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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Guideline study with acceptable restrictions (exposure at gestation day 6-15, necropsy on gestation day 21)

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes
Remarks:
Bushy Run Research Center
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Methyl vinyl ether (MVE)
Source: Union Carbide
Purity 99.5 %.(re-analysis)
Lot No. 281995

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Source: Charles River Lab
25 time-pregnant rats per dose level
acclimatisation: 2 weeks prior to mating
mean body weight range at gestation day 0 225-227 g
tap water and certified diet ad libitum (not during exposure)
dams housed singly
temperature: 66-77°F
day/night cycle: 12/12 h
rel. air humidity: 40-70%

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air
Details on exposure:
The rats were exposed to vinyl methyl ether vapour or filtered air for 6 h/d on gestational days 6 through 15 in stainless steel inhalation chambers. The chamber volume was 900 l, the airflow 200 l/min (13 changes/h). Target concentrations were 0; 5000; 10000 or 19500 ppm. Temperature and humidity were recorded.
MVE vapour introduced directly into the chambers from MVE cylinder (flowmeter control) after dilution with air.
Temperature: ranged from 21.1 to 22.4°C
Relative humidity: ranged from 37.4 to 46.2%
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of MVE in each chamber was monitored twice per hour during each 6-hour exposure by gas chromatography.
Details on mating procedure:
One male mated with one female; vaginal plugs controlled daily; gestation day (GD) 0 was the day the vaginal plug was detected
Duration of treatment / exposure:
GD 6-15
Frequency of treatment:
once daily, 6 hours/day
Duration of test:
GD 21
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0; 5000; 10000; 19500 ppm (12; 24; 47 mg/L)
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
0, 4946+-216, 10249+-465, and 19407+-796 ppm
Basis:
analytical conc.
average +- standard deviation
No. of animals per sex per dose:
25 pregnant dams
Control animals:
yes, concurrent vehicle
Details on study design:
Randomisation based on body weight at GD0

Examinations

Maternal examinations:
Twice daily clinical observations
Body weight GD 0, 6, 9, 12, 15, 18, 21
Food and water consumption measured GD 0-21 in 3-day intervals
The dams were necropsied on gestational day (gd) 21 and evaluated for body weight. Maternal liver, kidneys, and lungs were weighed and retained in fixative.
Ovaries and uterine content:
gravid uterine weight and pregnancy status determined;
Fetal examinations:
All fetuses were dissected from the uterus, counted, weighed, examined for determination of gender and for external variations and malformations (including cleft palate). Approximately one-half of the live fetuses in each litter were examined for visceral and craniofacial malformations and variations. The remaining one-half of the fetuses were examined for skeletal malformations and variations after staining with alizarin red S.
Statistics:
The data for quantitative continuous variables were intercompared for the exposure groups and the control group by use of Levene's test homogeneity of variance, by ANOVA, and by t-tests. T-tests were used if the ANOVA was significant to delineate which groups differed from the controls. If Levene's test indicated homogeneous variances, the groups were compared by ANOVA for equal variances. If Levene's test indicated heterogeneous variances, groups were compared by an ANOVA for unequal variances, followed, when appropriate, by t-tests. Frequency data were compared using Fisher's exact test. All statistical tests, except the frequency comparisons, were performed using computer software. The probability value of p<0.05 (two-tailed) was used as the critical level of significance for all tests.
Historical control data:
yes

