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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:
29 September 2015 - 29 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed according to OECD Guideline and under GLP conditions. Fully adequate for assessment.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
other: Pale yellow, viscous liquid
Details on test material:
Name: Terpenes and terpenoids, turpentine oil, alpha-pinene
fraction, oligomers CAS No. 70750-57-1
Batch/Lot No.: 1001135604
Description: Pale yellow, viscous liquid
Manufacture date: 19 May 2015
Expiry date: 19 May 2016
Purity: 100%
Storage condition: Frozen (≤ -15 C), protected from light
Safety Precautions: Routine safety precautions (lab coat, gloves, safety glasses and face mask) for unknown materials were applied to assure personnel health and safety.

Test animals

Species:
rat
Strain:
other: Hannover Wistar rats (Crl:WI(Han))
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D97633, from SPF colony
- Age at study initiation: Young adult female rats, nulliparous and non-pregnant, at least 11 weeks old
- Weight at study initiation: 182-232 g
- Fasting period before study: No
- Housing: Standard laboratory conditions during the study
- Diet and Water: The animals were provided with ssniff ® SM R/M-Z+H “Autoclavable Complete Feed for Rats and Mice – Breeding and Maintenance” (ssniff Spezialdiäten GmbH, D59494 Soest Germany) and tap water as for human consumption, ad libitum.
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.4-24.9ºC (range: 22 ± 3 °C)
- Humidity (%): 36-69 (range: 30 - 70 %)
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

STUDY SCHEDULE
Start of experiment : 29 September 2015 (first mating, when the females were at
least 11 weeks old)
Start of treatment : 05 October 2015 (first Gestation Day GD6)
End of treatment : 27 October 2015 (last Gestation Day GD19)
End of experiment : 28 October 2015 (last necropsy day)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Amount of vehicle (if gavage): A constant volume of 3 mL/kg body weight was administered to all dose groups, including the controls. The individual volume of the treatment was based on the most recent individual body weight of the animals.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Corn oil was selected based on the formulation and analytical trials in agreement with the Sponsor.
- Amount of vehicle (if gavage): 3 mL/kg body weight
- Lot/batch no. (if required): MKBQ9948V
- Manufacturer: Sigma-Aldrich Co.
- Expiry/Retest Date: 31 January 2016
- Storage: Room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
-The test item was formulated in the vehicle, corn oil at the appropriate concentrations according to the dose level and volume selected, in the Pharmacy of CiToxLAB Hungary Ltd.
- Formulations were prepared daily, fresh prior to administration to animals.
- Duplicate samples were taken from test item formulations (from the top, middle and bottom of the container) once during the first week of treatment.
Details on mating procedure:
- Oestrus cycle examination: shortly before start of pairing
- Pairing: After acclimatisation the females were paired according to their oestrus cycle with males in the morning for approximately 2 hours (1 male: 1 female) until at least 24 sperm positive females/group are attained.
- Proof of pregnancy: After the daily mating period, a vaginal smear was prepared and stained with 1% aqueous methylene blue solution. The smear was examined with a light microscope; the presence of a vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (GD0). Sperm positive females were separated and caged individually.
Duration of treatment / exposure:
Start of treatment : 05 October 2015 (first Gestation Day GD6)
End of treatment : 27 October 2015 (last Gestation Day GD19)
Frequency of treatment:
daily
Duration of test:
29 September 2015 (first mating, when the females were at least 11 weeks old)
Start of treatment : 05 October 2015 (first Gestation Day GD6)
End of treatment : 27 October 2015 (last Gestation Day GD19)
End of experiment : 28 October 2015 (last necropsy day)
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
nominal conc.
Control
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
nominal conc.
Low dose
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
nominal conc.
Mid dose
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
nominal conc.
High dose
No. of animals per sex per dose:
24
Control animals:
yes, concurrent no treatment
Details on study design:
Dose selection rational: In the DRF study doses of 100, 300 and 1000 mg/kg bw/day were administered by oral gavage to pregnant rats from GD6 to GD19. In the DRF study, 6 animals were examined in the control, 100 and 300 mg/kg bw/day dose groups, while 8 animals were examined in the 1000 mg/kg bw/day dose group. No maternal and/or foetal toxicity was detected at dose levels up to and including 1000 mg/kg bw/day. Based on the above results, doses of 100, 300 and 1000 mg/kg bw/day were selected for the main study.
Rational administration route: The oral route is a possible route of exposure to the test item in humans and is considered suitable to provide the systemic exposure required on this developmental toxicology study.

