Reaction mass of ethanol and propan-2-ol

Developmental toxicity / teratogenicity

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: Not a guideline study and publication does not report whether study performed according to GLP. However, data appears well documented and scientifically acceptable.

Data source

Reference

Reference Type:

publication

Title:

Prenatal ethanol exposure has differential effects on fetal growth and skeletal ossification

Author:

Simpson ME, Duggal S, & Keiver K

Year:

2005

Bibliographic source:

Bone 36: 521-532

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

Ethanol was added to the diets of female rats for six weeks (from 3 weeks prior to mating to gestational day (GD) 21) in an attempt to determine effects on fetal growth and skeletal development.

GLP compliance:

not specified

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethanol

EC Number:

200-578-6

EC Name:

Ethanol

Cas Number:

64-17-5

Molecular formula:

C2H6O

IUPAC Name:

ethanol

Details on test material:

- Name of test material (as cited in study report): ethanol
- Physical state: liquid

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, St. Constant, Quebec
- Age at study initiation: 3 months
- Weight at study initiation: mean 264 g (238-283 g)

ENVIRONMENTAL CONDITIONS
- Temperature-controlled rooms
- Photoperiod (hrs dark / hrs light): 12/12 (artificial lighting between 06.00-18.00)

Administration / exposure

Route of administration:

other: liquid diet

Vehicle:

unchanged (no vehicle)

Details on exposure:

no data

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

no data

Details on mating procedure:

Following 3 weeks of ethanol exposure, the virgin female rats were placed in breeding cages with unexposed males. Average time for mating was 3 days (range 1 - 5 days), and rats recieved lab chow ad libitum during this period. The appearance of a vaginal plug was considered day 1 of gestation (GD 1).

Duration of treatment / exposure:

Females exposed for six weeks (3 weeks prior to mating until GD 21)

Frequency of treatment:

Daily (liquid diet presented at 17.00)

Duration of test:

6 weeks

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
5.2
Basis:
actual ingested
g/kg bw/day

Doses / concentrations 2

Remarks:

Doses / Concentrations:
8.2
Basis:
actual ingested
g/kg bw/day

Doses / concentrations 3

Remarks:

Doses / Concentrations:
10.4
Basis:
actual ingested
g/kg bw/day

No. of animals per sex per dose:

Three test groups, fed diets containing 15% (E15; n=15), 25% (E25; n=15), and 36% (E36; n=26) ethanol-derived calories.
Four control groups, three of which were pair-fed (PF) isocaloric controls with maltose-dextrose replacing the ethanol at 15% (PF15; n=15), 25% (PF25; n=15), and 36% (PF36; n=25), and an additonal control group (C; n=31) receiving liquid control diet ad libitum.

Control animals:

other: Pair-fed (PF) isocaloric liquid diet (with the ethanol calories replaced by maltose-dextrose)... (see attached file)

Details on study design:

- Dose selection rationale: Dams were fed ethanol at doses to approximate low, moderate and high levels of maternal drinking.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: No

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly throughout experiment

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No data
- Number of corpora lutea: No data
- Number of implantations: No data
- Number of early resorptions: No data
- Number of late resorptions: No data
- Other: Number of fetuses per litter counted

Fetal examinations:

- External examinations: No data
- Soft tissue examinations: No data
- Skeletal examinations: Yes: [all per litter] The percent ossification of the fetal bones (scapula, humerous, ulna, radius, femur and tibia) were measured using a Zeiss dissecting microscope with a linear eyepiece raticule. The number of ossification centres was determined for the sternum, sacrum, and metatarsals.
- Head examinations: No data

Statistics:

Differences between the groups regarding the effects of diet and dose of ethanol were determined by one- and two-way ANOVAs followed by Newman-Keuls post hoc tests, except for the number of ossification centres which was determined by one- and two-way Kruskal-Wallis tests followed by a Tukey-like nonparametric mulitple comparison test. Comparisons among diets were analysed by seperate one-way Kruskal-Wallis tests for each of the exposure levels. Repeated measures ANOVAs were used to determine the effects of week of gestation on maternal ethanol intake (wk 0, 1, 2 and 3) and on peak blood ethanol concentrations (BEC). An unpaired t test was used to compare terminal BEC samples between E25 and E36 dams.