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Mortality: No mortality occurred during the study. Pregnancy rate was equivalent for all groups and ranged form 96 - 100 %. No females aborted or delivered early. At scheduled necropsy, one female each from 5000 and 10000 ppm group was not pregnant.
Clinical signs of toxicity: There were no exposure-related clinical signs observed in any exposure group.
Body weights: body weight decreases were noted as described below. p<0.05 indicates statistically significant differences compared to the control animals group. 19500 ppm group: slightly decreased on gd 9; (p<0.05) on gd 12 and 15; slightly on gd 18 and 21. 10000 ppm group: slightly decreased on gd 12; (p<0.05) on gd 15; 5000 ppm group: slightly decreased on gd 12; (p<0.05) on gd 15; Body weight gain was significantly reduced in the 19500 ppm group on most of the 3-days intervals, and in the 10000 and 5000 ppm groups on some of the 3-day intervals. Following the exposure period, body weight gain was comparable or slightly higher in the exposure groups when compared to the controls.
Food consumption was decreased in the 19500 and 10000 ppm groups during the exposure period. Food consumption was also reduced in the 5000 ppm group early in the exposure period (gd 6-9).
Maternal water consumption was increased, but the effect was not dose-related.
Terminal maternal body weight were slightly reduced, and corrected weight change was clearly reduced in all treatment groups. Gravid uterine weight and absolute and relative weights of lungs and kidneys were equivalent across groups. Absolute liver weights were slightly increased (2, 3, and 6 % for the 5000, 10000, and 19500 ppm groups, respectively. Increased relative liver weight was noted in all exposure groups.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOAEC
Effect level:
5 000 ppm
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOEC
Effect level:
5 000 ppm
Basis for effect level:
other: developmental toxicity
Dose descriptor:
LOAEC
Effect level:
10 000 ppm
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Reproductive parameters: there were no effect of exposure on gestational parameters including resorptions, pre- and postimplantation losses, percentages of live fetuses, and sex ratios. There was no effect of exposure on fetal body weight/litter.
Pups
There were no significant effects of exposure on the incidence of visceral, skeletal, or external malformations by category or in total malformations. An increased incidence of one common external variation, ecchymosis in the trunk region, was noted in the 19500 ppm group. In addition, several skeletal variations involving various regions of the skeleton were observed for the 19500 and 10000 ppm groups. These included: unossified and poorly ossified cervical centra, reduced number of (ossifying) caudal segments, unossified anterior arch of the atlas, and poorly ossified sternebra. In addition increased incidences of all unossified proximal phalanges (forelimb and hindlimb), and some unossified metatarsals (hindlimb) occurred only in the 19500 ppm group. Overall, maternal toxicity was observed at all concentration levels used in this study. No embryolethality or teratogenic effects were observed. A concentration-dependent profile of delayed skeletal development was observed for all MVE-exposed groups, considered as transient effects secondary to maternal toxicity.

Effect levels (fetuses)

Dose descriptor:
NOAEC
Effect level:
19 500 ppm
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Re-analysis of skeletal findings (Dow 2005):

1) Introduction: as two minor skeletal variations ("unossified anterior arch of the atlas" and "poorly ossified sternebrae 4") were statistically increased at the lowest exposure concentration, the authors of the inhalation study concluded that a no-observed-effect level (NOEL) could not be identified. However, they also acknowledged that "the biological significance of the findings (at 5000 ppm) is not certain, however, since several of these findings have been noted in fairly high incidences in control CD rat litters in this laboratory". While historical control data would have been extremely valuable to address the significance of the skeletal variations seen at 5000 ppm, unfortunately, such data were not provided in the study report. Nonetheless, a review of the study report revealed that the incidences of certain skeletal variations involving the same or neighboring bones were actually decreased at 5000 ppm relative to controls. Therefore, a re-analysis of the skeletal data was conducted using a more holistic approach to determine the overall effect of MVE exposure on these specific regions of the fetal skeletal system.

2) Method: all of the individual animal fetal skeletal data from the original study report were reviewed and certain variations involving either the same or neighboring bones were combined. A statistical analysis of the combined data was conducted using the Censored Wilcoxon test. The litter was the unit of analysis.

3) Result: re-analysis revealed that the combined incidence of all 3 arch variations (unossified, poorly ossified, bilobed) that initially had been reported separately (but which are very difficult to differentiate because of very subtle and transient differences) affected all litters (100 %) at all dose levels, and that 95.2 %, 88.6 %, 95.8 %, and 97 % of the fetuses were affected at the dose levels 0, 5000, 10000, and 19500 ppm, respectively. Therefore, there was no statistical significant difference between the dose groups.

Re-analysis of the other finding (poorly ossified sternebrae 4) involved ossification delays of sternebrae 1-6. The result is that the combined incidence of sternebrae variations (1-6) affected all litters (100 %) at all dose levels, and 83 %, 79.6 %, 82.1 %, and 84.5 % of the fetuses at the dose levels 0, 5000, 10000, and 19500 ppm, respectively. Therefore, there was no statistical significant difference between the dose groups.

4) Conclusion: the results of this re-analysis indicate a lack of a statistical or biologically significant effect on ossification of the atlas or sternebrae at any MVE exposure level evaluated in the study. This could possibly have been the result of an unusually low control incidence. Consideration of this re-analysis and the weight of evidence from the overall study findings suggest that the developmental toxicity NOEC for the study was 5000 ppm.

