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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1991
Report date:
1991

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Version / remarks:
before 27th June 2018
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OTS 798.4350 (Inhalation Developmental Toxicity Screen)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Methyl methacrylate
EC Number:
201-297-1
EC Name:
Methyl methacrylate
Cas Number:
80-62-6
Molecular formula:
C5H8O2
IUPAC Name:
methyl methacrylate
Test material form:
liquid
Specific details on test material used for the study:
- Supplier: Rohm and Haas Company
- Purity= 99.9 %

Test animals

Species:
rat
Strain:
other: Crl:CDBR
Details on test animals or test system and environmental conditions:
- 225 nulliparous female rats
- Source: Charles River Breeding Laboratories Inc., Kingston, NY
- Age at study initiation: 81 -88 days
- AGE AT TIME OF MATING: 88-95 days.
- Weight at study initiation: 183-240 grams upon arrival
- Fasting period before study:
- Housing: Animals were housed individually, except during mating, in suspended stainless-steel cages (7" x 8" x 13.5"). During exposures, females were housed individually in suspended stainless-steel, wire mesh cages (6" x 7" x 11").
- Diet (e.g. ad libitum): Food (Certified Purina Rodent Chow #5002 ad libitum except during exposures.
- Water (e.g. ad libitum): filtered tap water ad libitum except during exposures.
Neither feed nor water was available during the 6 hour exposure period.
- Acclimation period: 7 days prior to mating
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C
- Humidity (%): ranged from 40-60% during cohabitation and 63-80% during the exposure and postexposure
periods.
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hrs dark/ 12 hrs light was maintained

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
The test material exposure concentrations were generated by metering the test material with calibrated Fluid Metering Pumps (Fluid Matering Inc., Oyster Bay, NY) into 500 mL three-necked round bottom flasks (Lab Glass Inc., Vineland, NJ).
Exposures were whole body and were conducted in 2000 L stainless steel, glass and Plexiglas® chambers. Cage positions within the chamber were rotated daily. The temperature and relative humidity within the chambers during exposure were 22-24°C and 55-67%, respective.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of the test substance in the chambers was determined by the use of a Miran gas analyzer attached to a strip chart recorder. A probe was placed into the center of the chamber and the chamber atmosphere was drawn into the Miran A1 gas analyzer at a rate of 9.5 L/min. Each chamber was analyzed initially within 40 min. of the end of the t99 to insure that each chamber was within the accepted target range. Subsequently, each chamber was sampled every 120 min. A range of  plus or minus 10% of the target chamber concentration was maintained by making minor adjustments on the generator pump delivery rates whenever necessary.

Target concentrations [ppm] Analytical concentrations [ppm]
mean +/- SD
--------------------------------------------------------------------------------

100 98.8 ± 3.4
300 304.4 ± 9.1
1200 1178.1 ± 69.1
2000 2028.2 ±107.3
Details on mating procedure:
Females were mated with males overnight (one male:one female) and the presence of sperm in the vaginal smear was considered  gestation day 0. Mated females were exposed via inhalation to the test material for 6 hrs/day on gestation days 6 through 15 and then sacrificed on day 20.  
Duration of treatment / exposure:
6 - 15 day of gestation
Frequency of treatment:
6 hours/day
Duration of test:
20 d (dams were euthanized on gestation day 20)
Doses / concentrationsopen allclose all
Dose / conc.:
99 ppm
Remarks:
corresponding to 412 mg/m3 or 0.41 mg/L
Dose / conc.:
304 ppm
Remarks:
corresponding to1285 mg/m3 or 1.29 mg/L
Dose / conc.:
1 178 ppm
Remarks:
corresponding to 4900 mg/m3 or 4.9 mg/L
Dose / conc.:
2 028 ppm
Remarks:
corresponding to 8436 mg/m3 or 8.44 mg/L
No. of animals per sex per dose:
27 animals per group exposed; 22-25 pregnant  females per exposure group.
Control animals:
yes, sham-exposed
Details on study design:
- Other: The strain was selected because background development toxicity data exists as Rohm and Haas Company on this rat strain. The test material was given by inhalation since the respiratory route is a potential route of human exposure.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were observed daily for behavioral changes.
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily GD 0 - 20
BODY WEIGHT: Yes
- Time schedule for examinations: recorded on GD 0, 6, 8, 10, 13, 16 and 20.
Food consumption was measured for GD intervals 0-6, 6-10, 10-16 and 16-20.
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
A limited necropsy was performed on all adult female animals. During the necropsy, animals were ex
amined for evidence of pregnancy
and gross lesions of the abdominal and thoracic cavities. Each uterus was weighted.
Ovaries and uterine content:
On GD 20, all dams were asphyxiated with carbon dioxide, the thoracic and abdominal cavities were examined and the uterus was removed and weighed, and corpora lutea, implantation sites and resorptions were counted. The number of fetuses per litter was counted and position inside the uterus recorded. The uteri of apparently non pregnant rats were stained with a 10% ammonium sulfide solution to detect very early resorptions. All fetuses were weighed, examined for external alterations and the sex of each fetus was determined. 
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [all per litter]
- Head examinations: Yes: [half per litter ]

