Pin-2(3)-ene

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

OECD 443 study (K1): 

NOAEL for systemic toxicity = 6900 ppm for F0 ; NOAEL for systemic toxicity = 7300 ppm F1 Cohort 1A and 1B adult animals

(Mean achieved doses of 469 mg/kg bw/day for F0 males, 567 mg/kg bw/day for F0 females before pairing, 648 mg/kg bw/day for F1 males and 659 mg/kg bw/day for F1 females before pairing).

NOAEL for reproductive performance = 6900 ppm or 7300 ppm for the F0 and F1B parent animals and survival of the F1 and F2 offspring 

(mean achieved doses of 466 mg/kg bw/day and 1047 mg/kg bw/day during F0 gestation and lactation and 494 mg/kg bw/day and 1044 mg/kg bw/day during F1B gestation and lactation).

NOAEL for the growth of the F1 offspring (Days 1-21 of age) = 6900 ppm (mean achieved doses of 466 mg/kg bw/day and 1047 mg/kg bw/day) in F1 offspring and 7300 ppm (mean achieved doses of 494 mg/kg bw/day and 1044 mg/kg bw/day) in F2 offspring. 

 

Link to relevant study records

Reference

Endpoint:

extended one-generation reproductive toxicity - with F2 generation (Cohorts 1A, and 1B with extension)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 July 2020 to 20 October 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD 443 Guideline without deviations.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)

Version / remarks:

Adopted 25 June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Justification for study design:

SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS: as requested by ECHA in the final decision CCH-D-2114373437-42-01/F

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DRT / 1000082993
- Appearance: Colourless liquid.
- Expiration date of the lot/batch: 10 June 2021

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerated (2-8 °C) under nitrogen and kept away from light and humidity.

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on species / strain selection:

The rat was chosen as the test species because of the requirement for a rodent species by
regulatory agencies. The Crl:CD(SD) strain was used because of the historical control data
available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Limited.
- Age at study initiation: 26 to 32 days old.
- Weight at study initiation: Females 64 to 105g. Males 81 to 120g.
- Housing (see Table 1 below): Cages comprised of a polycarbonate body with a stainless steel mesh lid changed at appropriate intervals. Solid (polycarbonate) bottom cages were used throughout the study except during pairing. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
- Diet: SDS VRF1 Certified, powdered diet; Non-restricted (diet was removed overnight before blood sampling for hematology, blood chemistryand thyroid hormones and during the period of urine collection).The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes. Non-restricted (except during urine collection) and the bottles were changed at appropriate intervals. Certificates of analysis are routinely received from the water supplier.
- Acclimation period: 5 days.
- Environmental enrichment: During the acclimatization and appropriate study periods environmental enrichment in the form of Aspen wood based products (soft white untreated wood product) and a plastic shelter were available in each home cage.
Plastic shelters are not provided during pairing. Aspen wood based products and plastic shelters are not provided during lactation (from Day 20 after mating). From Day 20 after mating and throughout lactation, approximately two handfuls of paper shavings were provided to each cage as nesting material; this nesting material were changed at the same frequency as the cage bedding. See Table 2 below.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod: 12 h light : 12 h dark

IN-LIFE DATES: FROM: 15 July 2020 TO: 23 March 2021

Route of administration:

oral: feed

Vehicle:

corn oil

Remarks:

Ratio of the test item to corn oil of 5 to 1

Details on exposure:

RATIONALE FOR ROUTE OF ADMINISTRATION
Dietary, to simulate the conditions of possible human exposure.

DIET PREPARATION
- Rate of preparation of diet (frequency): No more than weekly.
- Mixing appropriate amounts with (Type of food): SDS VRF1(powdered) certified.
- Storage and stability of formulations: Frozen (-10 to -30ºC) until required for use; diets were removed from frozen storage very shortly before feeding. In Envigo Study No. CM17RD, treated diets were demonstrated to be stable for one day at 1000 ppm and two days at 15000 ppm at ambient temperature (15 to 25°C), and for 15 days following frozen storage (-10 to -30°C) at both concentrations.
- Method: On each occasion of the preparation of the premix the required amount of test item and corn oil (at a ratio of 5:1) were weighed into a suitable container. An amount of plain diet that approximately equaled the weight of test item was added and the mixturestirred together. A further amount of plain diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the premix was achieved or the mixture appeareddry. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test item was homogenouslydispersed in the diet. Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietaryconcentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer. For the control diet, an amount of diet was added directly to the corn oil and then prepared as indicated for the premix.

