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EC number: 216-653-1
CAS number: 1634-04-4
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).
addition, no adverse microscopic changes were observed in the gonads in
any of the sub-chronic or long-term toxicity studies.
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).
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
information from published literature does not change these conclusions.
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)).
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.
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
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).
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
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.
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.
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.
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,
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.
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.
description of key information:
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.
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.
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
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.
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.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
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