One half of the fetuses from each litter were examined for visceral alterations using the Staples' technique. Head alterations were recorded for these fetuses examined for soft tissue alterations using the technique of Barrow and Taylor (1969, J. Morphol., 127: 291-306). The carcasses of all fetuses  were stained with alizarin red S and examined for skeletal alterations. 
Statistics:
For the purpose of statistical evaluation, the litter was considered the experimental unit for fetal parameters. Pregnancy rate, clinical signs, maternal deaths, gross necropsy findings  and liters with total resorptions were statistically analyzed using the  Fisher's exact test. Maternal body weight data and feed consumption values were statistically analyzed using Dunnett's test when the one-way ANOVA was significant. The number of implantations, live fetuses, resorptions, corpora lutea, mean fetal body weight/litter, and incidence of fetal alterations were statistically analyzed using the Mann-Whitney U test. When more than 75% ties occurred, then Fisher's exact test was used in place of the Mann-Whitney U test to detect significant differences between groups.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were observed bevore, during, or after the exposures at any exposure levels tested.
The only clinical sign noted was a minimal (only noted once or twice in 6 of 7 affected females) increase in the incidence of scant feces at 2028 ppm.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
There were no test substance-related or spontaneous mortalities in any group.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment-related decreases on maternal body weight and feed consumption were noted at all exposure levels. The decreases in maternal body weight at 99 and 304 ppm were minimal and transient since they occurred only during the first 2 days of exposure and returned to control values by the next weighing period. In addition, maternal body weight gain over the entire treatment period (days 6-16 G) and corrected maternal body weight gain at 99 and 304 ppm were similar to the control values. The body weight and feed consumption values returned to control values for all groups during the post exposure period (GD 16-20). At 1178 and 2028 ppm, treatment-related effects included losses in maternal body and/or decreased body weight gain throughout the exposure period (GD 6 - 16) and decreased corrected maternal body weight gain (body weight gain minus gravid uterine weight).

Body weight during gestation in rats inhaling Methyl methacrylate on days 6 to 15 of gestation and euthanized on day 20:
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Concentrations No of dams Mean Body weight [g] ± SD
[ppm], 6 h/day on GD 0 on GD 6 on GD 8 on GD 10 on GD 13 on GD 16 on GD 20
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
0 25 244.7 ±3.13 285.6 ± 3.93 295.4 ±4.33 307.1 ±4.20 327.3 ±4.68 350.6 ±5.05 414.2 ±6.84
99 23 246.3 ±2.93 286.1 ± 3.56 289.9 ±4.01 304.8 ±3.99 325.4 ±4.31 351.8 ±4.72 416.6 ±6.87
304 22 248.7 ±2.62 286.2 ± 3.11 288.9 ±3.60 301.3 ±3.91 321.1 ±4.09 347.1 ±4.58 413.9 ±6.09
1178 23 244.9 ± 2.77 282.2 ± 3.53 278.1a ±3.96 289.0a ±3.94 308.3a ±4.04 334.6 ±4.88 400.4 ±6.80
2028 23 246.7 ± 2.85 286.4 ± 3.03 275.2a ±2.68 280.9a ±3.40 306.2a ±3.49 332.7a ±4.27 401.3 ±5.56
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
a Denote significant differences from the control (0 ppm), p < 0.05, respectively.


