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:
study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1984

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
intraperitoneal route of administration, treatment during days 6-15 of gestation, less than 20 females/group
GLP compliance:
no
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Provider: Eastman, Rochester, N.Y.

Test animals

Species:
rat
Strain:
Fischer 344
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Wilmington, Mass
- Housing: 3 per plastic cages containing woodchip bedding
- Diet (e.g. ad libitum): pelleted feed (Ralston Purina Co., St. Louis, Mo.), ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature : 72 ± 2°F
- Photoperiod (hrs dark / hrs light): 12-h light/dark cycle

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
physiological saline
Details on exposure:
All dose volumes were 2 mL/kg. Solutions were prepared daily in physiologic saline.
Analytical verification of doses or concentrations:
no
Details on mating procedure:
Two females were placed overnight with each male and checked for presence of sperm by vaginal wash the next morning. The day on which sperm was detected was considered d 0 of pregnancy.
Duration of treatment / exposure:
Days 6-15 of gestation
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
2.5 mg/kg bw/day (nominal)
Dose / conc.:
5 mg/kg bw/day (nominal)
Dose / conc.:
10 mg/kg bw/day (nominal)
No. of animals per sex per dose:
13 to 16
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Appropriate dose ranges for the three compounds were established during preliminary studies using groups of three or four pregnant animals. Parameters of toxicity observed in the higher dose preliminary treatment groups were maternal mortality and weight loss or markedly reduced weight gains and or convulsions (ranging from moderate to severe/resulting in mortality)
- The pregnant rats were sacrificed on gestation day 20 by overdose of halothane

Examinations

Maternal examinations:
The pregnant rats were weighed daily.
Ovaries and uterine content:
The uterus was opened and the numbers and positions of implants, dead fetuses, live fetuses, and resorptions were recorded
Fetal examinations:
Live fetuses were blotted dry, weighed, examined for external abnormalities, and divided into groups for visceral and skeletal examination (two-thirds and one-third of each litter, respectively). Fetuses for visceral examination were fixed in Bouin solution for at least 2 wk and were then examined by the freehand razor sectioning technique of Wilson (1965). The remaining fetuses were processed by an Alizarin red staining technique for skeletal examination (Dawson, 1926).
Statistics:
Maternal and fetal body weights and numbers of implants and resorptions were analyzed by the Student's t-test and are listed as mean ± standard deviation. Incidence of abnormalities was analyzed with Fisher's exact test. The litter was used as the experimental unit for those data that could be analyzed using either litter or fetus as the experimental unit, and a probability of p<0.05 was accepted as significant in all analyses.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Four of the pregnant females in the high-level treatment group convulsed following methylhydrazine injection on one or more occasions during the treatment period.
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Maternal weight gains were significantly less in all treatment groups during the early (days 6-10) part of the treatment period and significantly less for the two higher treatment groups during the latter (days 11-16) part of the treatment period (See Table 1). Significant decreases in weight gains for treatment groups were not seen after cessation (days 17-20) of treatment; however, overall weight gains were lower than those of the control group (-14%, -29% and -41% of controls in the 2.5, 5 and 10 mg/kg bw/day groups, respectively).
Food consumption and compound intake (if feeding study):
not examined
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 examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
not examined
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
not examined

Maternal developmental toxicity

Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
effects observed, treatment-related
Description (incidence and severity):
High incidence of resorptions in the two higher dose groups
Dead fetuses:
no effects observed
Changes in pregnancy duration:
not examined
Changes in number of pregnant:
no effects observed
Other effects:
not examined

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
2.5 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
clinical signs
body weight and weight gain
early or late resorptions

Maternal abnormalities

Abnormalities:
not examined

Results (fetuses)

Fetal body weight changes:
not examined
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
no effects observed
Changes in postnatal survival:
not examined
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Intermediate dose group had a moderate increase in abnormalities involving the eyes (anophthalmia and/or microophthalmia) but the higher dose group had only a slight increase in abnormalities involving the eye
Skeletal malformations:
no effects observed
Visceral malformations:
no effects observed
Other effects:
not examined