Examinations

Maternal examinations:
Clinical observations and mortality
- Morbidity and mortality: twice daily (at the beginning and end of each working day).
- Cage-side clinical observations: daily
- Detailed clinical observations: onset of treatment (GD6) then weekly.
- pertinent behavioural changes and signs of toxicity including mortality, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in gait,
posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), bizarre behaviour (e.g. selfmutilation, walking backwards), tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma.
- On GD13 and/or 14, the sperm positive females were examined for the presence of vaginal bleeding or “placental sign” (intrauterine extravasation of blood as an early sign of pregnancy in rat, which is considered to confirm implantation).

Body weight measurement
- The body weight of each animal was recorded on GD0, 3, 6, 8, 10, 12, 14, 16, 18 and 20

Food consumption measurement
- Food was measured on GD0, 3, 6, 8, 10, 12, 14, 16, 18 and 20. Food consumption was calculated for each interval, including GD6-20 and GD0-20.

Pathology
Caesarean section and necropsy
- Visceral examination: observation for structural abnormalities or pathological changes

Histopathology
Microscopic examination of three heads was performed to provide additional information for reporting.






Ovaries and uterine content:
- Ovaries and uterus examinations
- Cervix weight
- Early and late embryonic or foetal deaths, number of corpora lutea in each ovary
- Implantation sites in each uterine horn, the number of live foetuses, early and late embryonic death and foetal death, the number and percent of pre- and post-implantation losses
- Degree of resorption
- Placentas examinations (macroscopical examination)
Fetal examinations:
- Number of live foetuses
- Foetus weight (accuracy 0.01 g)
- External examination of the foetuses
- Examination of the great arteries
- The gender of foetuses was determined according to the anogenital distance.

For the foetuses subjected to skeletal examination, the abdominal region was opened and the viscera and skin of foetuses were removed and the cadaver and examined by means of a dissecting microscope.


Statistics:
Maternal Data:
- Clinical signs (by gestation day)
- Body weight and body weight gain: mean ± SD
- Corrected body weight on GD20 (body weight-gravid uterine weight) and corrected body weight gain: mean ± SD
- Food consumption: mean ± SD
- Pathology findings
- Gravid uterine weight

Caesarean Section and Necropsy Data:
- Number of corpora lutea: mean ± SD
- Number of implantations: mean ± SD
- Number and percent of live foetuses: mean ± SD
- Number and percent of intrauterine mortality: mean ± SD

Indices:
- Pre-implantation loss: %, group mean
- Post-implantation loss: %, group mean
Foetal Data:
- Sex distribution: %, group mean
- Foetal body weight (accuracy 0.01 g): mean ± S.D.
- External abnormalities/litter: %, group mean

Historical control data:
Control group, as well as with the relevant historical control data were included in all statistical calculations.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
No maternal toxicity was observed

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
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:
No embryotoxicity and/or teratogenicity was observed

Effect levels (fetuses)

open allclose all
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
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

DOSE FORMULATION ANALYSIS

Test item content of the dosing formulations was determined twice during the treatment period. All formulations were found to be in the range of 94-104% of nominal concentrations (33.3, 100 and 333 mg/mL) and were homogenous. Formulations had been shown to have good stability.

CLINICAL SIGNS AND MORTALITY

No test item related mortality and clinical signs occurred during the study at all dose levels.

MATERNAL BODY WEIGHT AND BODY WEIGHT GAIN

No toxicologically significant changes, adverse effects or effects considered related to test item administration were observed in the mean body weights or body weight gain values of the test item-treated evaluated dams when compared to control.

MATERNAL FOOD CONSUMPTION

There were no adverse, toxicologically significant effects considered related to test item administration, or relevant differences to the historical data available in the mean daily food consumption of the pregnant rats at any of the dose levels tested. No adverse effects were noted on the mean food consumption values of the evaluated animals in the test item treated groups when compared to control animals following daily administration to pregnant damns from GD6 to 20, at dose levels of up to and including 1000 mg/kg bw/day.

NECROPSY FINDINGS OF THE DAMS

No remarkable internal or external observations were recorded for any pregnant animals.