Details on mating procedure:

The F0 animals were paired on a 1:1 basis within each treatment group after 10 weeks of treatment and up to 2 weeks. The cohort 1B animals were paired on the same basis, approximately 10 weeks after selection. Sibling pairing was not permitted.
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm within vaginal smear.
- Day 0 of gestation: When positive evidence of mating detected.
- Male/female separation: Day when mating evidence detected.
- Pre-coital interval: Calculated for each female as the time between first pairing and evidence of mating.

Females showing no evidence of mating - following completion of the pairing period females were separated from the male and vaginal smearing continued for up to five days or until the first estrus smear is seen. If a female shows an estrus smear during this period, she was killed as soon as practically possible and subject to macroscopic examination.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 1000 to 15000 ppm were analyzed to assess the stability and homogeneity of the test item in the diet matrix (Covance Study Number CM17RD).
In Envigo Study No. CM17RD, treated diets were demonstrated to be stable for one day at 1000 ppm and two days at 15000 ppm at ambient temperature (15 to 25°C), and for 15 days following frozen storage (-10 to -30°C) at both concentrations.

Samples of each formulation prepared for administration in Weeks 1, 6, 7 and10 (F0 generation) and Week 1 and last week of treatment (F1 generation) were analyzed for achieved concentration of the test item.

Duration of treatment / exposure:

F0 ANIMALS: For 10 weeks before pairing until termination after litters were Day 28 of age.
F1 ANIMALS: From weaning until termination of respective cohort. Although direct exposure started at weaning on Day 21 of age, all offspring had potential indirect exposure in-utero and through the milk during lactation.
COHORT 1A : General toxicity and pathology of the tissues of the male and female reproductive systems. Treated from weaning to approximately 13 weeks of age.
COHORT 1B : Treated from weaning throughout pairing, gestation and lactation up to weaning of F2 litters.

Frequency of treatment:

Continuously. Animals were provided with fresh diet daily.

Dose / conc.:

0 ppm (analytical)

Remarks:

F0 : Group 1 (basal diet plus corn oil, to the same level as the Group 4 diet)

Dose / conc.:

2 000 ppm (analytical)

Remarks:

F0: Group 2 (test item)

Dose / conc.:

4 300 ppm (analytical)

Remarks:

F0 : Group 3 (test item)

Dose / conc.:

6 900 ppm (analytical)

Remarks:

F0 : Group 4 (test item)

Dose / conc.:

0 ppm (analytical)

Remarks:

F1 : Group 1 (basal diet plus corn oil, to the same level as the Group 4 diet)

Dose / conc.:

2 100 ppm (analytical)

Remarks:

F1 : Group 2 (test item)

Dose / conc.:

4 600 ppm (analytical)

Remarks:

F1 : Group 3 (test item)

Dose / conc.:

7 300 ppm (analytical)

Remarks:

F1 : Group 4 (test item)

No. of animals per sex per dose:

F0 generation: 25 animals/sex/dose
F1 generation: 20 animals/sex/dose
Spare animals were removed from the study room after treatment commenced.
See Table 3 below.

Control animals:

yes, plain diet

Details on study design:

