Registration Dossier

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: no guideline study, well described publication

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1984

Materials and methods

Principles of method if other than guideline:
The chemical N-Methylethanolamine was vaporized and administered to approximately 15 pregnant rats in one to three concentrations for 7 hr/day on gestation days 7 to 15, and dams were sacrificed on day 20. Fetuses were individually weighed, and two-thirds of them were fixed in Bouin's solution and examined for soft-tissue anomalies. The other one-third were fixed in alcohol, stained with Alizarin Red and examined for skeletal defects.
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
Virgin female and male Sprague-Dawley rats specified to be free of mycoplasma and Sendai virus and of internal and external parasites (Charles River Breeding Laboratories, Wilmington, MA) were acclimated to a 12-hr light-dark cycle (lights on at 6 am) and to a temperature of 24 + 2°C for 2 weeks. The humidity, not controlled, typically was in the range of 40 + 20%. Purina Lab Chow and tap water were available ad libitum except when pregnant animals were in exposure chambers. Bedding consisted of cleaned, heattreated sawdust from a local supplier (Absorb-Dri, Tasty Foods, Cincinnati, OH).
Females were placed alone in 38 x 33 x 17-cm polycarbonate cages with filter tops.

Administration / exposure

Route of administration:
inhalation: vapour
Type of inhalation exposure (if applicable):
whole body
Vehicle:
unchanged (no vehicle)
Details on exposure:
Pregnant females were transported from the animal quarters to the inhalation chambers in their home cages with filter tops (Hazleton Systems, Aberdeen, MD). They were placed individually in 13 x 25 x 189-cm stainless steel wire mesh cages within exposure chambers.
Air flow through the chambers provided approximately four air changes per minute.
Exposures were conducted sequentially in one or two chambers, with a third chamber for sham exposure of control subjects.
Control animals were placed in similar chambers for the same hours as the exposed animals; a pooled group of controls (N = 34) served as the comparison group for the first three chemicals examined. Another group of 15 controls served as the comparison group for the last two chemicals examined, as these groups were exposed at a later time (approximately 6 months later) than the first three.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations within the exposure chambers as measured by the infrared analyzer were relatively close to those obtained from gas chromatography (Table 2).
Details on mating procedure:
Males weighing over 300 g were placed individually into a cage with three females weighing 200 to 300 g. Vaginal smears were taken each morning, and the presence of sperm marked day zero of gestation.
Duration of treatment / exposure:
7 hours (animals were left in the chamber for at least one additional hour blow-off time after vapor generation terminated)
Frequency of treatment:
Exposures, as outlined above, were conducted 7 hr/day, and the animals were left in the chamber for at least one additional hour blow-off time after vapor generation terminated. They were then returned in their individual housing cages to the animal quarters, where water bottles were replaced. Exposures were conducted on gestation days 7-15.
Duration of test:
Exposures were conducted on gestation days 7-15.
15 days of gestation.
On day 20 of gestation, dams were sacrificed.
Doses / concentrations
Remarks:
Doses / Concentrations:
150.0 +/- 15.2 ppm
Basis:
other: vapor generated, by gas chromatography
No. of animals per sex per dose:
approximately 15 pregnant rats
Control animals:
yes
Details on study design:
Controls: three solvents were compared with a pooled group (N = 34) of sham-exposed controls, and the remaining two were compared with a group of 15 controls.

Examinations

Maternal examinations:
Feed and water intake and maternal weight were recorded weekly (i.e., on days 7, 14, and 21); any other signs of maternal toxicity were noted daily.
On day 20 of gestation, the females were individually weighed and euthanized by chloroform asphyxiation.
Ovaries and uterine content:
The entire uterus was removed and numbers of resorption sites (classified as early, middle or late) and live fetuses wvere determined.
Fetal examinations:
Fetuses were serially removed, blotted of excess fluids, weighed, examined for external malformations and external sex determined.
One third of the fetuses were randomly selected and placed in 95% ethanol, and the remainingfetuses were placed in Bouin's solution. After being in
the Bouin's solution for at least 1 week, these fetuseswere examined for visceral abnormalities using Wilson's razor blade sectioning technique. The viscera wereexamined with the aid of a dissecting microscope. A representative sample of sections with malformations was identified by dam number and saved in 70% alcohol.