CAESAREAN SECTION AND PREGNANCY DATA

Pregnancy data

Ninety six females (24 in each group) were mated in the study. The number of confirmed pregnant, evaluated dams was 23, 24, 23 and 22 in the Control, Low, Mid and High dose groups, respectively

Intrauterine mortality, corpora lutea and implantation sites

The mean number of corpora lutea and the mean number of implantation sites were comparable with the controls in all treated groups. No adverse effect was observed in pre-implantation loss of the test item treated groups when compared to the control. The early and the late embryonic loss did not differ significantly from the control in the test item treated groups. There was no significant difference in the post-implantation loss between the test item treated and control groups. The total intrauterine mortality was comparable with the control, although there was one animal in the High dose group with 100% total intrauterine mortality. There was no statistically significant difference in foetal death compared to the control.

Viable foetuses and their sex distribution

The mean number of viable foetuses was comparable with the control mean. There was no toxicologically significant difference in the sex distribution of foetuses between the control and treatment groups.

Evaluation of placentae

There were no toxicological significant differences in the external morphology of placenta at evaluation of the treated groups compared to controls.

The weight of foetuses in the Low, Mid and High (100, 300 and 1000 mg/kg bw/day, respectively) did not differ significantly from the control mean value, when evaluated by both litter mean and group mean.

EVALUATION OF THE FOETUSES

The total number of retarded foetuses (with a weight of < the concurrent control mean- 2SD) in the Mid dose group was statistically higher than in the Control group. However, there was no clear dose-response as the incidence of body weight retarded foetuses was similar in the Control, Low and High dose groups. The number of affected litters were comparable between all groups, the difference was caused by two litters containing 100% and 50% body weight retarded foetuses in the Mid dose group. This statistical difference was not considered to be related to test item, rather an incidental finding.

Foetal External Abnormalities and visceral examination:

In summary, all abnormalities observed at visceral examination in this study were considered as incidental findings; they corresponded with the concurrent study control data, with historical control (HC) data or were considered to be spontaneous events unrelated to treatment.

Skeletal examination:

All of the skeletal findings above occurred at an incidence within the current historical control (HC) range or the concurrent study control data, thus they were considered as incidental.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study, the following no-observed-adverse-effect levels were derived: NOAEL maternal toxicity: 1000 mg/kg bw/day NOAEL embryotoxicity: 1000 mg/kg bw/day NOAEL foetotoxicity: 1000 mg/kg bw/day NOAEL teratogenecity: 1000 mg/kg bw/day.
Executive summary:

The aim the study was to assess the effects of the test item Terpenes and terpenoids, turpentine oil, alpha-pinene fraction, oligomers CAS No. 70750-57-1 on the embryonic and foetal development (including the organogenesis period) of Hannover Wistar rats in their first pregnancy.

The dams (one control and three test item treated groups) were treated daily by oral (gavage) administration, at dose levels of 100, 300 and 1000 mg/kg bw/day. 

There was no unscheduled mortality or treatment related clinical signs in the study. No test item related effect was observed on body weight, body weight gain, corrected terminal body weight, corrected body weight gain / corrected net body weight gain or animal food consumption during the study. There were no toxicologically significant differences, or test item related-changes in the reproductive parameters examined in the conditions of this study. The number of corpora lutea, implantation sites and pre-implantation loss mean values were comparable in the Control or test item treated groups at all of the dose levels tested.

There were no effects considered related to test item administration on the early and late embryonic loss, post-implantation (total resorption, including the early and late embryonic loss) or total intrauterine mortality in the test item-treated dams evaluated. No remarkable internal or external observations were recorded for any pregnant animals during necropsy. The mean number of viable foetuses in all test item treated groups was comparable with the control mean. The sex distribution of foetuses did not differ significantly between the control and treatment groups both for mean and absolute numbers. There were no toxicologically significant differences in the placenta of the test item treated groups. The litter mean and group mean weights of foetuses in the Low, Mid and High dose groups did not differ significantly from the control mean. The number of body weight retarded foetuses (with a weight of < the concurrent control mean -2SD) in the Mid dose group was statistically higher than in the Control group, although the incidence of body weight retarded foetuses was similar in the Control, Low and High dose groups. All abnormalities observed at external, visceral or skeletal examination in this study were considered as incidental findings since they occurred at a incidence similar to the concurrent study control data, within the historical control (HC) incidence range, or were considered to be spontaneous events unrelated to treatment.

Based on the results of this study, the following no-observed-adverse-effect levels were derived: NOAEL maternal toxicity: 1000 mg/kg bw/day NOAEL embryotoxicity: 1000 mg/kg bw/day NOAEL foetotoxicity: 1000 mg/kg bw/day NOAEL teratogenecity: 1000 mg/kg bw/day.