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Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no study available
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
28 560 mg/m³
Species:
rat
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

MTBE has been tested for effects on fertility in one- and two-generation studies in Sprague-Dawley rats. Reproductive function was not affected adversely and no pathological changes were observed during microscopic examination of gonad tissue from exposed animals. Out of these studies, the two-generation study was chosen as a key study (Bevan et al., 1997a).

In addition, no adverse microscopic changes were observed in the gonads in any of the sub-chronic or long-term toxicity studies.

In the one-generation study (Bio/dynamics, Inc, 1984), no effects on fertility and no parental toxicity were observed. Pup viability indices at birth were comparable for control and treated (250, 1000 and 2500 ppm (893, 3570 and 8925 mg/m3)) groups for the F1a generation, but there was a significant (p<0.05) decrease in pup viability indices at birth in the two highest dose groups of the F1b-litter. The viability percentage of the control group of the F1b litter was 99.0% while it was 97.6% for the F1a litter. This may have skewed the significance seen. Moreover, no adverse effect on the survival of the pups was seen in any dose group of the second litter. There was also a significant reduction in pup survival index in pre-cull days 0-4 at 250 ppm and 1000 ppm. Although this may be of toxicological significance, it should be noted that there was no statistically significant difference observed in the highest concentration group or in the parallel F1b-litter. In addition, there was no significant change in the survival indices during the lactation period (days 4-21).

In the two-generation study, there were general toxicity signs at 3000 and 8000 ppm (10710 and 28560 mg/m3) in both generations of parental animals (NOAEC parental toxicity: 400 ppm (893mg/m3)). No significant changes were reported regarding fertility even at the highest tested concentration, resulting in a NOAEC of 8000 ppm (28560 mg/m3) for effects on fertility (the highest concentration tested). Some postnatal toxicity (lowered body and body weight gains in both the F1 and F2 litters) was observed in the offspring of both generations (at 3000 and 8000 ppm), but only in the presence of maternal toxicity. In conclusion, the one- and two-generation studies show that MTBE did not cause effects on fertility.

New information from published literature does not change these conclusions.

Billitti et al. (1999) administered MTBE by oral gavage to CD-1 male mice at doses of 400, 1000 and 1500 mg/kg bw on days 1, 3 and 5 of a 7-day study. An abstract of this investigation initially reported a small but significant increase in gross disruption of the seminiferous tubules at the 1500 mg/kg bw dose. After de Peyster et al. (2008) had conducted a similar follow up experiment and found no effect up to 2000 mg/kg bw, Billitti et al. published their study with more details and a final conclusion of no MTBE treatment-related effects (Billitti et al. (2005)).

Studies by Almeida and Hall (2004) and Li et al. (2008) reported effects on the male reproductive system in mice and rats after oral subacute exposure. Almeida and Hall (2004) had reported a statistically significant increase in testes weight (both combined) at all dose levels and increased seminal vesicle weight at 800 and 8000 ug/L in BALB/c mice (n=5/dose). Seminiferous tubule diameter increased with drinking water concentration of MTBE but apparently was not statistically greater. Serum testosterone appeared to be greatly decreased at 800 and 8000 although these may not have been statistically different.

However, the study of Almeida and Hall (2004) is only available as an abstract and therefore the relevance of that study can only be considered with regards to whether similar findings are available in full (peer-reviewed) published studies or official study reports. The low number of mice used in the experiment also gives a particularly low statistical power.

In response to the findings reported by Almeida and Hall (2004), de Peyster et al. (2008) attempted to repeat the study by using the same protocol. However, de Peyster et al. (2008) could not replicate the findings reported by Almeida and Hall (2004).

In a gavage study by Li et al. (2008) significant adverse effects in the reproductive system of Sprague-Dawley rats were observed during 2-week and 4-week exposures at 400, 800 and 1600 mg/kg bw/day by gavage including: a significant increase in the percentage of abnormal sperm; an irregular and disordered arrangement of the seminiferous epithelium indicated by a histopathological examination; changed serum levels of testosterone, luteinizing hormone (LH) and follicle stimulating hormone (FSH); and decreased levels of mRNA and of androgen binding protein (ABP).