Fetuses were examined for skeletal defects by using a modified Staples technique. They were fixed in 95% alcohol, eviscerated and macerated in 2% KOH/Alizarin Red S solution. The fetuses were further macerated and cleared in the appropriate solutions of 2% KOH/glycerin (60:40, 40:60, 20:80) and stored in 100% glycerin. A crystal of thymol was added to each storage vial to retard fungal growth. Storage vials were individually identified by dam number.
Statistics:
Numbers of implants and proportions of resorptions were independently analyzed by using a Kruskal-Wallis test corrected for ties, with subsequent multiple comparisons to determine where the differences occurred. Analysis of pup weights involved a mixed model analysis of covariance (with the number of live pups in the litter as the covariate) using maximum likelihood estimation. The model was mixed, since there was both within-litter and between-litter variation. Subsequently, pairwise comparisons between the pooled control group and each treatment group were performed. Incidence oftotal defects and of total variants were compared using a Kruskal-Wallis test with multiple comparisons with the litter as the experimental unit and the level of significance at p< 0.05.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:not examined

Details on maternal toxic effects:
At 150 ppm N-Methylethanolamine (mean concentration from 28 silica gel tubes, one per day, analyzed in duplicate = 150.0 ppm), no maternal or
fetal toxicity was observed.

Effect levels (maternal animals)

Dose descriptor:
NOAEC
Effect level:
150 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
At 150 ppm N-Methylethanolamine (mean concentration from 28 silica gel tubes, one per day, analyzed in duplicate = 150.0 ppm), no maternal or
fetal toxicity was observed.

Effect levels (fetuses)

Dose descriptor:
NOAEC
Effect level:
150 ppm (nominal)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Low vapor pressure also prevented our generating high concentrations of 2-MAE. At 150 ppm 2-MAE (mean concentration from 28 silica gel tubes, one per day, analyzed in duplicate = 150.0 ppm), no maternal or fetal toxicity was observed (Tables 8-10).

Finally, the lack of teratogenic response of 2-methylaminoethanol was interesting and from a mechanistic or theoretical point of view, would merit follow up using a different route of exposure. At first glance, one might expect that its biotransformation would be similar to that of 2-ME. However, our results of no maternal or fetal toxicity at 150 ppm 2-MAE suggest that this may not be the case; since the amine is likely more lipidsoluble and less water-soluble than the methoxy portion, the absorption and excretion of the 2-MAE is likely quite different from that of 2-ME. Thus it would be of interest to see if a higher dose of 2-MAE would be teratogenic, though a route other than inhalation would be required, since the vapor concentration we used was near the saturation point.' This lack of teratogenicity at three times the concentration of a teratogenic level of its structurally similar glycol ether, points to a relatively strict structural requirement to produce teratogenic effects.

We observed that embryotoxicity decreases as alkyl chain length increases, similar to observations with testicular atrophy.

Applicant's summary and conclusion

Conclusions:
In this study N-Methylethanolamine showed neither maternal nor fetal toxicity effects.
Executive summary:

The chemical N-Methylethanolamine was vaporized and administered to approximately 15 pregnant rats in one to three concentrations for 7 hr/day on gestation days 7 to 15, and dams were sacrificed on day 20 of gestation. Fetuses were individually weighed, and two-thirds of them were fixed in Bouin's solution and examined for soft-tissue anomalies. The other one-third were fixed in alcohol, stained with Alizarin Red and examined for skeletal defects. As overall result for the substance N-Methylethanolamine it can be stated that neither maternal nor fetal toxicity effects can be concluded in this study.