DOSE SELECTION
Dietary concentrations of 0, 2500, 5500 and 8500 ppm were selected in conjunction with the Sponsor based on the findings from a preliminary reproductive study conducted at these laboratories (Covance Study no. VW31CD). In the preliminary study, in rats treated at 3000, 6000 or 12000 ppm, mean body weights from Day 9 of gestation and body weight gain throughout gestation were statistically significantly lower than Controls in the 12000 ppm groups (mean bodyweight: -14% than Controls at Day 21 of gestation; body weight gain during gestation: -25% than Controls). Body weights remained lower than Controls throughout lactation at this dose level. The effects on bodyweight at 12000 ppm were associated with statistically lower food consumption during gestation and lactation (-17% than Controls). Similarly, statistically significantly lower body weights than Controls were recorded in offspring of the 12000 ppm group (Day 1: -13% and -14% for males and females, respectively; Day 21: -24% for males and females). Also, body weight gain during Days 1-21 of age was statistically significantly lower than Controls (-26% in both sexes). In addition, diet exposure of pups to 12000 ppm led to a test item intake of 1548 and 1447 mg/kg bw/day in males and females, respectively, which is approximately 50% greater than the recommended maximum dose of 1000 mg/kg bw/day. Therefore, the dose level of 12000 ppm was considered too high as a top dose for the main study. In a conservative approach, the top dose of 8500 ppm was chosen in order to ensure that female offspring are exposed to the highest recommended dose of 1000 mg/kg bw/day. The dietary concentration of 2500 ppm was anticipated to be the NOAEL and 5500 ppm corresponds to the middle dose between low and high doses.
However, the formulation chemistry investigations performed during the study revealed lower than nominal dietary concentrations of alpha-pinene multiconstituent offered to both the F0 and F1 generations in all treated groups. To ensure the integrity and validity of the study, at the end of the study the dietary concentrations offered to the F0 and F1 were calculated based on the mean dietary concentrations determined in the formulation chemistry investigation (performed in Weeks 1, 6, 7 and 10 of the F0 generation and Week 1 and last week of treatment of the F1 generation).
It was determined that the F0 generation, from the start of treatment through to weaning of the F1, was treated at 2000, 4300 or 6900 ppm, and the F1 generation, following weaning, was treated at 2100, 4600 or 7300 ppm (Groups 2, 3 and 4, respectively).

ANIMAL GROUP SELECTION
- Allocation to treatment groups (F0 generation): Each F0 animals were allocated by sex, after a period of acclimatization. Animals showing signs of ill health were excluded. Animals at the extreme of the weight range were not selected if alternatives were available. At commencement of the study the body weight of animals did not exceed ±20% of the mean for each sex.
Where possible, two male and two female were selected from each selected litter and were allocated to each of the two cohorts. If more were required, up to two males and two females were selected from each selected litter. Selected animals were microchipped on Day 18 to 21 of age and separated from littermates on Day 21 of age. Up to two male and two female F1 offsprings per group were retained as spares, to provide potential replacement in the event of any mortality. These spares had body weights and clinical signs recorded weekly and were terminated after commencement of the F1 generation.

- Selection of Offspring to form F1 Generation: The selection of F1 animals was performed nominally on Day 18 to 21 of age. The offspring with the lowest within-litter identification per sex from each selected litter were selected to form the F1 generation, after exclusion of grossly atypical animals.

ANIMAL REPLACEMENT
For the F0 Generation; one male (Group 2; No. 44) was euthanized for welfare reasons on Day 6 of study, due to a mis located identity microchip and was replaced on Study Day 8 with a Spare male (No. 102) from the same batch of animals. For Cohort 1B; Group 3F, animal number 733 died on Day 5 of the formal F1 generation as a result of an accident and the animal was replaced. No data is reported for this animal.

Positive control:

No

Parental animals: Observations and examinations:

MORTALITY: Yes
- A viability check was performed near the start and end of each working day. Animals were killed for reasons ofanimal welfare where necessary.A complete necropsy was performed in all cases.

CLINICAL OBSERVATIONS & DETAILED CLINICAL OBSERVATIONS: Yes
F0 and F1 animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time inrespect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate. During the acclimatization period, observations of the animals and their cages were recorded at least once per day.
A detailed physical examination was performed on each animal to monitor general health.
- Time schedule: Once each week.
After mating of F0 and F1 Cohort 1B females: Days 0, 5, 12, 18 and 20 after mating and Days 1, 7, 14 and 21 of lactation.

