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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:
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:
study report
Title:
Unnamed
Year:
1987
Report Date:
1987

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Groups of 21 pregnant rats dosed from day 6 to 15 of gestation inclusive. Control group received distilled water (vehicle) throughout the same period.
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River U.K. ltd.
- Age at study initiation: 9 - 11 weeks
- Weight at study initiation: 196 - 238 g
- Housing: in RC1, RB3 and R83 modified cages. All cages consisted of high density polypropylene bodies with lids of stainless steel grid. RC1 and RB3 modified cages had stainless steel grid floors and were suspended in batteries over trays covered with absorbent crepe paper which was changed on alternate weekdays (daily during pairing). During gestation females were housed in RB3 cages with solid polypropylene floors and the autoclaved wood shavings provided as bedding were renewed at least twice weekly.
Cages were distributed on the racking to equalise, as far as possible, environmental influences amongst the groups. At various stages of the study the maximum number of rats per cage was:
no. of rats cage type
acclimatisation 0 M, 5 F RC1
mating 1 M, 1 F RB3 modified
gestation 0 M, 1 F RB3
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 °C, range 18 - 25 °C
- Humidity: 55 %, range 40 - 70 %
- Photoperiod (hrs dark / hrs light): 12 h light - 12 h dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: solution in distilled water prepared freshly each day

VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg
Details on mating procedure:
Females were paired on a one-to-one basis with stock males of the same strain. Each morning following pairing, the trays beneath the cages were checked for ejected copulation plugs and a vaginal smear was prepared from each female and examined for the presence of spermatozoa.
The day on which a sperm positive vaginal smear or at least three copulation plugs were found was designated as day 0 of gestation.
Duration of treatment / exposure:
From day 6 to 15 of gestation inclusive.
Frequency of treatment:
Daily.
Duration of test:
On day 20 of gestation, females were killed by inhaled CO2.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
control
Dose / conc.:
1.5 mg/kg bw/day (actual dose received)
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Dose / conc.:
15 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
21 females
Control animals:
yes, concurrent vehicle

Examinations

Maternal examinations:
Clinical signs
All animals were examined daily throughout the study and any visible signs of reaction to treatment were recorded, with details of type, severity, time of onset and duration.

Maternal bodyweight
Females were weighed on days 0, 3, 6 to 16 inclusive, 18 and 20 of gestation.

Food consumption
Food consumption was recorded on days 0, 3, 6, 9, 12, 16, 18 and 20 of gestation.

Terminal studies
On day 20 of gestation, the females were killed by inhaled carbon dioxide for examination of their uterine contents. Each animal was first examined macroscopically for evidence of disease or adverse reaction to treatment and specimens of abnormal tissues
were retained.
The reproductive tract, complete with ovaries, was dissected out and the following recorded:
a) number of corpora lutea in each ovary;
b) number of implantation sites.
c) number of resorption sites (classified as early or late);
d) number and distribution
Ovaries and uterine content:
The reproductive tract, complete with ovaries, was dissected out and the following recorded:
a) number of corpora lutea in each ovary;
b) number of implantation sites.
c) number of resorption sites (classified as early or late);
d) number and distribution
Fetal examinations:
External examination at necropsy
Each foetus was weighed, sexed and examined for any external abnormalities. Individual placental weights and placental abnormalities were recorded.

Internal examination at necropsy
Neck, thoracic and abdominal cavities of approximately half of each litter were dissected and examined. All foetal abnormalities were recorded and the offspring eviscerated prior to fixation in industrial methylated spirit (74 o.p.).

Internal examination by free-hand serial sectioning
The remaining half of the foetuses in each litter were placed in Bouin's fixative. After a period of fixation they were examined by the Wilson free-hand serial sectioning technique (Wilson, J.G. Teratology: Principles and Techniques, p.251, University of Chicago Press, 1965).

Skeletal examinations
Eviscerated foetuses were processed and stained with Alizarin-red, using a modification of the Dawson staining technique (Tesh, .J.M., Ph.D. Thesis, Faculty of Veterinary Science, University of Liverpool, 1958), and the skeletons were examined.
Statistics:
The significance of suggestive inter-group differences was tested using appropriate statistical tests, each of which has been specified where used. Differences with an associated probability of P<0.05 were considered to be statistically significant.