Applicant's summary and conclusion

Conclusions:
Maternal toxicity: LOAEC = 5000 ppm or 12 mg/L (lowest dose tested).
Developmental toxicity: NOEC = 5000 ppm or 12 mg/L.
Teratogenicity: NOAEC = 19500 ppm or 47 mg/L (highest dose tested).
Executive summary:

Four groups, each consisting of 25 timed-pregnant CD rats, were exposed to vinyl methyl ether (MVE) vapor or filtered air for 6 hours/day on gestational days (gd) 6 through 15. Target concentrations of MVE vapor were 0 (control), 5000, 10000, and 19500 ppm. Average analytical concentrations (± S.D.) were 4946 (± 216), 10249 (± 465), and 19407 (± 796) ppm, respectively. Clinical observations were made daily. In addition, during the actual exposures, each group of dams was observed from outside their respective exposure chambers for overt clinical signs. Body weights were measured on gd 0, 6, 9, 12, 15, 18, and 21, and body weight gains were calculated. Maternal food and water consumption were measured at 3-day intervals throughout gestation. The dams were necropsied on gd 21 and evaluated for body weight, gravid uterine weight, and pregnancy status. Maternal liver, kidneys, and lungs were weighed and retained in fixative. All fetuses were dissected from the uterus, counted, weighed, examined for determination of gender and for external variations and malformations (including cleft palate). Approximately one-half of the live fetuses in each litter were examined for visceral and craniofacial malformations and variations. The remaining one-half of the fetuses were examined for skeletal malformations and variations after staining with alizarin red S.

No mortality occurred during the study. The pregnancy rate was equivalent for all groups and ranged from 96 to 100%. No females aborted or delivered early. At scheduled necropsy, one female each from the 5000 and 10000 ppm groups was not pregnant. There were no exposure-related clinical signs observed in any exposure group. Gestational body weight and body weight gains were reduced during the exposure period in all exposure groups. Food consumption was decreased in the 10000 and 19500 ppm groups during the exposure period. Food consumption was also reduced in the 5000 ppm group early in the exposure period (gd 6-9). The relative liver weight was increased in all exposure groups. There were no effects of exposure on gestational parameters including resorptions, pre- and post-implantation losses, percentages of live fetuses, and sex ratios. There was no effect of exposure on fetal body weights/litter. There were no effects of exposure on the incidence of visceral, skeletal, or external malformations. An increased incidence of one common external variation, ecchymosis in the trunk region, was noted in the 19500 ppm group. In addition, several skeletal variations involving various regions of the skeleton were observed for the 10000 and 19500 ppm groups. These included: unossified cervical centra #5 and #6, unossified and poorly ossified cervical centrum #7, reduced number of (ossifying) caudal segments, unossified anterior arch of the atlas, and poorly ossified sternebra #4. In the 5000 ppm group, the incidences of unossified anterior arch of the atlas and poorly ossified sternebra #4 were also increased. In addition, increased incidences of all unossified proximal phalanges (forelimb and hindlimb), and some unossified metatarsals (hindlimb) occurred only in the 19500 ppm group.

All variations characteristic of delayed ossification of the anterior arch of the atlas or sternebrae (e .g., unossified, poorly ossified and bilobed) were combined and statistically analyzed using the Censored Wilcoxon test (Dow, 2005).The combined analysis of unossified, poorly ossified and bilobed anterior arch of the atlas revealed a 100 % litter incidence in all groups, with fetal incidences of 95.2, 88.6, 95.8, and 97.0 % in the 0, 5000, 10000, and 19500 ppm groups, respectively. Similarly, combined analysis of variations characteristic of delayed sternebrae ossification showed respective litter incidences of 100, 100, 100, and 96 .0%, with fetal incidences of 83.0, 79.6, 82.1, and 84.5 %. None of these differences were statistically significant. Furthermore, the high incidence in controls indicates that these minor skeletal variations were not biologically significant.

Based on this re-analysis, 5000 ppm is considered to be the developmental toxicity NOEC for the study. Based on the results, maternal toxicity was observed at all concentration levels used in this study. No embryolethality or teratogenic effects were observed. A concentration-dependent profile of delayed skeletal development was observed for mid and high dose groups, considered as transient effects secondary to maternal toxicity. In conclusion,the LOAEC for maternal toxicity was 5000 ppm or 12 mg/L (lowest dose tested). The NOEC for developmental toxic effects was 5000 ppm and the NOAEC for teratogenic effects was 19500 ppm or 47 mg/L (highest dose tested).