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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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1986
Report Date:
1986

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: EPA Health Effects Test Guidelines 560/6-82-001
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories (Michigan)
- Age at study initiation: 91 days approx.
- Weight at study initiation: 220 g approx.
- Fasting period before study: no data
- Housing: individually
- Diet (e.g. ad libitum): ad libitum (Purina certified rodent chow #5002)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: 20 days minimum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 72°C +/-3°F
- Humidity (%): 40% or above
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hr light / 12 hr dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% aqueous methylcellulose
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
A specified amount of the test article DPG was weighed into a glass weigh boat for each group, transferred to a mortar and ground with the vehicle until a slurry was obtained. The slurry was transferred to a volumetric flask via a series of vehicle rinses. Vehicle was then added in sufficient quantity to achieve the appropriate concentration for each dose group. The flasks were inverted several times and stirred for 5 to 10 minutes. The mixtures were then transferred to amber dosage jars. The test mixtures were prepared fresh daily.
A dosage volume of 10 ml/kg was used for all dosage levels.

VEHICLE
- Justification for use and choice of vehicle (if other than water): 0.5% aqueous methylcellulose (methocel)
- Lot/batch no. (if required): 820-7112-A
- Purity: no data
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No data
Details on mating procedure:
The animals were paired for mating in the home cage if the male.
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1 M /1 F
- Length of cohabitation: until prove of pregnancy
- Verification of same strain and source of both sexes: yes
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
Duration of treatment / exposure:
days 6-15 of gestation
Frequency of treatment:
once daily
Duration of test:
15 days
Doses / concentrations
Remarks:
Doses / Concentrations:
5, 25 or 50 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
25 females / dose
Control animals:
yes, concurrent vehicle
Details on study design:
No

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes, each morning
DETAILED CLINICAL OBSERVATIONS: Yes, daily during all the gestation
BODY WEIGHT: Yes , on gestation days 0, 6, 9, 12, 16 and 20
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
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: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
Each fetus was individually weighed, sexed and tagged for identification.
- External examinations: Yes: all per litter (eyes, palate, external orifices)
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: No data
Statistics:
All analyses were conducted using two-tailed tests for a minimum significance level of 5% comparing the treatment group to the control group; all means have been presented with standard deviations. The number of animals (N) used to calculate the means has been provided on the individual data tables. All statistical tests were performed by a Digital Computer with appropriate programming as referenced below.
1. The fetal sex ratios were compared by the Chi-square test with Yates’ correction factor.
2. The numbers of litters with malformations and variations were compared by Fisher's Exact Test.
3. The numbers of early and late resorptions, dead fetuses and post-implantation losses were compared by the Mann-Whitney U-test.
4. Mean numbers of corpora lutea, total implantations, viable fetuses, mean fetal and maternal body weight at each interval and maternal body weight
gains were analyzed by a one-way analysis of variance, and Dunnett's test.
Indices:
No
Historical control data:
No

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Alopecia on the forepaws and forelegs was observed in one a nimal in the 50 mg/kg/day group prior to dosing on gestation day 6 and in all study groups during the treatment period, with an increased incidence and duration noted in the 50 mg/kg/day group. During the treatment period, hair loss was extensive in the 50 mg/kg/day group in the pelvic, abdominal, thoracic, urogenital, inguinal, dorsal back and tail areas. All animals in this dose group were lethargic and had tachypnea and decreased limbtone during the treatment period and with one exception all animals were prostrate and ataxic. A few animals were hypersensitive to the touch, salivated and had piloerection during the treatment period. Clonic convulsions, lacrimation, clear nasal discharge, dried red material around the nose, red urogenital discharge and yellow urogenital matting were observed as single incidences in the 50 mg/kg/day group. Lethargic behavior, salivation prior to dosing, hair loss in the pelvic and abdominal areas and dried brown material around the mouth were each noted once in different animals in the 25 mg/kg/day group and may be related to treatment with DPG. No clinical signs of toxicity were observed in the 5 mg/kg/day group.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Mean maternal body weight gain in the 50 mg/kg/day dose group was significantly decreased at all intervals during the treatment period. The most severe decrease (p < 0.01) occurred during the last four days of treatment (gestation days 12-16). The mean body weight gain in the 50 mg/kg/day group was very slightly increased after the treatment period (gestation days 16-20) when compared to the vehicle control group. This resulted in significantly decreased (p < 0.01) body weight gains for the entire gestation period (days 0-20). Group mean body weights were slightly decreased on gestation day 9 and significantly decreased at p < 0.01 on gestation days 12, 16 and 20 in the 50 mg/kg/day group. Mean body weight gain in the 25 mg/kg/day group was very slightly decreased during the overall treatment period (gestation days 6-16) when compared to the vehicle control group. This effect may be related to treatment as there was also a very slight increase in body weight gain following treatment. However, mean body weights in the 25 mg/kg/day group were comparable to the vehicle control group throughout gestation. Body weights and body weight gains in the 5 mg/kg/day group were not affected by treatment with DPG.
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