BODY WEIGHT: Yes
F0 Males: Day that treatment commenced, each week, before necropsy
F0 Females: Day that treatment commenced, each week until mating detected, days 0, 6, 14 and 20 after mating, days 1, 4, 7, 14, 21 and 28 of lactation and before necropsy.
F1 selected animals: Days 21, 22, 25 and 28* of age, then each week.
F1 Cohort 1B females on Days 0, 6, 13 and 20 after mating and Days 1, 4, 7, 14, 21 and 28 post-partum. Before necropsy.
*Only applicable before formal commencement of the F1 generation at nominal 4 Weeks of age (Day 28 of age ±2 days).

FOOD CONSUMPTION: Yes
- F0 Males and Females: Daily, from the day that treatment commenced. Food consumption was not recorded for males and females during the period when paired for mating (Days 71 to 77), but recommenced for males on Day 78.Daily, for females after mating and during lactation.
- Selected F1 generation: Cohort 1A: daily from Day 1 of the Formal F1 generation until termination. Cohort 1B: daily from Day 1 of the Formal F1 generation until paired for mating. Daily, for females after mating and during lactation.Daily for F1B males during Study Days 106 to 108. From these records the mean or daily consumption per animal (g/animal/day) was calculated for each relevant phase.

HEMATOLOGY, PERIPHERAL BLOOD (F0 and F1A generation): Yes
Blood samples were collected after overnight withdrawal of food. Sampling was performed on the morning after overnight collection of urine. These animals were therefore deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Blood samples were collected under light general anesthesia (isoflurane) from sublingual vein for :
- F0 Adults: Ten male and nine female (one female (No. 283) was found dead on LD 22) animals per group
- F1 Cohort 1A: Ten male and ten female animals per group
All samples (0.5 mL) were examined for the following characteristics:
1) Using EDTA as anticoagulant: Hematocrit*, Hemoglobin concentration, Erythrocyte count, Reticulocyte count, Total leucocyte count, Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes and Large unstained cells), Platelet count, Mean cell hemoglobin*, Mean cell volume, Mean cell hemoglobin concentration*
2) Using citrate as anticoagulant: Prothrombin time, Activated partial thromboplastin time
* Derived values calculated in ClinAxys.

BLOOD CHEMISTRY (F0 and F1A generation): Yes
Blood samples were collected after overnight withdrawal of food. Sampling was performed on the morning after overnight collection of urine. These animals were therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of blood sampling procedures. Blood samples were collected under light general anesthesia (isoflurane) from sublingual vein for :
- F0 Adults: Ten male and nine female (one female (No. 283) was found dead on LD 22) animals per group
- F1 Cohort 1A: Ten male and ten female animals per group
All samples (0.7 mL) were examined for the following characteristics, using lithium heparin as anticoagulant: Alkaline phosphatase, Alanine amino-transferase, Aspartate amino-transferase, Gamma glutamyl transpeptidase, Glucose, Total bilirubin, Bile acids, Total cholesterol, Creatinine, Urea, Total protein, Albumin, Albumin/globulin ratio, Sodium, Potassium, Chloride, Calcium, Inorganic phosphorus.

BIOMARKERS - TSH AND T4 (F0 and F1 generation): Yes
F0 adults: Ten male and nine female (one female (No. 283) was found dead on LD 22) animals per group
F1 offspring: Ten litters per group - pooled litter sample Day 4 of age#; Ten male and ten female animals per group on Day 22 of age (from as many litters as possible).
F1 adults (1A): 10 animals/sex/group (approx 13 weeks of age)
- Conditions: Adults: Following overnight deprivation of food; Offspring Day 4 and Day 22 of age: no overnight deprivation of food.
- Sample site/Volume: Adults: Sublingual vein/1.0 mL; Offspring Day 22 of age: Orbital sinus/1.0 mL; Offspring Day 4 of age: Decapitation/maximum possible.
- Anesthesic: Adults and offspring on Day 22 of age: Isoflurane. Offspring Day 4 of age: not required.
- Maximum no of samples per occasion: F0 adults, F1 offspring Day 22 of age and F1A adults: 80 per analyte; F1 offspring Day 4 of age: 40 for T4 only.
- Overall maximum no. of samples for analysis: T4: 280 and TSH: 240

