Registration Dossier

Toxicological information

Developmental toxicity / teratogenicity

Currently viewing:

Administrative data

Endpoint:
developmental toxicity
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From April 28 to August 5, 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to OECD Guideline 414 with a minor deviation as only 2 doses were tested but up to the limit dose of 1000 mg/kg/day
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report Date:
1992

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
only 2 doses tested but up to the limit dose of 1000 mg/kg/day
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Name of test material (as cited in study report): Camphene
Substance type: Technical grade
Physical state: Vitreous, colourless, waxy-crystalline solid
Analytical purity: 78%
Lot/batch No.: 54/90
Stability under test conditions: 12 months at 25 °C; solution in sesame oil is stable for 4 hours
Storage condition of test material: Cool in a well-closed container
Solubility: medium solubility in sesame oil

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Lippische Versuchstierzucht, Hagemann GmbH, Germany
- Stock: Tif: RAI f (SPF)
- Age at study initiation: 54 days
- Weight at study initiation: 180-190 g
- Fasting period before study:
- Housing: Individually housed in MAKROLON cages type III
- Diet (e.g. ad libitum): ALTROMIN 1314 (ALTROMIN GmbH, Lage/Lippe, Germany), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): 50 ± 15%
- Photoperiod (hours dark / hours light): 12 hours dark / 12 hours light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sesame oil, DAB 10
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Test solutions were freshly prepared in sesame oil each day immediately before dosing

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test material was soluble in sesame oil
- Amount of vehicle (if gavage): 5 mL/kg bw
- Lot/batch no. (if required): 2009
- Source: Henry Lamotte, Bremen, Germany
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Concentrations: 50 and 200 mg/mL suspension
- Sampling interval: Sampled at start and termination of treatment
- Sample storage conditions before analysis: Samples were deep-frozen at -20 °C or cooler until dispatch
- Analytical method: Gas chromatography with FID detector
Details on mating procedure:
- Impregnation procedure: Cohoused
- If cohoused:
M/F ratio per cage: 1:1
Length of cohabitation: Overnight
- Proof of pregnancy: Sperm in vaginal smear referred to as Day 0 of pregnancy. If the smear was negative, mating was repeated.
- Fertile (proved) male rats of the same breed were repeatedly employed for mating, at the earliest 3 days after successful copulation.
Duration of treatment / exposure:
Day 6-15 of pregnancy
Frequency of treatment:
Once daily
Duration of test:
20 days
Doses / concentrations
Remarks:
Doses / Concentrations:
250 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
20 pregnant females per dose (plus 5 females per dose as reserve)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Doses were selected on the basis of a pilot study. In this limit test study, camphene (1000 mg/kg bw/day) was administered to 3 pregnant rats from Day 6 to 15 of pregnancy. Except for a slight transient reaction after the first dosing, 1000 mg/kg bw/day was well-tolerated by the dams and did not influence the prenatal development.
- Rationale for animal assignment (if not random): Rats were assigned to their respective group according to their mating day i.e. in a cyclic way following the listing of positive findings in the vaginal smear.

Examinations

Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cage side observations were included: Behaviour, external appearance, mortality and faeces

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Daily

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation Day 20 under ether
- Organs examined: Ovaries and uteri
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes

Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations and location of fetuses in the uterus: Yes
- Number and size of resorptions: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Other: Fetuses and placentae count, sex and viability of fetuses, weight of ovaries, weight and length of fetuses and weight of placentae
Fetal examinations:
- External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [half per litter]
- Skeletal examinations: Yes: [half per litter]
Statistics:
- Comparison of malformation and variation rates was carried out using R.A. Fisher’s exact test (p ≤ 0.05).
- Homogeneity of variances was tested by the Bartlett chi-square test followed by a one-way analysis of variance (ANOVA).
- When the results indicated a significant difference among groups Dunnett test (p ≤ 0.01) was used to compare the experimental groups with the control group.
Indices:
- Resorption rate in % = (Resorptions / implantations) X 100
- Malformation rate in % = (malformed fetuses / fetuses) X 100
- Variation rate in % = (fetuses with variations / fetuses) X 100
- Pre-implantation loss in % = (corpora lutea-implantations / corpora lutea) X 100
- Post-implantation loss in % = (implantations-living fetuses / implantations) X 100
- Conception rate in % = (number of pregnant animals / number of animals mated) X 100
Historical control data:
Yes; summarized results of teratology studies in Sprague-Dawley rats (1988-1991) were attached as Appendix-4 to the study