Details on maternal toxic effects:
In all groups treated with DPG, foetal sex ratios, the mean numbers of viable foetuses, implantation sites and corpora lutea were comparable to the vehicle control group. Mean foetal weights in the 5 and 25 mg/kg/day groups were comparable to the vehicle control. Mean foetal weight in the 50 mg/kg/day group was significantly reduced (p < 0.05) when compared to the vehicle control group. Mean postimplantation loss was slightly increased in the 5 mg/kg/day group due to one female with twelve early resorptions. This increase was not considered biologically meaningful since the effect was not observed at the 25 mg/kg/day dose level. An increase in mean post-implantation loss was also apparent in the 50 mg/kg/day group. One female in the 50 mg/kg/day had all five of the late resorptions occurring in this study, which may be a secondary effect of maternal toxicity. Internal gross necropsy findings for females sacrificed at the scheduled laparotomy such as cystic ovaries, pitted kidneys, white foci or nodules on the lungs and hydronephrosis are considered normal for animals of this strain and age and could not be attributed to the compound.

Effect levels (maternal animals)

open allclose all
Dose descriptor:
NOAEL
Effect level:
5 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Dose descriptor:
LOAEL
Effect level:
25 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Maternal abnormalities

Abnormalities:
not specified

Results (fetuses)

Details on embryotoxic / teratogenic effects:
The infrequent occurrence of foetal malformations observed in this study was not indicative of a response to treatment with DPG; each study group, including the control, had one foetus with malformations. One foetus in the control group had multiple anomalies including vertebral agenesis, mandibular microagnathia, a dome-shaped head and microphthalmia. Situs inversus was observed in one foetus in the 5 mg/kg/day group, anophthalmia and internal hydrocephaly were observed in one foetus in the 25 mg/kg/day group and a thread-like tail with anal atresia was observed in one foetus in the 50 mg/kg/day group. Developmental variations observed in the DPG groups were similar to those in the control group except for an increase in the number of fetuses with unossified sternebrae (#5 and/or #6), reduced ossification of the thirteenth ribs, 25 presacral vertebrae and bent ribs in the 50 mg/kg/day group. Reduced ossification would be expected in view of the foetal body weight inhibition at this dose level. The increased number of foetuses with bent ribs in the 50 mg/kg/day dose group is probably associated with maternal toxicity. Although three foetuses from one dam in the 25 mg/kg/day group had bent ribs, the incidence is within the range of our historical control data. In addition, maternal toxicity was slight at this dose level and foetal body weight inhibition was not apparent. The expression of bent ribs at the 25 mg/kg/day dose level was not considered compound-related.

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fetotoxicity
Dose descriptor:
LOAEL
Effect level:
50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: fetotoxicity
Dose descriptor:
NOAEL
Effect level:
> 50 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity
Remarks on result:
not determinable due to absence of adverse toxic effects

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
In conclusion, DPG induced severe maternal toxicity at a dose level of 50 mg/kg/day. Fetotoxicity was also expressed at this dose level by a significantly reduced mean fetal body weight and by an increase in fetal variations. A dose level of 25 mg/kg/day was considered a marginal "no-effect" level for maternal toxicity; a fetotoxic response was not apparent. A dose level of 5 mg/kg/day was considered a "no-effect" level.
Executive summary:

Potential maternal, embryotoxic and teratogenic effects of DPG were evaluated in this study in rats. DPG was admixed in 0.5% aqueous Methocel and administered orally by gavage to three groups of 25 bred Charles River COBS CD female rats as a single daily dose from days 6 through 15 of gestation. Dose levels of5, 25 and 50 mg/kg/day were selected. For comparative purposes, 25 control females were concurrently dosed with 0.5% aqueous Methocelon a comparable regimen at 10ml/kg/day. Throughout gestation, all females were observed twice daily for toxicity and body weights were recorded at appropriate intervals. On day 20 of gestation, all surviving females were sacrificed for Cesarean section; fetuses were weighed, sexed and examined for external, skeletal and soft tissue anomalies and developmental variations.No unscheduled deaths occurred in any study group. Severe clinical signs oftoxicity, decreased maternal body weights and body weight gains, a slight increase in postimplantation loss and a significantly decreased mean fetal weight were evident inthe50mg/kg/day dose group. A slight increase in fetuses with reduced ossification (associated with reduced fetal weights) and an increase in bent ribs (attributed to maternal toxicity in this group) were observed at the 50 mg/kg/day dose level. Scattered, infrequent clinical findings and a slightly reduced body weight gain over the treatment period (gestation days 6-16) occurred at the 25 mg/kg/day dose level.The 5 mg/kg/day group was comparable to the vehicle control group in all parametersmeasured. The infrequent occurrence and nature of the malformations were not indicative of a teratogenic response in any dose group.