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Gross pathological findings:
no effects observed

Maternal developmental toxicity

Pre- and post-implantation loss:
no effects observed
Total litter losses by resorption:
no effects observed
Early or late resorptions:
no effects observed
Dead fetuses:
no effects observed
Other effects:
no effects observed
Description (incidence and severity):
Mean placental weights were similar in all groups.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
pre and post implantation loss
total litter losses by resorption
early or late resorptions
dead fetuses
Dose descriptor:
NOAEL
Effect level:
13 mg/kg bw/day (nominal)
Based on:
act. ingr.
Basis for effect level:
clinical signs
mortality
body weight and weight gain
food consumption and compound intake
organ weights and organ / body weight ratios
gross pathology
pre and post implantation loss
total litter losses by resorption
early or late resorptions
dead fetuses

Maternal abnormalities

Abnormalities:
no effects observed
Localisation:
ovary
placenta

Results (fetuses)

Fetal body weight changes:
no effects observed
External malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Two grossly abnormal foetuses were seen at necropsy, one in group 2 (1.5 mg/kg/day) with no lumbar spine, agenesis of the tail and imperforate anus and one in group 4 (15 mg/kg/day) with exencephaly, open eye and spina bifida occulta.

Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Examination of foetuses after skeletal processing showed that the incidence of incomplete ossification of the supra-occipital bone in group 4 foetuses (15 mg/kg/day) was significantly higher than in the concurrent control group (P<0.01) and was also considerably above the background control range. The incidences in groups 2 and 3 (1.5 and 5.0 mg/kg/day) were slightly above the background control range but were not significantly different from the concurrent control value. The incidences of incomplete ossification of the interparietal and hyoid bones in groups 3 and 4 (5.0 and 15.0 mg/kg/day) were marginally increased compared with the concurrent controls, but all values were well within the background control ranges. Other ossification parameters showed slight intergroup differences.
Visceral malformations:
effects observed, non-treatment-related
Description (incidence and severity):
Examination following free-hand serial sectioning also revealed a small number of foetuses with more complex morphological changes, viz.
group 1 (control) - one foetus with slight hydrocephaly.
group 2 (1.5 mg/kg/day) - one foetus with a small diaphragmatic hernia.
group 3 (5 mg/kg/day) - one foetus with a retro-oesophägeal right subclavian artery

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
external malformations
skeletal malformations
visceral malformations
Dose descriptor:
NOAEL
Effect level:
13 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
not specified
Basis for effect level:
fetal/pup body weight changes
external malformations
skeletal malformations
visceral malformations

Fetal abnormalities

Abnormalities:
effects observed, non-treatment-related
Localisation:
external: tail
external: anus
skeletal: skull

Overall developmental toxicity

Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
Oral administration of test substance at doses of 1.5, 5.0 and 15.0 mg/kg/day to pregnant rats during organogenesis was without significant adverse effects upon maternal performance, litter parameters and growth and survival in utero. Ossification of foetal posterior cranial bones was slightly reduced, most obviously at 15.0 mg/kg/day, but there were no adverse morphological changes that were considered to be related to treatment.
Executive summary:

Method

Effects of test substance on progress and outcome of pregnancy was assessed in sexually mature rats of the CD strain. Groups of 21 pregnant rats were dosed by gavage at dose levels of 1.5, 5.0 or 15.0 mg/kg bw/day from day 6 to day 15 of gestation inclusive. Control animals received the vehicle, distilled water, throughout the same period.

All females were killed on day 20 of gestation for examination of their uterine contents.

Results

General condition of the dams was unaffected by treatment, and no deaths occurred. Bodyweight gain of females in all treated groups was similar to that of controls. Food intake was unaffected by treatment. There were no adverse effects of treatment upon litter size, post-implantation survival or foetal and placental weights. At 15 mg/kg/day there was a statistically significant increase in the incidence of foetuses with incomplete ossification of the supra-occipital bone and there was a lesser, non-significant increase at 5.0 and 1.5 mg/kg/day.

There were marginal increases in the incidences of incomplete ossification of the interparietal and hyoid bones at 5 and 15 mg/kg/day, but these values remained within the background control ranges. There were no

morphological changes that were considered to be related to treatment.

In conclusion, no significant adverse effects upon maternal performance, litter parameters and growth and survival in utero were seen in rats upon dosing during organogenensis. Ossification of the foetal posterior cranial bones was slightly reduced, most obviously at 15.0 mg/kg/day, but there were no adverse morphological changes that were considered to be related to treatment.

On these bases, NOAEL = 15 mg/kg/day, equivalent to 13 mg/kg a.i..