However, the study by Li et al. (2008) also reports no significant difference in epididymis weight or sperm number sperm. The study by Li et al. (2008) also contains an oxidative stress experiments, where results indicated an increased maleic dialdehyde (MDA) content, implying a raised peroxide level, and that the total antioxidant ability in serum was significantly increased. This finding was especially strong at 1600 mg/kg bw/day MTBE. In the 2-week treatment, at 1600 mg/kg bw/day, the mRNA level of 8-oxoguanine DNA glycosidase (OGG1) was significantly decreased, and the mRNA level of the extra-cellular form of superoxide dismutase (SOD(EX)) was significantly increased.

The effects on the seminiferous epithelium of the male reproductive system reported by Li et al. (2008) were not observed in the histopathology of the inhalation two-generation study with Sprague-Dawley rats at higher levels and no other effects on fertility were observed in this two-generation study (Bush Run Research Center, 1991). This negative results of two-generation study are considered the most complete study for assessing effects on fertility. Therefore, the observed effects in the recent oral study by Li et al. (2008), for which the functional consequences on fertility were not measured, are not considered relevant to classification for reproductive toxicity.

The findings of Li et al. (2008) were further considered in a review on MTBE potential for reproductive and developmental toxicity by Li and Han (2011), where the authors consider that the mouse model may be more appropriate to compare with humans than the rat model and note that no significant effects to mouse testes or other reproductive organs have been induced by MTBE. The authors conclude that MTBE is unlikely to pose to reproductive or developmental hazards to humans. Detailed evaluation of the endocrine related findings in the study by Li et al. (2008) is limited by the ages of the rats not being reported, where this may have a confounding effect on the endocrine related endpoints reported.

Increases in testosterone metabolism-specific cytochrome P450 levels were noted at high doses following oral gavage treatment of male rats with MTBE for 15 and 28 consecutive days (Williams & Borghoff, 2000). There was a 2.0-fold increase in CYP2B1/2 in rats treated with 1000 mg/kg bw/day MTBE for 28 days and with 1500 mg/kg bw/day for 15 and 28 days (6.5 and 2.9-fold respectively). CYP1A1/2, CYP2A1 and CYP2E1 activities were increased 1.5-, 2.4- and 2.3-fold, respectively, in high dose, 15-day treated rats. CYP2E1 was also increased 2.0-fold in high-dose rats treated for 28 days. CYP3A1/2 was increased 2.1-fold and UDP-glucuronosyltransferase activity 1.7-fold in high-dose, 28-day treated rats. The authors postulate that decreased serum testosterone levels observed in some MTBE-treated rats may therefore be the consequence of enhanced testosterone metabolism and subsequent clearance (Williams & Borghoff, 2000). However, the authors note that the changes in enzymes were minimal compared to known inducers of specific CYP enzymes and only evident at high MTBE exposures (Williams & Burghoff, 2000).

As discussed in section 5.6.3 on repeated dose toxicity, changes in testosterone levels in male rats is not consistent across studies. Where changes in testosterone levels have been seen, these are only in very high exposure levels and may be the result of effects other than P450 activity. The potential relevance of P450 activity to endocrine modulation is considered further in section 5.10.3 on specific investigations. Based on the other reproductive toxicity studies available, the observed changes in P450 activity and testosterone do not give rise to observable (adverse) effects.

In an investigative study on MTBE on mouse spermatogenic cells in vitro , there were no significant differences between control and treatments of 5 to 1000 ppm MTBE at the same time point (Li & Han, 2006). Exposure at 3000 ppm MTBE could however exert toxic effects directly on spermatogenic cells (Li & Han, 2006) and altered spermatogenic cell morphology, and reduced size and number. Some cells were killed or lost cellular integrity and became necrotic. Treatment at all levels including 3000 ppm did not alter ploidy distributions or DNA histograms. Only the lower exposure treatment levels of the in vitro assay are considered potentially relevant to in vivo studies.

 

Short description of key information: 

Based on the available data, MTBE is not considered toxic to fertility in rats and mice. Therefore, no DNEL has to be derived for this endpoint. There are no indications from the available data that dams are more sensitive regarding systemic effects compared to animals exposed in the repeated dose toxicity studies.

