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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
Study period:
1985
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Remarks:
A published study which contains sufficient experimental detail to be able to judge it as reliable. Basic experimental detail provided.

Data source

Reference
Reference Type:
publication
Title:
Teratological assessment of methanol and ethanol at high inhalation level in rats.
Author:
Nelson, B., Brightwell, W., MacKenzie, D., et al.
Year:
1985
Bibliographic source:
Fundam Appl Toxicol 5:727-736.

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
low number of pregnant females, longer exposure period.
Principles of method if other than guideline:
Method: other
GLP compliance:
not specified
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): ethanol absolute-200 proof
- Analytical purity: 96.5%
- Impurities (identity and concentrations): Analysis for water and benzene detected none.
- Ethanol is the in vivo hydrolysis product of ethyl acetate.: (Morris, J.B. (1990): Toxicol. Appl. Pharmacol. 102, 331 - 345; Gallaher, E.J., Loomis, T.A. (1975): Toxicol. Appl. Pharmacol. 34, 309 - 313; Section 5.10 Records 15 and 16).

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories
- Age at study initiation: not stated
- Weight at study initiation: 200-300g
- Fasting period before study: no
- Housing: 3 per cage in stainless-steel cages except whilst in chamber
- Diet (e.g. ad libitum): purina or comparable-grade lab chow ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period:1-2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24
- Humidity (%): 20-60
- Air changes (per hr):
- Photoperiod (hrs dark / hrs light):12/12

Administration / exposure

Route of administration:
inhalation
Type of inhalation exposure (if applicable):
whole body
Vehicle:
other: air in chamber
Details on exposure:
0.5m3 Hinner-type exposure chambers under negative pressure. Controls were placed in similar cage as the exposed animals with adjacent exposure chamber for the same hours.
- Method of holding animals in test chamber: Females were placed into 13 X 25 X 18-cm compartments in stainlesssteel wire-mesh caging within the exposure chambers.
- Method of conditioning air: The vapor generation equipment was housed above the exposure chambers in glove boxes whlen were maintained under negative pressure to prevent any cage of contaminants into the room. Reagent-grade methanol (Matheson, Coleman, and Bell Manufacturing Chemists. Cincinnati. Ohio) or reagent-grade (absolute-200 proof ethanol (AAPER Alcohol and Chemical Co. Louisville. Ky.) was placed into a flask. A low-flow purn,: RP model lab pump; Fluid Metering Inc. Oyster Bal N.Y.) circulated liquid from the reservoir flask into a 10-ml syringe contained within the flask such that the syringe was constantly overflowing. Thus the syringe provided a constant head of chemical for a second pump (controlled by a micrometer adjustment) which injected the specified amount of liquid into a three-way valve which was attached to a Greensmith impinger. Heated compressed air was introduced through the second inlet of the threeway valve. Alcohol evaporation was controlled by regulating the preheating of compressed air. The impinger provided increased contact time between the air and the liquid to assure total evaporation. In generation of high concentrations, glass beads were also placed at the bottom of the impinger to further increase the heat transfer area between the alcohol and the compressed air. This vapor and air mixture was introduced into the chamber air flow prior to positioning of the orifice plate. The turbulence resulting from the pressure drop created by the orifice plate provided uniform mixing of the vapor and air before the mixture entered the chamber
- Air flow rate: Air flow through the chambers provided approximately one air change per minute.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Two methods used: Continuously by a Miran 1A general purpose infrared analyzer (Wilkes/Foxboro Analytical), on an hourly basis; and concentration samples taken from chamber atmosphere by charcoal tube. Sampling times 10-30 mins. 5-10 samples/week. Analysed by NIOSH 1977b-No. S-56 Method with slight modifications.
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- Housing of pregnant females: individually into 30 X 34 X 17-cm polycarbonate cages having autoclavable polyester filter covers. Bedding consisted of cleaned. heat-treated sawdust from a local supplier (Absorb-Dri. from Tasty Foods, Cincinnati, Ohio).
Duration of treatment / exposure:
7 hours per day in exposure chamber on gestation days 1-19. Animals left in the chambers for degassing for approximately half an hour after vapor generation terminated.
Frequency of treatment:
daily (7 days/week)
Doses / concentrationsopen allclose all
Dose / conc.:
10 000 ppm
Dose / conc.:
16 000 ppm
Dose / conc.:
20 000 ppm
No. of animals per sex per dose:
not explicitly stated but from other data in the study report believed to be approximately 16.
Control animals:
not specified
Details on study design:
Sex: female
Duration of test: Days 1-19 of gestation

Examinations

Maternal examinations:
- No organs from dams examined at necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: Most females were also weighed each morning for the first week of exposure and weekly thereafter.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Weekly

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Weekly

POST-MORTEM EXAMINATIONS: Yes, euthanized by CO2 asphyxiation
- Sacrifice on gestation day # 20

OTHER:
- Blood levels of ethanol
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: implants and resorptions were recorded as was litter weight.
- Other: vaginal smears were taken.
Fetal examinations:
Foetuses were examined externally and internally for malformations;
- Soft tissue examinations: Yes: half per litter were placed into Bouin's solution and subsecuentlv examined for visceral malformations and variations using a razor blade cross-sectioning technique (Wilson, 1965).
- Skeletal examinations: Yes: half per litter were randomly selected, placed into 80% ethanol and subsequently eviscerated, macerated in 1.5% OH stained in alizarin red S and examined.
Statistics:
Statistical Method: Multivariate analysis, Kruskal-Wallis test, analysis of variance and Fisher's exact test.
Indices:
Number of pregnant animals per dose group, Litter sizes/weights, Sex ratios.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The lower two concentrations of ethanol seemed to cause hyperactivity after exposure, whilst the high dose caused complete narcosis by the end of the exposure (described as severe toxicity).
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Maternal weight gains were not affected by treatment
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Food consumption was significantl lowered in the high-dose group during week 1 of exposure.

