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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2-generation
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP compliant near-guideline study; minor restrictions; fully adequate for assessment

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1997

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
No specific guideline was reported. However, the study was generally well conducted.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
tert-butyl methyl ether
EC Number:
216-653-1
EC Name:
tert-butyl methyl ether
Cas Number:
1634-04-4
Molecular formula:
C5H12O
IUPAC Name:
propane, 2-methoxy-2-methyl-
Constituent 2
Reference substance name:
methyl tert-butyl ether
IUPAC Name:
methyl tert-butyl ether
Details on test material:
-Name of test material (as cited in study report): MTBE
-Physical state: liquid
-Analytical purity: at least 99% (determined by gas chromatography)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
-Source: Charles River Breeding Laboratories
-Age at study initiation: 6 weeks
-Housing: individually
-Diet: ad libitum
-Water: ad libitum

ENVIRONMENTAL CONDITIONS
-Temperature (°F): 66-77
-Humidity (%): 40-70
-Air changes (per hr): 14
-Photoperiod: (hrs dark/ hrs light): 12/12

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: Air
Details on exposure:
All exposures were conducted in 4.3 m3 stainless-steel and glass inhalation chambers. Airflow in each chamber was approximately 1000 L/min (14 air changes per hour). For the generation of atmospheres, liquid MTBE was metered from a piston pump into a heated glass evaporator. The temperature was maintained at the lowest level sufficient to vaporize liquid. The resulting vapor was carried into the chamber by a countercurrent air stream that entered the bottom of the evaporator.
Details on mating procedure:
During the mating period, one male was co-housed with one female selected randomly within the same treatment group for a maximum of 7 days. After 7 days, the females of those pairs that were not confirmed as having successfully mated were placed with males of other unmated pairs within the same treatment group for the remainder of the 21-day mating period. Mating was confirmed by observation of a copulatory plug or by the presence of sperm in a vaginal rinse. The day on which mating was confirmed was designated as the female’s Day 0 of gestation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Atmosphere was analyzed once every 25 minutes during each 6 hours of exposure using a Perkin-Elmer gas chromatograph equipped with a flame ionization detector.
Duration of treatment / exposure:
Males: 10 weeks before mating until delivery of the F1-litters
Females: 10 weeks before mating, during 21-day mating period, gestation and lactation starting Day 5 and until the day of sacrifice, which followed the weaning of the offspring
Frequency of treatment:
6 hours/day, 5 days/week
Details on study schedule:
The exposure started when the rats were about 6 weeks old and lasted for 10 weeks before mating. Exposure of the females continued through the 21-day mating period, gestation and lactation starting on Day 5 and continued until the day of sacrifice, which followed the weaning of the offspring. Male exposure continued until the delivery of the F1 litters. The new parents were randomly selected from the F1-litters at weaning on postnatal day 28 (PND 28), which was also the day they started receiving exposure. The exposure procedure was the same as it was for the P-animals.
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
400, 3000 and 8000 ppm of MTBE
Basis:
other: target concentration
Remarks:
Doses / Concentrations:
402 ± 8, 3019 ± 56 and 8007 ± 137 ppm of MTBE
Basis:
analytical conc.
No. of animals per sex per dose:
25
Control animals:
yes

Examinations

Parental animals: Observations and examinations:
Viability was observed twice a day, detailed clinical examinations were performed once daily and body weights were recorded weekly. Female body weights were recorded before mating, during gestation and in the post-natal days until weaning. Food consumption was recorded weekly and for females at every 3-4 days of gestation.
Litter observations:
Litters were examined twice daily. On PND 0, 1, 4, 7, 14, 21 and 28, litters were counted, weighed and sexed and checked for abnormalities. On PND 4, the size of each litter was reduced by random selection to yield as nearly as possible four male and four female pups per litter. Culled pups were euthanized and given an external examination (including an examination for cleft palate). Abnormal pups were given a gross post-mortem examination.
Postmortem examinations (parental animals):
All adult F0 and F1 animals used for mating went through a post-mortem examination (gross necropsy). Pituitary, testes, epididymis, prostate and seminal vesicles, vagina, uterus, ovaries and respiratory tract were examined microscopically from all parent animals of the control and high-dose group as well as tissues with gross lesions.

Livers from F1- animals were weighed and those of the control and high-dose group were examined microscopically.
Postmortem examinations (offspring):
On PND 4, culled pups were euthanized and given an external examination (including an examination for cleft palate). Abnormal pups were given a gross post-mortem examination.
Statistics:
The unit of comparison was the male, the female (during the pre-mating exposure period), the pregnant female or the litter. Results of quantitative continuous variables were intercompared for the three treatment groups and one control group by use of Levene’s test for equal variances, analysis of variance (ANOVA), and t-tests. Non-parametric data were statistically evaluated using the Kruskal-Wallis test followed by the Mann-Whitney U test for pairwise comparisons when appropriate. Frequency data were compared using the Fisher’s exact test.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
effects observed, treatment-related

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Details on results (P0)

Toxic effects observed in parent F0 animals: Hypoactivity and lack of startle reflex were observed in the parental rats at 3000 and 8000 ppm. Parental animals at 8000 ppm were also ataxic. The 8000 ppm dose group males had a significantly reduced body weight throughout the 10-week pre-mating period. In the beginning of the mating period, the reduction in male bodyweight was almost 14%. There was also a significant reduction in the bodyweight gain in that group during the first 7 weeks. In the first three pre-mating weeks, male food consumption was reduced on average by 11%. Female body weights did not differ from controls in the pre-mating period but the body weight gain was significantly increased in the 8000 ppm group during post-natal days 21-28. The overall difference in lactation-period weight gain was more than two-fold (p<0.05).