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
2.5 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
external malformations

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related
Localisation:
external: eye
Description (incidence and severity):
Intermediate dose group had a moderate increase in abnormalities involving the eyes (anophthalmia and/or microophthalmia) but the higher dose group had only a slight increase in abnormalities involving the eye

Overall developmental toxicity

Developmental effects observed:
no

Any other information on results incl. tables

Table 1: Effects of methylhydrazine given intraperitoneally on days 6 -15 of gestation in the rat

 

Methylhydrazine dose (mg/kg bw/day)

Effect

0

(13 litters)

2.5

(15 litters)

5

(15 litters)

10

(16 litters)

Gestation weight gain of females (g)a

 

 

 

 

Days 6-10

8 ± 5

3 ± 3b

2 ± 4b

- 3 ± 3b

Days 11-16

21 ± 5

19 ± 10

15 ± 5b

12 ± 8b

Days 17-20

30 ± 6

29 ± 10

25 ± 10

26 ± 12

Litter data

 

 

 

 

Implants/littera

7.8 ± 3.6

8.8 ± 3.4

7.3 ± 2.5

7.6 ± 3.4

Viable fetuses/littera

6.8 ± 4.0

7.5 ± 3.4

6.2 ± 2.7

6.1 ± 3.9

Fetal weighta

3.1 ± 0.3

3.3 ± 0.3

3.1 ± 0.3

3.2 ± 0.3

Litters with ≥ 33% fetuses resorbed

0

2

3

3

Incidence of abnormalities:

 

 

 

 

Litters (fetuses) affected

 

 

 

 

Gross examc

2(2)

1(1)

3(4)

2(2)

Soft-tissue exam

1(1)d

2(2)e

6(9)e

3(4)f

Skeletal examg

0(0)

1(1)

1(1)

2(2)

aMean ± standard deviation.

bSignificant at ≤ 0.05 level.

cAII nanoids (≤ control mean fetal weight minus 3 standard deviations).

dOne fetus with anophthalmia and hydrocephalus.

eAII anophthalmia or severe microophthalmia.

fHydronephrosis and dilated ureter in 1 fetus, hydrocephalus in another, and 2 fetuses with anophthalmia.

gAll unfused ossification centers of vertebrae.

Applicant's summary and conclusion

Conclusions:
All data that might suggest an embryotoxic or teratogenic effect were obtained with doses producing significant/marked maternal toxicity (marked decrease in bodyweight gain at the 2 higher doses and convulsions at the highest dose)
Executive summary:

Pregnant Fischer female rats were treated daily with methylhydrazine intraperitoneal injections during gestation Days 6 -15 at 2.5, 5 and 10 mg/kg bw/day. The pregnant rats were weighed daily. The uterus was opened and the numbers and positions of implants, dead fetuses, live fetuses, and resorptions were recorded. Live fetuses were blotted dry, weighed, examined for external abnormalities, and divided into groups for visceral and skeletal examination (two-thirds and one-third of each litter, respectively).

Four of the pregnant females in the high-level treatment group convulsed following methylhydrazine injection on one or more occasions during the treatment period. Maternal weight gains were significantly less in all treatment groups during the early (days 6-10) part of the treatment period and significantly less for the two higher treatment groups during the latter (days 11-16) part of the treatment period (See Table 1). Significant decreases in weight gains for treatment groups were not seen after cessation (days 17-20) of treatment; however, overall weight gains were lower than those of the control group (-14%, -29% and -41% of controls in the 2.5, 5 and 10 mg/kg bw/day groups, respectively). High incidence of resorptions in the two higher dose groups was observed. Intermediate dose group had a moderate increase in abnormalities involving the eyes (anophthalmia and/or microophthalmia) but the higher dose group had only a slight increase in abnormalities involving the eye.

All data that might suggest an embryotoxic or teratogenic effect were obtained with doses producing significant/marked maternal toxicity (marked decrease in bodyweight gain at the 2 higher doses and convulsions at the highest dose). Therefore NOAEL for maternal toxicity and developmental toxicity were both identified at 2.5 mg/kg bw/day.