Effects on developmental toxicity

Description of key information
Based on the available data, developmental effects were only observed at maternal toxic concentrations. Based on the available data classification for developmental toxicity is not warranted. The long-term DNEL (repeated dose toxicity) will also prevent the occurrence of developmental toxic effects. Furthermore, there are no indications from the available data that dams are more sensitive regarding systemic effects compared to animals exposed in the repeated dose toxicity studies.
Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
1 428 mg/m³
Quality of whole database:
Studies on rat, mouse and rabbit available.
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

Developmental toxicity has been assessed in rat, mouse and rabbit. The four available well-conducted studies are all considered key.

There were no adverse developmental effects observed in Sprague-Dawley rats exposed to MTBE via inhalation at exposures up to 2500 ppm (8925 mg/m3) (Conaway et al, 1985). Reduced food consumption was noted during the treatment interval (days 9-12) at all concentrations (250, 1000 and 2500 ppm) but there was no accompanying effect on body weight and this finding is therefore not considered adverse.

Two studies are available that have examined the developmental toxicity of MTBE in pregnant CD-1 mice after exposure via inhalation. In the investigation of Bevan et al. (1997b), pregnant mice were exposed to 1000, 4000 and 8000 ppm MTBE vapour on gestation days 6-15. Reduced foetal body weight and skeletal abnormalities were noted at 4000 ppm (14280 mg/m3) while at 8000 ppm (28560 mg/m3) there was a statistically significant increase in the incidence of cleft palate and alterations in several gestation parameters (post-implantation loss, percentage of dead foetuses/litter). Both of these exposure concentrations caused severe toxicity to dams, giving an overall NOAEC of 1000 ppm (3570 mg/m3) for both maternal and foetal toxicity in this study. In an investigation reported by Conaway et al (1985), sternebrae malformations were observed in CD-1 mice at all tested concentrations (250, 1000 and 2500 ppm (893-8925 mg/m3), gestation days 6-15). The authors did not consider this effect treatment related since there was no increase in rib/vertebrae defects, which are commonly associated with fused sternebrae. Moreover, no such malformation was reported by Bevan et al. (1997b) in CD1 mice exposed to up to 8000 ppm MTBE vapour.

No developmental toxicity was demonstrated in rabbits up to a concentration of 8000 ppm (28560 mg/m3). At this concentration maternal toxicity was observed (Bevan et al., 1997).

In an investigative study, pups of Fisher 344 rats gavaged with MTBE at 500 to 1500 mg/kg bw/day from day 6 of organogenesis through to 10 days post parturition were examined for histological changes in vasculature (Kozlosky et al., 2013). The study also included in vitro assays using isolated rat brain endothelial cells were cultured on Matrigel and exposed to MTBE (1.25-80mM) and mouse brain Matrigel plug implants were exposed to MTBE at 34.0mM. No organ toxicity or histological changes to pup vasculature were observed in F344 rats treated with MTBE by oral gavage.

In the in vitro components of the study, MTBE (0.34-34.0 mM) exposure elicited a dose-dependent reduction in tube formation (LOAEL 0.34 mM) in rat brain endothelial cells (Kozlosky et al., 2013). In the mouse implantation assay MTBE (34.0 mM) completely inhibited vessel invasion into plugs containing endothelial cell growth supplement compared with control plugs containing this supplement alone. The authors note that these effects on developing vasculature may holds implications for possible medical surgery applications (Kozlosky et al., 2013). However such localized extreme concentrations are not achievable in vivo through oral, inhalation or dermal routes of exposure and therefore not relevant to human health risk assessment under the REACH Regulation.

Based on the available data, classification of MTBE for developmental toxicity is not needed.

In the presence of maternal toxicity, developmental effects were observed at concentrations from 4000 ppm in CD-1 mice (NOAEC: 1000 ppm (3570 mg/m3)). In the two-generation study with rats (see section regarding effects on fertility), some postnatal toxicity (lowered body weights and body weight gains in both the F1 and F2 litters) was observed in the offspring of both generations at 3000 and 8000 ppm (10710 and 28560 mg/m3), but also only in the presence of maternal toxicity. Therefore, the 8-h TWA of 50 ppm (178.5 mg/m3) derived by the SCOEL (2006) based on repeated dose toxicity data (and used as the starting point for the inhalation and dermal DNEL derivation) will also protect against the occurrence of developmental toxicity.

Justification for classification or non-classification

Based on these available data, MTBE is not considered to be a reproductive or developmental toxicant. In accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for reproductive and developmental toxicity.