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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:
June 1985 to July 1988
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1991

Materials and methods

Test guideline
Qualifier:
no guideline followed
Principles of method if other than guideline:
33 positively mated female Sprague-Dawley rats per group were administered zinc naphthenate in corn oil by oral gavage. The dosage levels were as follows: 0, 94, 188, and 938 mg/kg bw/day. A vehicle control group was also used during the study. The pregnant females were treated with the test substance day 6 of gestation throughout day 15 of gestation.
GLP compliance:
yes
Remarks:
US-GLP (Title 21, Code of Federal Regulations (CFR), 1990 rev, Part 58, Good Laboratory Practice for Nonclinical Laboratory Studies)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: tarry

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS - CRL:COBS-CD-(SD)BR colony animals
- Source: Charles River Breeding Laboratories
- Age at study initiation: 9 to 12 weeks
- Weight at study initiation: 208 - 265 g
- Housing: animals were housed in hanging-type cages 20 cm wide, 20 cm high and 30 cm deep for the one-on-one mating procedure. Following mating, females were housed three per unit in hanging wire cages 40 cm wide, 16 cm high and 35 cm deep.
- Diet (ad libitum): a certified pesticide-free rodent chow
- Water (ad libitum): water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.3 to 23.9 °C
- Humidity: 50 ± 5%
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Remarks:
Mazola
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Zinc naphthenate was mixed with corn oil to make a 250 mg/mL solution.

VEHICLE
- Amount of vehicle (if gavage): vehicle controls received 3.75 mL/kg/ day of corn oil.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
not stated
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused: one male with one female
- Proof of pregnancy: the occurrence of copulation was established by daily (morning) inspection for sperm plugs on the pad under the cage. A positive finding set day 0 of gestation.
- The mating procedure continued until there were at least 33 positively mated females in each dose group.
- Positively mated female rats were housed individually.
Duration of treatment / exposure:
day 6 of gestation and continued through day 15 of gestation
Frequency of treatment:
daily
Duration of test:
20 days
No. of animals per sex per dose:
33 mated female rats
Control animals:
yes, concurrent vehicle
Details on study design:
PILOT STUDY
The mating procedure consisted of housing one male with one female rat. The occurrence of copulation was established by daily (morning) inspection for sperm plugs on the pad under the cage. A positive finding set day 0 of gestation. Thirty-six positively mated female rats were housed individually, and assigned among five treatment and one control dosage group. Dosages of zinc naphthenate selected as fractions of the previously determined oral ALD, (7500 mg/kg) were 1875, 938, 469, 235 and 118 mg/kg /day. Single daily doses of the compound, in a 250 mg/ml corn oil solution, were administered by gavage beginning on day 6 of gestation and continued up to and including day 15 of gestation. The control group received the vehicle only, (7.50 mL/kg) on a comparable regimen. Individual daily doses were based on the maternal animal's body weight on day 6 of gestation.
All females were observed daily for changes in appearance and behaviour. A gross necropsy was performed on all rats which died before the scheduled sacrifice day. All females were weighed on gestation days 0, 6, 10, 13, 16 and 20. On the morning of the twentieth day of gestation, each female (dam) was sacrificed by carbon dioxide (CO2) inhalation and the uterus and ovaries exposed by laparotomy. The number and location of viable foetuses, nonviable foetuses, resorptions, total implantations, and corpora lutea were recorded. The dams were examined for gross pathological changes before being discarded. Foetuses were individually weighed, measured, sexed, and examined for external anomalies before being discarded. These foetal examinations were conducted to screen for potential foetal toxicity and/or teratogenicity.
The lowest dosage level significantly affecting group maternal weight gain or producing other outward signs of group maternal toxicity was chosen as the highest dosage level for the teratology study.

Results:
All females receiving zinc naphthenate, 1875 mg/kg/day, were pregnant. Only one of these dams lived for the duration of the study. However, none of her conceptuses were viable. Observable maternal toxic signs at this dosage level consisted of lethargy, brown/urine-stained urogenital areas, red
nasal discharge, and generalized alopecia.
All pregnant females receiving zinc naphthenate, 938 mg/kg/day, lived for the duration of the study. Pups derived from these dams were externally normal and were not statistically different from the control pups in either weight or length. Observable maternal toxic signs at this dosage level consisted of moderate amounts of generalized alopecia.
All pregnant females receiving zinc naphthenate, 469 mg/kg/day, 235 mg/kg/day and 118 mg/kg/day lived for the duration of the study. Maternal rats at these dosage levels were asymptomatic. Their pups were externally normal.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: all females were observed daily for clinical and behavioural deviations from normal.

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: animals were weighed on days 0, 6, 10, 13, 16 and 20 of gestation.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): No
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
Females (dams) were sacrificed by CO2 inhalation.

