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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:
From 21.04.1992 to 06.10.1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Test method according to OECD guideline 414 and GLP, but only two dose levels were tested.

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 two dose levels were tested)
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: solid
- Physical state: vitreous, colourless waxy-crystalline
- Analytical purity: 78 %
- Lot/batch No.: Op. 54/90
- Stability under test conditions: For 12 months at 25 ºC; the solution in sesame oil, DAB, is stable for 4 hours.
- Storage condition of test material: Cool in a well-closed container

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Lippische Versuchstierzucht, HAGEMANN GmbH, D-4923 Extertal 1
- Age at study initiation: ca. 54 days old
- Weight at study initiation: 180 - 190 g
- Housing: The dams were kept singly in MAKROLON cages type III.
- Diet (e.g. ad libitum): ALTROMIN 1314 (supplied by: ALTROMIN GmbH, P.O.Box 285, D-4937 Lage/Lippe) served as food, ad libitum.
- Water (e.g. ad libitum): Tap water, ad libitum.
- Acclimation period: Seven days

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

IN-LIFE DATES: From: 28.04.1992 To: 05.08.1992

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: sesame oil, DAB 10
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance-vehicle mixtures were freshly prepared each day immediately before dosing.
Volume of administration: 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the determination of the concentration in the substance-vehicle preparations, 2 samples of 10 mL (each one for the concentrations 50.0 mg/Camphene/mL suspension and 200.0 mg Camphene/mL suspension) were analyzed at the start and at the end of treatment.
The determination of the concentrations was performed by gaschromatography using a FID detector.
Details on mating procedure:
Fertile ('proved') 4 - 12 months old male rats of the same breed served as partners. They were repeatedly employed, at the earliest three days after successful copulation. The female breeding partners were randomly chosen.
Matings were monogamous.
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Duration of treatment / exposure:
From the 6th to 15th day of pregnancy
Frequency of treatment:
Daily
Duration of test:
20 days (from day 0 to day 20 of pregnancy)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
250 mg/kg bw/day
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
nominal conc.
No. of animals per sex per dose:
20 pregnant female rats per group (plus 15 reserve animals)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
During a pilot study using 3 pregnant rats from the same strain as selected for the main experiment, a Camphene dose-Ievel of 1000 mg/kg b.w., by gavage, was administered from day 6 to 15 of pregnancy. 1000 mg/kg b.w. is the highest requested dose according to the
OECD method 414 (limit test for embryotoxicity).
Except for a slight transient reaction after the first dosing, 1000 mg/kg b.w. was well-tolerated by the dams. 1000 mg/kg b.w. did not influence the prenatal development. Based on the afore-described results, 250 mg and 1000 mg Camphene/kg b.w./day, by gavage, treatment from the 6th to 15th day of prenancy were selected by the sponsor as doses for the main experiment.

Examinations

Maternal examinations:
Daily checks (once in the morning and once as late on the day as practicable) were performed on behaviour, external appearance, mortality and faeces.
Body weight was ascertained daily - always at the same time in the morning - and these measurements were also used for calculating the daily amount of test compound to be administered.
Body weight change: The difference between the body weight on the day of weighing and the body weight on the day -4 weighing was calculated.
Food consumption was determined daily by weighing the residue. Intake of drinking-water was observed daily.
Ovaries and uterine content:
On the 20th day of gestation the surviving rats were laparotomised under ether narcosis. The ovaries and uteri were removed and examined. To check for possible drug effects a dissection was carried out which included macroscopic examination of internal organs. A full macroscopic inspection was carried out in the prematurely deceased dams as soon as possible after their exitus.
Fetuses were removed and the following examinations performed:
A count was taken of fetuses and placentae.
Sex and viability of fetuses were determined. Animals are said to be viable when they are found alive (spontaneous breathing, spontaneous movement).
Number and size of resorptions were determined.
Corpora lutea in the ovaries, implantations and location of fetuses in the uterus were determined.
The uterus weight was determined (with and without fetuses), weight of the ovaries was determined.
Weights and length of fetuses and weight of the placentae were determined (fetuses were considered as runts if their weight was less than 70% of the
mean litter weight).
Fetal examinations:
Fetuses were inspected externally for damages, especially for malformations.
Fetuses were dissected and the number and type of possible variations (incl. retardations) or malformations was determined macroscopically:
In 50% of the number of fetuses/L thorax and peritoneal cavity (without damage to ribs and sternum) were opened; location, size, and condition of the
internal organs were determined. Then the skeletons were stained with Alizarin according to DAWSON, the skeletal system was examined. Determination of the number and an examination of retardations, variations as well as malformations was carried out.
In 50% of the number of fetuses/L body sections were made and examined for variations according to WILSON.
Statistics:
The comparison of malformation and variation rates was carried out using R.A.FISHER's exact test (p < = 0.05). All other data were evaluated in the following way:
Homogeneity of variances were tested by the Bartlett chi-square test, and - if the variances were homogeneous - a one-way analysis of variance was applied. When the results indicated a significant difference among groups the DUNNETT test (p <= 0.01) was used to compare the experimental groups with the control group.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
No substance-related mortality was observed. Six of the 20 dams treated with 1000 mg/kg bw/day showed reduced motor activity and salivation after first dosing, two of them salivation after second dosing. The reactions occurred within 5 - 20 min after administration and lasted for 20 - 60 min, 1-2 hrs or 2-6 hrs. No clinical signs were observed in the remaining high-dosed and the low-dosed dams. Body weights remained within the normal range, body weight gain showed no influence of the test compound, also. Transient impairment of the food consumption by the highest tested dose (1000 mg/kg b.w.) was observed on the 7th, 8th and 9th gestation day by 6%, 22% and 10%, respectively. Treatment did not influence drinking-water consumption. No substance-related pathological changes were detected at autopsy.