Indices:

Ponderal index (fetal weight/length3) calculated to examine effect of ethanol on relationship between fetal body weight and length.

Historical control data:

no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Overall, there was no statistically significant difference in maternal weight gain between the ethanol (E15, E25 and E36) and corresponding pair-fed (PF15, PF25 and PF36) dams. However, there was a significant decrease in weight gain at the E36, compared to E15 and E25 dams.

Effect levels (maternal animals)

1

Dose descriptor:

LOAEL

Effect level:

8 200 mg/kg bw/day (actual dose received)

Basis for effect level:

other: developmental toxicity

2

Dose descriptor:

NOAEL

Effect level:

5 200 mg/kg bw/day (actual dose received)

Basis for effect level:

other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
No significant differences in the number of fetuses per litter between the groups were apparently observed.

Maternal ethanol intake from the E36 mothers resulted in fetuses which were statistically significantly lighter than the pair-fed (PF36) isocaloric controls, the ad libitum controls, and compared to the E15 and E25 groups. In addition, fetuses from the E36 dose groups were significantly shorter than the ad libitum controls and the E25 and E15 groups. The ponderal index was not significantly affected by diet or dose, indicating that the ethanol-induced restriction in growth did not alter the relationship between body weight and length. No significant effects on fetal growth (body weight or length) were seen in the fetuses of mothers exposed to E15 or E25 compared to the PF15 or PF25 isocaloric controls, respectively, or compared to the ab libitum controls.

In the E25 group, a statistically significant effect on fetal bone ossification was seen for the ulna, radius, tibia, and scapula, but not the sternum, metatarsals, sacrum, femur or humerus, when compared to the PF25 isocaloric controls. No significant differences between ossification at the E15 compared to the PF15 isocaloric controls were seen for any of the bones sites, although a statistically significant effect at the E15 was found for the radius compared with the ab libitum controls only.

Ethanol induced a statistically significant delay in the development of body weight and ossification of individual bones, particularly at the E36 dose group. The radius and scapula showed the greatest delay at the E25 and E36 groups, followed by the ulna and tibia.

Taken together, the data suggest that prenatal ethanol exposure (at 8.2 g/kg bw/day and above), caused a delay in the early development (ossification) of the fetal skeleton (by 0 - 0.5 days). No skeletal malformations or variations were reported.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Prenatal ethanol exposure affected fetal skeletal ossification at exposure levels lower than those required to affect fetal body weight and length, although the significance of these changes for long-term bone health is unknown. No statistically significant effects on fetal growth or ossification were seen in the fetuses of dams ingesting about 5.2 g ethanol/kg bw/day from 3 weeks prior to mating to GD 21.

Executive summary:

Groups of female Sprague-Dawley rats were given liquid diets with 15, 25, or 36% ethanol-derived calories (E15, E25, and E36 groups, respectively), or without ethanol (pair-fed isocaloric (PF15, PF25, PF36) or ad libitum (C) control) for 3 weeks prior to mating, and throughout 21 days of gestation.

Prenatal ethanol exposure at 36% ethanol-derived calories (E36; about 10.4 g ethanol/kg bw/day) decreased fetal body weight and length, and skeletal ossification (a developmental delay), compared with pair-fed (PF36) and ad libitum controls at GD 21. Significant effects on ossification, but not body weight or length, were seen at E25 (corresponding to a dose of about 8.2 g ethanol/kg bw/day), compared to PF25 isocaloric controls. The NOAEL for this study can be considered to be 5.2 g ethanol/kg bw/day, as at this dose level (E15), no significant effects on fetal growth or ossification were seen compared to the PF15 isocaloric controls. A delay in the development of body weight and skeletal ossification was seen in the ethanol-treated (E25 and E36) fetuses on GD21, compared to the ab libitum controls. There were no skeletal malformations or variations (other than the delay in ossification) reported for any of the ethanol-treated groups.

The data indicate that ethanol has differential effects on fetal weight and skeletal development, and that the skeletal sites differ in their sensitivity to ethanol.