The reproduction data, such as pregnancy rates, gestation days and mating and fertility indices showed no meaningful differences from the control.

Toxic effects observed in parent F1 animals: Hypoactivity and lack of startle reflex were observed in the parental rats at 3000 and 8000 ppm. Parental animals at 8000 ppm were also ataxic. In the adult animals, the highest dose group of F1-males had about 15% less weight gain (p<0.01) during the first two weeks, accompanied by a reduced absolute body weight throughout the pre-mating period. Although no decrease in weight gain was noted with either male or female rats in the 3000 ppm exposure group, they still had significantly reduced body weight in the first 3-4 pre-mating weeks. Similarly to the F0 parent animals, no differences were noted in female body weights during the gestation period. However, the high-dose group had a significantly increased (4-fold) body weight during PND 14-21 and the lactation period. Nevertheless, the food consumption was significantly (p<0.01) reduced by approximately 10% during PND 7-14 in these females. Gross examination at necropsy revealed statistically significantly increased relative liver weights in both sexes of the F1-animals at 8000 ppm and in males at 3000 ppm. However, no treatment-related histopathological changes were noted.

Pregnancy incidence, mating and fertility and gestation indices were within the control values.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEC
Remarks:
(parental toxicity)
Effect level:
400 ppm
Sex:
male/female
Basis for effect level:
other: Based on general toxicity signs (reduced bodyweight, hypoactivity, blepharospasms) at 3000 and 8000 ppm in parental animals.
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
NOAEC
Remarks:
(fertility)
Effect level:
8 000 ppm
Sex:
male/female
Basis for effect level:
other: No significant changes in the reproduction parameters.
Remarks on result:
other: Generation: P and F1 (migrated information)
Dose descriptor:
other: NOAEC developmental toxicity
Effect level:
400 ppm
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Remarks on result:
other: Generation: F1 and F2 generation (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings:
no effects observed

Details on results (F1)

Developmental toxicity observed in F1 litters: There was a statistically significant (p<0.01) increase of dead pups in the 8000 ppm group. This followed from the death of an entire litter of 16 pups. However, there was no significant change in the survival index of the F1 generation. The high-dose group male and female offspring body weights were significantly lower than the controls during PND 14-28. The female offspring of the 3000 ppm exposure group had significantly lower body weights also on Day 14. In general, when compared to controls, no significant differences were evident in the number of pups stillborn relative to live ones, judging by the survival indices on Days 4, 7, 14, 21, 20 or the lactation index.

Developmental toxicity observed in F2 litters: As in the F1-offspring, the F2-pups also had a significantly increased incidence of dead pups on PND 4 in the 8000 ppm exposure group. Again, in a manner similar to F1-pups, the F2-offsping had no significant difference in the survival indices when compared to controls. Male body weights of the 3000 and 8000 ppm dose groups were statistically significantly (p<0.05) reduced when compared to the control group. Weight loss was seen in males of the 8000 ppm group during PND 7-28 and in the males of the 3000 ppm group during PND 14-28.

Effect levels (F1)

Dose descriptor:
NOAEC
Generation:
F1
Effect level:
400 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
other: CNS depression

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on metabolism studies which demonstrate that MTBE is metabolized to tertiary butyl alcohol in vivo, data from the two-generation study with MTBE is relevant for evaluation of reproductive and developmental toxicity of tertiary butyl alcohol.

There were no adverse effects on mating performance, fertility or reproductive organs in a two-generation whole-body inhalation study in which Sprague-Dawley rats were exposed to MTBE at target exposure concentrations of 400, 3000 and 8000 ppm. General signs of toxicity were observed at 3000 and 8000 ppm in both generations of parental animals. Some developmental 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. The NOAEC for parental toxicity and developmental toxicity was 400 ppm while the NOAEC for reproductive toxicity was 8000 ppm. Because these data demonstrate that MTBE is not a reproductive or developmental toxicant, it is reasonable to conclude that the metabolite tertiary butyl alcohol is also not a reproductive toxicant and is also not selectively toxic to the developing fetus. MTBE, and by extension tertiary butyl alcohol, is not classified for “Developmental or Reproductive Toxicity” according to GHS.
Executive summary:

In a two-generation reproductive toxicity study, MTBE was administered to 25 Sprague-Dawley rats/sex/group by whole body inhalation at target concentrations of 0, 400, 3000 and 8000 ppm for 10 weeks prior to mating. Males continued exposure until delivery of the F1-litters. Females continued exposure during mating, gestation and lactation until weaning of the offspring. There were no adverse effects on any reproductive parameters including pregnancy rates, gestation days, and mating and fertility indices. Toxic effects including clinical signs of CNS depression, and reduced body weight and/or body weight gain were observed in the F0 and F1 parental generations at 3000 and 8000 ppm. Signs of developmental toxicity such as lowered body weight and body weight gains were observed in the F1 and F2 litters when parents were exposed to 3000 or 8000 ppm but adverse effects occurred only in the presence of maternal toxicity. MTBE and by analogy its metabolite tertiary butyl alcohol are not considered to be reproductive or developmental toxicants.