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day
Basis for effect level:
other: maternal toxicity

Results (fetuses)

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

Details on embryotoxic / teratogenic effects:
1000 mg Camphene/kg b.w./day caused a slight but not significant (at p < = 0.01) increase of the resorption rate and - consequently - of the post-implantation loss (resorption rate and post-implantation loss substance-treated group: 11.5%, control group: 5.2%). No further influence on the prenatal development was detected. All other fetal parameters were within the normal range of the control group. External macroscopic inspection, examination of soft tissue (WILSON's method) and skeletal examination (staining of the skeleton according to DAWSON's method) revealed no substance-related variations and/or retardations. One malformed fetus at 1000 mg/kg (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). No dead fetuses occurred in the substance treated and control dams.

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Under the present test conditions, the NOEL for the dams and for the fetal organism was 250 mg Camphene/kg bw/day. Camphene did not possess teratogenic properties.

Applicant's summary and conclusion

Conclusions:
Under the present test conditions, the NOEL for the dams and for the fetal organism was 250 mg Camphene/kg bw/day.
Camphene did not possess teratogenic properties.
Executive summary:

Pregnant Sprague-Dawley rats were orally treated with the test substance by gavage during the 'critical' phase of organogenesis (daily from the 6th to 15th day of pregnancy). Test method was according to OECD guideline 414. Tested concentrations were 0, 250 and 1000 mg/kg bw/day, using sesame oil as vehicle. 20 pregnant rats were used in each group. Their development during the gestation period is observed. On day 20 of pregnancy the animals were laparotomised and examined macroscopically for implantation sites, resorptions in the uterus and for condition of the fetuses.

Influence on the dam:

No substance-related mortality was observed. Six of the 20 dams treated with 1000 mg/kg bw/day showed reduced motor activity and salivation after first dosing, two of them salivation after second dosing. The reactions occurred within 5 - 20 min after administration and lasted for 20 - 60 min, 1-2 hrs or 2-6 hrs. No clinical signs were observed in the remaining high-dosed and the low-dosed dams. Body weights remained within the normal range, body weight gain showed no influence of the test compound, also. 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 substance-related pathological changes were detected at autopsy.

Influence on the fetus:

1000 mg Camphene/kg bw/day caused a slight but not significant (at p < = 0.01) increase of the resorption rate and - consequently - of the post-implantation loss (resorption rate and post-implantation loss substance-treated group: 11.5%, control group: 5.2%). No further influence on the prenatal development was detected. All other fetal parameters were within the normal range of the control group. External macroscopic inspection, examination of soft tissue (WILSON's method) and skeletal examination (staining of the skeleton according to DAWSON's method) revealed no substance-related variations and/or retardations. 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). No dead fetuses occurred in the substance treated and control dams.

Under the present test conditions, the NOEL for the dams and for the fetal organism was 250 mg Camphene/kg bw/day. Camphene did not possess teratogenic properties.