Change in weight during gestation in rats inhaling Methyl methacrylate on days 6 to 15 of gestation and euthanized on day 20:
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Concentrations No of dams Mean body weight gain ± SD on GD [g]
[ppm], 6 h/day on GD 0 - 6 on GD 6 - 8 on GD 8 - 10 on GD 10-13 on GD 13-16 on GD 6-16 on GD 16-20
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
0 25 41.0 ±1.86 9.8 ±0.83 11.6 ±0.58 20.3 ±0.94 23.2 ±1.76 65.0 ±2.33 63.6 ±2.75
99 23 39.7 ±2.36 3.8a ±1.39 15.0 ±1.90 20.5 ±1.28 26.4 ±1.52 65.7 ±2.83 64.8 ±3.81
304 22 37.5 ±2.05 2.7a ±1.09 12.4 ±1.09 19.8 ±1.04 26.0 ±1.30 60.9 ±2.62 66.8 ±2.54
1178 23 37.3 ±1.91 -4.1a ±-1.41 10.9 ±0.91 19.3 ±1.09 26.3 ±1.76 52.4a ±2.56 65.8 ±3.15
2028 23 39.7 ±1.08 -11.2a ±0.88 5.7a ±2.24 25.4a ±1.62 26.5 ±1.99 46.4a ±2.35 68.6 ±2.12
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
a Denote significant differences from the control (0 ppm), p < 0.05, respectively.


Summary of Uterine and Net body weights in rats inhaling Methyl methacrylate on days 6 to 15 of gestation and euthanized on day 20:
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
Concentrations No of dams Gravid Uterus weight ± SD [g] Corrected Mean body weight ± SD on GD 20 [g] Net weight change ± SD from GD 6 [g]
[ppm], 6 h/day
------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
0 25 82.7 ±3.81 331.5 ±4.77 45.8 ±2.21
99 23 82.7±5.76 333.9 ±5.08 47.9 ±2.76
304 22 86.4 ±3.75 327.5 ±4.71 41.3 ±2.73
1178 23 79.9 ±5.04 320.5 ±5.04 38.3 ±3.04
2028 23 84.5 ±3.62 316.8 ±3.60 30.4a ±2.25
-----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
a Denote significant differences from the control (0 ppm), p < 0.05, respectively.

Corrected body weight: terminal body weight at GD 20 minus Uterine weight
Net weight change from GD 6: Corrected body weight minus day 6 body weight
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Treatment related decrease waere noted on feed consumption between days 6-10 G at all exposure levels teasted. Decteases in feed consumption continued between days 6-10 G at 304, 1178, and 2028 ppm. Feed consumption of all treated groups returend to control value during post-treatment period (days 16-20 G)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not specified
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Description (incidence and severity):
There were no abortions observed in any of the treated dose groups.
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The pre- and postimplantation loss was unaffected by treatment.
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
In the study, there was one dam high-dose dam with total litter loss. This observation is considered as not treatment-related.
Early or late resorptions:
no effects observed
Description (incidence and severity):
In the study, there were no early or late resorptions observed.
Dead fetuses:
no effects observed
Description (incidence and severity):
There were no dead fetuses found in the uterine.
Changes in pregnancy duration:
not specified
Description (incidence and severity):
There was a Caesarian section performed on GD 20.
Changes in number of pregnant:
no effects observed
Details on maternal toxic effects:
Details on maternal toxic effects:
No animals died and no treatment-related clinical signs were noted for the dams in the 99, 304 or 1178 ppm groups. Scant feces was noted in the 2028 ppm group throughout the exposure period (GD 6-15). Treatment-related decreases on maternal body weight and feed consumption were noted at all exposure levels. The decreases in maternal body weight at 99 and 304 ppm were minimal and transient since they occurred only during the first 2 days of exposure and returned to control values by the  next weighing period. The body weight and feed consumption values returned to control values for all groups during the post exposure period  (GD 16-20). At 1178 and 2028 ppm, treatment-related effects included losses in maternal body and/or decreased body weight gain throughout the exposure period (GD 6 - 16) and decreased corrected maternal body weight gain. The gross necropsy evaluations did not indicate any treatment-related effects and there were no treatment-related differences between the control and treated groups in any reproductive parameter.  