Any rats found dead or moribund during the course of the study were submitted for gross necropsy.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes; this weight was subtracted from the terminal female body weight in order to determine absolute body weight gain/loss during gestation.
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of foetuses: Yes
Fetal examinations:
All foetuses were removed from the uterus and assigned a number, starting from the dam's upper right horn and proceeding to the dam's upper left horn. Weight, as well as gross observation and sexing of foetuses were recorded.
- External examinations: No
- Soft tissue examinations: Yes; even-numbered foetuses
- Skeletal examinations: Yes; odd-numbered foetuses
- Head examinations: No

Further observations:
- Number of runts was determined
Statistics:
- Statistical analyses were performed on maternal, litter, and foetal data. Only those differences between treated and control group values which were significant at p < 0.05 were reported. Analyses of foetal data were performed based on the litter as the experimental unit.
- Maternal body weight and body weight gain were analysed using a one-way analysis of variance followed by Dunnett's test.
- The number of corpora lutea, implantations and live foetuses per litter were analysed using the t-test.
- Percentage data, which included percent female (sex ratio), resorptions, malformations, variations and normal foetuses per litter, were transformed by the
angular transformation and analysed using a t-test.
- The percent of litters which contained a runt, resorption, dead foetus, malformation or variation was analysed using chi-square and the square root of chi-square. The percent of litters which contained all normal foetuses was analysed in the same manner.
- Foetal body weights were analysed by a nested one-way analysis of variance.
Indices:
The following group parameters were calculated or counted without statistical analysis (Palmer, 1978)*:
- Fertility index = (pregnant animals/positively mated animals at terminal sacrifice) x 100
- Gestation index = (viable litters/pregnant animals) x 100
- Index of alive foetuses = (alive foetuses/total foetuses) x 100
- Resorption index = (total number of resorptions/total number of implantations) x 100
- Malformation index = (total number of foetuses with malformations/total number of foetuses) x 100
- Variation index = (total number of foetuses with variations/total number of foetuses) x 100

* Reference:
A.K. Palmer, Handbook of Teratology, Vol IV, Chapter 8, "The Design of Subprimate Animal Studies," pp. 215-253, Plenum Press, New York, New York, 1978.
Historical control data:
not stated

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Dams receiving 938 mg/kg/day of zinc naphthenate had a significantly greater number of implantation sites on the average than control and lower dosage groups. Dams in that group also had a significantly higher average number of resorptions. Differences in those two parameters netted a result of no overall difference in foetuses per dam.
One pregnant female from each of the groups receiving zinc naphthenate at levels of 188 mg/kg/day and 938 mg/kg/day died from dosing errors.
Premortem signs for dams receiving zinc naphthenate at 938 mg/kg/day included brown-stained urogenital areas, red nasal and oral exudate, generalized alopecia, and lethargy.
Control dams and dams receiving zinc naphthenate at 188 mg/kg/day or 94 mg/kg/day were asymptomatic during the course of the study.
Maternal body weights and body weight gains for zinc naphthenate-treated rats, 938 mg/kg/day, were significantly lower on gestation day 10 then for any of the other dosage groups. However, by gestation day 13 up until the time of necropsy on day 20, there was no significant difference between maternal body
weights or body weight gain among any of the dosage groups.
There was a notable increase in the percentage of litters with resorptions among dams receiving 938 mg/kg/day of zinc naphthenate.

Effect levels (maternal animals)

Key result
Dose descriptor:
NOAEL
Effect level:
188 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
Foetal variations were significantly more prevalent in groups receiving zinc naphthenate, 188 and 94 mg/kg/day.
Foetuses from dams receiving zinc naphthenate at 938 mg/kg/day had significantly lower body weights, on the average, than control foetuses or foetuses of the lower dosage groups. Variances between foetal parameters of control and zinc naphthenate, 188 mg/kg/day and 94 mg/kg/day, were not significant.
No dose relationship was established for foetal variations, although, foetuses from dams receiving zinc naphthenate, 188 mg/kg/day, had a significantly higher incidence of variants than corn oil controls. Foetuses from dams receiving 938 mg/kg/day of zinc naphthenate showed a slightly higher incidence of variation than controls. There was no trend toward delayed ossification among foetuses from 938 mg/kg/day litters.

Effect levels (fetuses)

Key result
Dose descriptor:
NOAEL
Effect level:
188 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
fetal/pup body weight changes

Fetal abnormalities

Key result
Abnormalities:
effects observed, non-treatment-related

Overall developmental toxicity

Key result
Developmental effects observed:
no

Applicant's summary and conclusion

Conclusions:
NOAEL maternal: 188 mg/kg bw/d
NOAEL development: 188 mg/kg bw/d
Clinical signs of toxicity and a statistically significant lower gestational body weight were observed in maternal animals of the high dose group receiving 938 mg/kg bw/d.
Foetuses from dams receiving the highest dose level (938 mg/kg bw/d) had a significantly lower body weight, and there was a notable increase in the percentage of litters with resorptions in the high dose group. Other foetal and litter parameters were not affected by maternal treatment.
No treatment-related foetal variations or malformations were determined in pups of all dose groups.
In conclusion, oral administration of zinc naphthenate to female rats during the major period of organogenesis did not result in teratogenic effects. Development of foetuses was only affected at the high dose level where signs of maternal toxicity were observed.