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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
other: On-going experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
read-across: supporting information

Data source

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

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Type:
impurity
Test material form:
liquid
Details on test material:
Batch No. : FAB-03082016
Purity : 96.5% (sum of the two main constituents)
Name of test material (as cited in study report): alpha-pinene multiconstituent
Physical state: colourless liquid
Storage Conditions: +2°C to +8°C, under nitrogen and protected from light
Expiry Date: 02 August 2017

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
- Age at study initiation: days
- Weight at study initiation: g for males; g for females
- Housing:
- Diet:
- Water:
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES
Not available yet

Administration / exposure

Route of administration:
oral: feed
Details on exposure:
DIET PREPARATION

- Frequency of preparation: not available yet
- Storage of formulation: not available yet
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: After a minimum of two weeks of treatment, males and females were paired on a one-to-one basis from within the same treatment group for a period of up to two weeks.
- Proof of pregnancy: Day on which evidence of mating was found (Ejected copulation plugs in cage tray and sperm in the vaginal smear) was designated Day 0 of gestation.
- After successful mating each pregnant female was caged: 1/cage
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
F0 animals:
Males - for a minimum of four weeks.
Females - for 15 days before pairing until Day 6 after birth of F1 generation.
F1 animals: No direct treatment
The F1 generation received no direct administration of the test substance; any exposure to the test substance or metabolites was through the mother to the offspring in utero and/or through the milk.
Frequency of treatment:
Continuously
No. of animals per sex per dose:
10
Details on study design:

- Rationale for animal assignment: Randomly allocated on arrival. Before Day 1 of treatment, animals were weighed and tattooed. On Day 1 (before dosing) variations in body weight of animals were confirmed to be within ±20% of the mean for each sex.
Positive control:
None

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage-trays were inspected daily for evidence of ill-health amongst the occupant(s).
During the acclimatisation period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: A detailed physical examination was performed on each animal to monitor general health at the following time points:
F0 males: Once each week
F0 females: Once each week before pairing, on Days 0, 7, 14 and 20 after mating and on Days 1 and 7 of lactation.

BODY WEIGHT: Yes
- Time schedule for examinations:
F0 males: Before dosing on the day that treatment commenced (Day 1), weekly thereafter and before necropsy.
F0 females: Before dosing on the day that treatment commenced (Day 1), weekly before pairing, on Days 0, 3, 7, 10, 14, 17 and 20 after mating, on Days 1, 4, and 7 of lactation and before necropsy.

FOOD CONSUMPTION: Yes
- The weight of food supplied, that remaining and an estimate of any spilled was recorded as follows:
F0 males: Weekly before pairing.
F0 females: Weekly before pairing, on Days 0-2, 3-6, 7-9, 10-13, 14-16 and 17-19 after mating and on Days 1-3 and 4-6 of lactation.
From these records the mean weekly or daily consumption per animal (g/animal/week or g/animal/day) was calculated for each phase.

WATER CONSUMPTION: Yes
- Fluid intake was assessed by daily visual observation.

OTHER:
PARTURITION OBSERVATIONS AND GESTATION LENGTH:
Duration of gestation: Time elapsing between the detection of mating and commencement of parturition.
Parturition observations: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.
Oestrous cyclicity (parental animals):
Dry smears: Daily smears were taken for 15 days before pairing, using cotton swabs moistened with saline. Smears were subsequently examined to establish the duration and regularity of the oestrous cycle.
Wet smears: After pairing with the male, daily smearing was continued using pipette lavage, until evidence of mating was observed.
Litter observations:
PARAMETERS EXAMINED
Clinical observations: Examined at approximately 24 hours after birth (Day 1 of age) and then daily thereafter for evidence of ill health or reaction to treatment; these were on an individual offspring basis or for the litter as a whole, as appropriate.
Litter size: Daily records were maintained of mortality and consequent changes in litter size from Days 1 to 7 of age.
Sex ratio of each litter: Recorded on Days 1, 4 and 7 of age.
Individual offspring body weights: F1 offspring - Days 1, 4 and 7 of age.

GROSS EXAMINATION OF DEAD PUPS: Yes
Postmortem examinations (parental animals):
SACRIFICE
All adult animals were killed by carbon dioxide asphyxiation.
Male animals: All surviving F0 males were killed during Week 5 of treatment.
Maternal animals:
- F0 females surviving until the end of the scheduled study period were killed on Day 7 of lactation.
- F0 females that failed to produce a viable litter were killed on Day 25 after mating.

GROSS NECROPSY
All adult animals were subject to a detailed necropsy.
- After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.
- The number of uterine implantation sites were checked after staining with ammonium sulphide (modification of the Salewski staining technique (Salewski 1964)), for females failing to produce a viable litter only.

ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for adult animals killed at scheduled necropsy as listed in the table 7.8.1/1.

HISTOPATHOLOGY
Fixation: Tissues were routinely preserved in 10% Neutral Buffered Formalin with the exception of those detailed below:
Testes Initially, in modified Davidson’s fluid.
Histology: Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: All adult animals of Groups 1 and 4 killed at scheduled termination.
Abnormalities only: All adult animals.
Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.
Light Microscopy: Tissues preserved for examination were examined as follows:
Terminal sacrifice:
All adult animals in Groups 1 and 4: All specified in Table 7.8.1/1
All adult animals from all groups: Abnormalities

For the assessment of the testes, a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. The examination was conducted in order to identify treatment related effects such as missing germ cell layers or types, retained spermatids, multinucleate or apoptotic germ cells and sloughing of spermatogenic cells in the lumen. Any cell- or stage-specificity of testicular findings was noted.
For the assessment of the ovaries a qualitative evaluation of one section from each ovary was made.
Postmortem examinations (offspring):
SACRIFICE
All offspring were killed by an intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
F1 generation offspring
- Offspring at scheduled termination: Examined externally; those offspring deemed normal were discarded without further macroscopic examination. Any externally abnormal offspring were examined internally and any abnormal tissues were retained.
- Premature deaths (before Day 7 of age): Missing offspring and those grossly autolysed or grossly cannibalised could not be examined. All other offspring dying before Day 7 of age were subject to fresh macroscopic examination, where possible (external and internal); this also included an assessment for the presence of milk in the stomach, where this was possible.
Abnormal tissues retained.
Statistics:
See section “Any other information on materials and methods incl. tables”
Reproductive indices:
Percentage mating = (Number animals mating / Animals paired) x 100
Conception rate (%) = (Number animals achieving pregnancy / Animals mated) x 100
Fertility index (%) = (Number animals achieving pregnancy / Animals pairing) x 100
Gestation index (%) = (Number of live litters born / Number pregnant) x 100
Offspring viability indices:
Post-implantation survival index (%) = (Total number of offspring born / Total number of uterine implantation sites) x 100
Live birth index (%) = (Number of live offspring on Day 1 after littering / Total number of offspring born) x 100
Viability index (%) = (Number of live offspring on Day 7 after littering / Number of live offspring on Day 1 after littering) x 100

Results and discussion

Results: P0 (first parental animals)

Reproductive function / performance (P0)

Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed

Effect levels (P0)

Based on:
test mat.
Remarks on result:
not measured/tested
Remarks:
Ongoing study

Results: F1 generation

Effect levels (F1)

Remarks on result:
not measured/tested

Results: F2 generation

Effect levels (F2)

Remarks on result:
not measured/tested

Overall reproductive toxicity

Reproductive effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Study currently on-going.
Executive summary:

A reproduction toxicity study in rats is currently on-going according to guideline OECD 421.