Effect levels (maternal animals)

open allclose all
Dose descriptor:
LOEC
Remarks:
maternal toxicity
Effect level:
ca. 0.41 mg/L air (analytical)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEC
Remarks:
maternal toxicity
Effect level:
8.44 mg/L air (analytical)
Based on:
test mat.
Remarks on result:
other: no adverse effects observed

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
There were no treatment-related effects on fetal body weight in males, females and both sexes combined at any dose level tested.
Reduction in number of live offspring:
no effects observed
Description (incidence and severity):
Reproductive parameters in rats inhaling Methyl methacrylate on days 6 to 15 of gestation and euthanized on day 20:
--------------------------------------------------------------------------------------------------------------------------------------
Concentrations No of litters No. of live fetuses per litter
[ppm], 6 h/day
------------------------------------------------------------------------------------------------------------------------------------
0 25 13.88
99 23 13.87
304 22 14.86
1178 23 13.70
2028 23 14.65
-----------------------------------------------------------------------------------------------------------------------------------
Changes in sex ratio:
no effects observed
Description (incidence and severity):
There were no treatment-related changes in the sex ratio observed at any dose group.
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
The mean number of pups per litter and litter weight were unaffected by treatment.
Changes in postnatal survival:
no effects observed
Description (incidence and severity):
The pups were euthanised shortly after the Caesarian section.
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related increase were detected in the type of external, visceral, or skeletal malformations.
Spontaneous malformations detected in the study included duplicated hypothalamus in a control fetus, hydrocephaly in a fetus at 304 ppm and an omphalocele in one fetus and enlarged adrenal glands in two fetuses at 1178 ppm was not considered to be treatment related as at 2028 ppm no increase was observed.
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
The significant increase in skeletal developmental variations at 1178 ppm was not detected at 2028 ppm, the highest doese tested. The slightly, not statistically significant number of fetuses with partial or unossified sternebra at 304 ppm was not considered a result of treatment since no dose response was demonstrated.
Visceral malformations:
no effects observed
Description (incidence and severity):
There were no visceral malformations observed in any of the dose groups.
Other effects:
not specified
Details on embryotoxic / teratogenic effects:
Details on embryotoxic / teratogenic effects:
 Fetal body weight was not affected by exposure to MMA vapors. The fetal external, visceral and skeletal examinations did not show any treatment related effects.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEC
Remarks:
fetotoxicity
Effect level:
8.44 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: corresponding to 2028 ppm; no substance related effects observed
Key result
Dose descriptor:
NOAEC
Remarks:
teratogenicity
Effect level:
8.44 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Remarks on result:
other: corresponding to 2028 ppm; no substance related effects observed

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Mean measured concentrations (± SD) within the chambers for the 0, 100, 300, 1200 and 2000 ppm groups were 98.8 (±3.4), 304.4 (±9.1), 1178.1  (±69.1) and 2028.2 (±107.3) ppm, respectively.

Applicant's summary and conclusion

Conclusions:
Exposure by inhalation to methyl methacrylate concentrations up to 8.44 mg/L (2028 ppm) resulted in no embryo or fetal toxicity or malformations even at exposure levels that resulted in maternal toxicity.
Executive summary:

In a developmental toxicity study on rats acc. OECD 414 by inhalation pregnant CRl: CD Br rats were exposed to methyl methacrylate at concentrations of 0 (control), 99, 304, 1178 and 2028 ppm on days 6-15 of gestation. A maternal no observed level was not demonstrated since losses in maternal body weight or decreases in maternal body weight gain and decreases in maternal feed consumption were noted at all exposure levels tested. No embryo or fetal toxicity was evident and no increase in the incidence of malformations or variations was noted at exposure levels up to the highest dose of 2028 ppm. Therefore toxicity to the conceptus was not evident even at exposure levels that resulted in overt maternal toxicity.