overall, these investigations demonstrate that ETBE has no adverse effects on reproduction when administered by oral gavage at doses up to 1000 mg/kg bw/day over two generations.

Short description of key information:
ETBE did not cause effects on fertility in a two-generation study. The NOAEL for adult toxicity was 250 mg/kg bw/day based on body weight and absolute/relative organ (liver and kidney) weight changes at higher treatment levels. The NOAEL for reproductive effects was 1000 mg/kg bw/day, the highest dose tested.

Effects on developmental toxicity

Description of key information
ETBE did not cause developmental toxicity. The NOAEL for maternal toxicity was 300-500 mg/kg bw/day (decreased maternal body weight gain) and 1000 mg/kg bw/day (the highest dose tested) for developmental toxicity. No treatment-related malformations were observed.
Additional information

Prenatal developmental toxicity by the oral route was studied in Sprague-Dawley rats and New Zealand White rabbits. The studies were performed in agreement with OECD Guideline 414 and under GLP.

No change in foetal viability, foetal weights, placental weights or any other reported parameters were noted following gavage administration of ETBE to pregnant SD rats on GD 5-19 (Centre International de Toxicologie, 2004b). In general foetal skeletal findings were recorded in all groups without any dose-relationship and/or statistical significance and/or occurred at incidences consistent with historical control background data. The single exception was a significant increase in the incidence of unossified 4th metacarpal at the highest dose; however, when expressed on a foetus/litter basis, it was not statistically significant and within historical control data. Cartilage was generally present suggesting that this was due to slightly delayed ossification rather than to a persistent alteration. In addition, the findings were not associated with any significant delay in ossification of other bones and the total incidence of skeletal variations in treated animals did not increase. Therefore, the effect was concluded not to be of toxicological relevance. The NOAEL for developmental toxicity was 1000 mg/kg bw/day (the highest dose tested) and the NOAEL for maternal toxicity was 500 mg/kg bw/day (decreased maternal body weight gain).

In a second rat oral gavage study (Chemicals Evaluation and Research Institute, 2008b), maternal performance and necropsy findings were unremarkable, although salivation was noted immediately after dosing in all groups (most common in the high dose group). Cesarean section data revealed that the number of corpora lutea, implantations, ovary weights, uterus weights, number of live foetuses, sex ratio and foetal body weights were comparable, irrespective of treatment status. There were no significant differences in the incidence of visceral or skeletal malformations or variations, although the incidence of lumbar rib (a variation) was significantly increased at 1000 mg/kg bw/day. This finding was considered developmentally insignificant as it is transient (i.e. disappears soon after birth: Chernoff et al., 1991; Foulon et al., 2000) and the incidence (19% of foetuses affected versus 2.9% of controls) was within the historical range for this strain of rat . The maternal and foetal NOAELs from this study were therefore 1000 mg/kg bw/day.

Chernoff, N, Rogers, JM, Turner, CI and Francis, BM (1991). Significance of supernumerary ribs in rodent developmental toxicity studies: Postnatal persistence in rats and mice. Fund. Appl. Toxicol. 17, 448-453

Foulon, O, Jaussely, C, Repetto, M, Urtizberea M and Blacker, AM (2000). Postnatal evolution of supernumerary ribs in rats after a single administration of sodium salicylate. J Appl. Toxicol. 20, 205-209.

In an oral gavage developmental toxicity study using New Zealand rabbits (Bozo Research Center Inc., 2008; Asano et al., 2011), food intake was reduced up to GD 14 by approx. 25-30% in dams given 1000 mg ETBE/kg bw/day, and body weight gain decreased by around one half on GD 2-28, however caesarean section data (i.e. the number of corpora lutea and implantations, uterine weight, number of live foetuses, sex ratio, foetal body weight, and an absence of external malformations) were comparable to that of the controls. There were no differences in the incidence of visceral or skeletal malformations or variations, with any findings present occurring in control and treated groups with no obvious dose relationship present. The maternal and developmental NOAELs from this study were 300 and 1000 mg/kg bw/day, respectively.

Information on the developmental toxicity of ETBE summarised in this dossier has been the subject of a published expert review (de Peyster, 2010) which concluded that the available data indicate that ETBE does not appear to be selectively toxic to the foetus in the absence of other manifestations of general toxicity. This was based, in part, upon results from the rat and rabbit studies discussed above together with evidence showing that tert-butanol (a primary metabolite of ETBE) and MTBE (a structural homologue) revealed no appreciable effects on embryofoetal development in rats and rabbits at non-maternally toxic doses. Reductions in survival (Safety Research Institute for Chemical Compounds, 2008) or increased pup mortality (Centre International de Toxicologie, 2004b) recorded in the available one- and two-generation reproduction studies, respectively, were linked to the occurrence of total litter loss affecting a small number of dams and appeared secondary to maternal toxicity, although the possibility of effects linked to exposure through maternal milk could not be excluded completely.

Overall, these investigations demonstrate that ETBE has no adverse effects in two species when administered by oral gavage at doses up to 1000 mg/kg bw/day during pregnancy.

Justification for classification or non-classification

In accordance to Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for effects on fertility or developmental toxicity.

Additional information