Maternal developmental toxicity

Number of abortions:
not specified
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
The number of implantations were 14-16/litter in all ethanol-treated groups and 15/litter in the control group. The number of corpora lutea were 14-16/litter.
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not specified
Description (incidence and severity):
Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): not specified
Changes in number of pregnant:
effects observed, treatment-related
Description (incidence and severity):
The number of pregnant per dose level were 15/15, 15/15, 15/16 and 14/16 in the control, low, medium and high dosage groups. The effect was slight but significant in the high dose group.
Details on maternal toxic effects:
Blood alcohol levels ranged from 0.02 to 0.03 mg/ml at 10000 ppm, 0.42 to 0.84 mg/ml at 16000ppm and 1.48 to 1.93 mg/ml at 20000 ppm. Measurements were made on non-pregnant rats and represent the ranges of the average values measured at days 1, 10 and 19.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
16 000 ppm
Basis for effect level:
clinical signs
food consumption and compound intake
Dose descriptor:
LOAEL
Effect level:
20 000 ppm
Basis for effect level:
clinical signs
food consumption and compound intake

Maternal abnormalities

Abnormalities:
no effects observed

Results (fetuses)

Fetal body weight changes:
no effects observed
Description (incidence and severity):
LItter weights were not significantly affected by ethanol treatments. Weights of males (but not females) were reported as significantly lowered although the tabulated data does not indicate this as statistically significant (% reduction 2.4%, 4.5%, 3.2% L/M/H dose respectively) and suggests that there was a 7.8% drop in female weights in the high dose group. As the changes were below 10% they may be treatment related are not considered adverse.
Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
Skeletal malformations:
no effects observed
Description (incidence and severity):
Grossly visible abnormalities are given in detail but the frequency of each did not differ significantly between groups. The only finding was urinary - hydronephrosis but this was not seen in the mid dose group (no dose response)
Visceral malformations:
no effects observed
Description (incidence and severity):
No significant findings other than those seen spontaneously in control animals.
Other effects:
effects observed, treatment-related
Description (incidence and severity):
More litters contained abnormal foetuses in the 20,000 ppm group compared to th concurrent contrl but differences were only of borderline statistical significance and the rate of skeletal malformations in this group was similar to those seen in the control from the first experiment.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
no effects observed

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Skeletal malformations

 

Control 1

10,000ppm

16,000ppm

Number of litters (foetuses) observed

15 (99)

15 (100)

15 (107)

 

 

 

 

Cranial –abnormal nasal

1(1)

 

 

Cranial - Shortened maxilla

1(1)

 

 

Cranial – split basisphenoid

 

 

 

Vertebral – fused thoracic centra

 

 

 

Vertebral – fused cervical arches

1(1)

 

 

Ribs – rudimentary cervical

1(1)

 

2(2)

Ribs – wavy fused

2(2)

 

 

Ribs - missing

 

 

1(1)

 

 

 

 

Total malformations

4(4)

0

2(3)

 

 

Control 2

10,000ppm

Number of litters (foetuses) observed

15 (90)

14 (92)

 

 

 

Vertebral – Lordosis

 

1(1)

Ribs – rudimentary cervical

 

2(2)

Ribs – wavy fused

 

2(3)

 

 

 

Total malformations

0

4(5)

 

Visceral malformations

 

Control 1

10,000ppm

16,000ppm

Number of litters (foetuses) observed

15 (107)

15 (106)

15 (114)

 

 

 

 

Urinary - hydronephrosis

0

2(2)

0

 

 

Control 2

20,000ppm

Number of litters (foetuses) observed

15 (99)

14 (97)

 

 

 

Urinary - hydronephrosis

0

4(4)

Applicant's summary and conclusion

Conclusions:
No definite evidence of malformations due to ethanol exposure were seen although the incidence of abnormal changes at the highest concentration was of borderline statistical significance. There was clear maternal toxicity at this concentration however.
Executive summary:

Pregnant female rats were exposed to ethanol by inhalation at concentrations of 10000, 16000, or 20000ppm in a chamber for 7 hours per day on gestation days 1 -19. On day 20 the animals were euthanized and their fetuses examined. There was no definite increase in malformations at any level of ethanol exposure. There was clear maternal toxicity evident at the highest dose (narcosis, food intake reduction).

Synposis

NOAEL (maternal toxicity) :16,000ppm (30,400mg/m3)

NOAEL (teratogenicity): 20,000ppm (38,000mg/m3)