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
- Mortality: No treatment-related mortality was observed.
- Clinical symptoms: At 1000 mg/kg bw/day, 6/20 dams showed reduced motor activity and salivation after first dosing, two of them salivation after second dosing. The reactions occurred within 5-20 minutes after administration and lasted for 20-60 minutes, 1-2 hours or 2-6 hours. No clinical signs were observed in the remaining high-dosed and the low-dosed dams.
- Body weight, body weight change: Body weights remained within the normal range, body weight gain showed no influence of the test material.
- Food consumption: Transient impairment of the food consumption by the highest tested dose (1000 mg/kg bw/day) was observed on the 7th, 8th and 9th gestation day by 6%, 22% and 10%, respectively. Food consumption had normalised from the 10th gestation day onwards.
- Drinking-water consumption: Treatment did not influence drinking-water consumption.
- Autopsy findings: No treatment-related pathological changes were detected at autopsy.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
250 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:yes

Details on embryotoxic / teratogenic effects:
- Mortality: No dead fetuses observed in control or treatment groups.
- Uterus and placentae weights, conception rate, mean number of corpora lutea and implantation sites, pre-implantation loss and number of viable fetuses, sex distribution of fetuses and weight of fetuses: No treatment-related effects
- Number of resorptions and consequently the post-implantation loss: Slightly but not significantly (at p ≤ 0.01) increased at 1000 mg/kg bw/day
- External macroscopic examination: No treatment-related effects; one malformed fetus at 1000 mg/kg bw/day (shifted and fused dorsal, lumbar and coccygeal vertebrae, bilateral crossed legs, stump tail, omphalocele) belongs to the spontaneous range as to type and number of affected fetuses (control: one fetus with stump tail)
- Soft tissue examination: No treatment-related effects; very common variations (uni- or bilateral dilated renal pelvis, haemorrhages of the liver, 4th cerebral ventricle enlarged) were observed in all groups without any dose-response relationships
- Skeletal examination: No treatment-related effects; variations (accessory 14th ribs, wavy ribs and bipartite/misaligned sternum) were observed in all groups and/or retardations (incomplete or missing ossification of hyoid, skull, vertebral bodies and/or sternebrae) were found biologically non-significant

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Table 1: Examination of the influence on the pregnant rat and the fetus by oral administration summary of maternal clinical symptoms

 

Test Group 1: Control

Test Group 2: 250 mg/kg bw/day

Test Group 3: 1000 mg/kg bw/day

Dams evaluated

n =

20

20

22#

No remarkable observations

n =

20

20

14

%

100

100

63.6

Reduced motility

n =

0

0

6

%

0

0

27.3

Salivation

n =

0

0

7

%

0

0

31.8

Opisthotonus

n =

0

0

1*

%

0

0

4.5

Tonoclonic convulsions

n =

0

0

1**

%

0

0

4.5

* deceased dam no. 53; ** * deceased dam no. 56; # dams no. 53, 56 died prematurely due to regurgitation and aspiration of the test substance

Applicant's summary and conclusion

Conclusions:
In conclusion, the no-observed-effect level (NOEL) for maternal and fetotoxicity of camphene administered orally (gavage) was considered to be 250 mg/kg bw/day in Sprague-Dawley rats. Therefore, camphene is not considered to be teratogenic.
Executive summary:

In a prenatal developmental toxicity study performed in accordance with OECD guideline 414 and in compliance with GLP, camphene in sesame oil was administered through gavage to groups of Sprague-Dawley pregnant rats (20/dose) at dose levels of 0 (vehicle control), 250 and 1000 mg/kg bw/day from Day 6 to 15 of pregnancy. Dams were observed twice daily for behaviour, external appearance, mortality and faeces. Body weights, food and water consumption were noted daily. Caesarean sections were performed on Day 20 of pregnancy and the ovaries and uterine contents were recorded. Foetuses were weighed and examined for external, visceral and skeletal malformations.

 

No treatment-related mortality was observed at any dose level. At 1000 mg/kg bw/day, 6/20 dams showed reduced motor activity and salivation after first dosing, two of them salivation after second dosing. The reactions occurred within 5-20 minutes after administration and lasted for 20-60 minutes, 1-2 hours or 2-6 hours. Body weights remained within the normal range and body weight gain showed no influence of the test material. Transient impairment of the food consumption by the highest tested dose (1000 mg/kg bw/day) was observed on the 7th, 8th and 9th gestation day by 6%, 22% and 10%, respectively. Treatment did not influence drinking-water consumption. No treatment-related pathological changes were detected at autopsy. No dead fetuses observed in control or treatment groups. No treatment-related effects were observed on uterus and placentae weights, conception rate, mean number of corpora lutea and implantation sites, pre-implantation loss and number of viable fetuses, sex distribution of fetuses and weight of fetuses. External macroscopic examination, examination of soft tissue and skeletal examination revealed no treatment-related biologically and/or statistically significant variations/retardations. Number of resorptions and consequently the post-implantation loss were increased slightly (statistically non- significant) at 1000 mg/kg bw/day.

The no-observed-effect level (NOEL) for maternal and fetotoxicity of camphene administered orally (gavage) was considered to be 250 mg/kg bw/day in Sprague-Dawley rats. Therefore, camphene is not considered to be teratogenic.