URINALYSIS (F0 and F1A generation): Yes
Urine samples were collected after overnight withdrawal of food and water atthe following occasion:
- F0 Adults: Ten male and nine female (one female (No. 283) was found dead on LD 22) animals per group
- F1 Cohort 1A: Ten male and ten female animals per group
Using manual methods: Clarity/colour (appearance), Volume, pH, Specific gravity.
Using semi-quantitative methods (Clinitek): Glucose, Ketone, Bilirubin (bile pigments), Blood pigments.
Using quantitative automated methods (Cobas 6000): Protein (total and concentration), Sodium (total and concentration), Potassium (total and concentration), Chloride (total and concentration)
The deposit, obtained from centrifugation, were examined microscopically for epithelial cell, leucocyte, erythrocytes, crystals, casts, spermatozoa and other abnormal components.

Oestrous cyclicity (parental animals):

DRY SMEARS
- F0 females only: For 15 days before pairing, using cotton swabs.
- Cohort F1A: For two weeks from approximately Day 75 of age, using cotton swabs.
WET SMEARS:
- F0: After pairing, using pipette lavage, until evidence of mating confirmed. For four days before scheduled termination (nominally Days 25 to 28 post partum). Females that failed to litter were retained and smeared for four days starting on the day on which the first batch of ‘true’ Day 25 post partum females started smearing, and were then killed with that first batch of females.
- Cohort F1B: For at least three days prior to the start of the necropsy phase and on the day of termination. Using pipette lavage.
- Cohort F1A: Following onset of vaginal patency until first cornified (estrus) smear was recorded. For at least three days prior to the start of the necropsy phase and onthe day of termination. Using pipette lavage.

Sperm parameters (parental animals):

Cohort 1A:
Immediately after scheduled sacrifice of each F1 Cohort 1A male and collection of blood, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed.
Vas deferens (from left side): Sperm sample (where possible at least 200) assessed for motility using a computer assisted sperm analyzer (CASA). Each animal in each group. A manual assessment of sperm morphology were performed. Each animal in Groups 1 and 4. In group 2 and 3, fixed samples were retained for possible future assessment.

Cauda epididymis (from left side): the cauda epididymis were weighed and homogenized and the number of sperm were counted using a computer assisted sperm analyzer (CASA). Each animal in Groups 1 and 4. In group 2 and 3, samples frozen were retained for possible future assessment.

Testis (from left side): the testis were homogenized and the number of homogenization-resistant spermatids were counted using a computer assisted sperm analyzer (CASA). Each animal in Groups 1 and 4. In group 2 and 3, samples frozen were retained for possible future assessment.

Litter observations:

CLINICAL OBSERVATIONS: Yes
Animals were observed approximately 24 hours after birth (Day 1 of age) and then daily for evidence of ill-health or reaction to maternal treatment. Each offspring were examined daily.
On Day 1 of age all offspring receive a qualitative assessment of body temperature, state of activity and reaction to handling. The weaning of offspring was in Day 22 of age.

LITTER SIZE: Yes
- Schedule: Daily records were maintained of mortality and consequent changes in litter size on Days 1-21 of lactation. On Day 4 of age, litters containing more than ten offspring were reduced to ten by random culling, leaving where possible, five male and five female offspring in each litter. Culled offspring were examined.

SEXE RATIO: Yes
Recorded Days 1, 4 (before and after culling) and Day 21 of age.

INDIVIDUAL OFFSPRING BODY WEIGHT: Yes
Recorded on Days 1, 4 (before culling), 7, 14, 21 and 22 of age (day of necropsy).

WEANING OF OFFSPRING: The dam was removed from the litter cage and offspring were weaned on Day 21 of age.

ANO-GENITAL DISTANCE: Offspring on Day 1 of age.

NIPPLE COUNT: Male offspring on Day 13 of age.

SEXUAL MATURATION: Yes
- Males: Sexual maturation was assessed by daily examination from Day 38 of age until balano-preputial separation occurred. Body weight was recorded on the day of completion of separation.
- Females: Sexual maturation was assessed by daily examination from Day 28 of age until vaginal opening occurred. Body weight was recorded on the day of vaginal opening. For Cohort 1A: a wet smear was taken daily from the day of vaginal opening until first estrus was detected.

Postmortem examinations (parental animals):

SACRIFICE
- F0 males: After weaning of the F1 animals, after confirmation that no further mating required.
- F0/F1B Females: Day 28 post partum.
- F0/F1B females failing to produce a viable litter and those with total litter loss: Terminated with first cohort of females with live litters.
- Method: Animals 14 days and older: Carbon dioxide. Each animal were subsequently exsanguinated.

MACROSCOPIC PATHOLOGY
A complete gross necropsy were performed on all animals, including surplus offspring culled on Day 4 of age and Day 22 of age unselected offspring. Any abnormality in the appearance or size of any organ and tissue was recorded and the required tissue samples preserved in appropriate fixative.
For F0 and F1 Cohort 1B females the implantation site count was recorded. For females of Cohort 1A, counts were performed for the number of ovarian follicles and corpora lutea. The organs weighed, tissue samples fixed and sections examined microscopically.

ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together, unless specified in the relevant pathology procedures table. Requisite organs were weighed for animals killed at scheduled intervals.

FIXATION
For F0 animals, unselected F1 animals (including decedent offspring), Cohorts 1A, 1B and F2 offspring (including decedents).
- Standard: 10% Neutral Buffered Formalin. Where possible, carcass retained for decedent offspring ≤ 21 days of age.
- Testes (F0 and F1 adults only): modified Davidson’s fluid.
- Eyes: Davidson’s fluid.

HISTOLOGY
Processing to slide:
- Full List: All adult animals killedor dying prematurely. F0/Cohort 1A: All terminal adult animals of Groups 1 and 4 killed at a scheduled interval.
- Reproductive Organs: from F0 animals in Groups 2 and 3 that showed reduced fertility. This included males that failed to sire a pregnancy and females that were not pregnant or females with a litter death.
- Abnormalities only: F0/Cohort 1A: All terminal adult animals of Groups 2 and 3 killed at a scheduled interval.Cohort 1B: All animals.

Processing to block:
- Reproductive Organs: Cohort 1B: All animals.

Staining:
- Routine staining: 4-5 µm sections stained with haematoxylin and eosin.
- Special staining: None scheduled.

IMMUNOPHENOTYPING OF SPLEEN LEUCOCYTES
- Necropsy procedures: Ten males and ten females per group from F1 Cohort 1A were selected for immunophenotyping. Where possible, one male or one female was assigned from each selected litter.The whole spleen was weighed. After weighing, a 3-5 mm mid transverse section was removed and retained for histopathological evaluation. The remaining portions of the spleen was then weighed, placed in to a vial of chilled Hank’s Balanced Salt Solution (HBSS) and held in wet ice until processing for analysis.
- Each sample were analysed for the following cell types: T cells, B cells, NK cells, CD4+ et CD8+ T cells, Monocytes and Neutrophils.

LIGHT MICROSCOPY
Tissues preserved for examination were examined as follows:
- Premature deaths: All animals from all groups for all specified tissues.
- Scheduled kill: F0/F1A: All animals from groups 1 and 4 for all specified tissues; All animals from groups 2 and 3 for abnormalities only and reproductive organs for F0 groups 2 and 3 with suspect fertility.
F1B: All animals from all groups for abnormalities only.

- Right testis: A detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. Any cell- or stage-specificity of testicular findings was noted.
- Ovaries: F0 - Qualitative evaluation of one section from each ovary.
F1 Cohort 1A - Qualitative evaluation of five sections from each ovarywith quantitative assessment of primordial follicle and small growing follicle populations as well as evaluation of corpora lutea numbers in one section from each ovary.
- Vagina: The stage of vaginal estrus was evaluated based on vaginal epithelial morphology (and appearance of the uterus and endometrial glands).

The pathology procedures was detailed in the Tables 4, 6 and 7 below.

Postmortem examinations (offspring):

SACRIFICE
- Unselected offspring: culled on Day 4 and Day 22 of age.
- Cohort 1A adult animals: Scheduled kill on approximately Week 13 of age.
- Cohort 1B adult animals: Scheduled kill on approximately Week 17 of age (after weaning of F2 offspring).
- Method: Animals 14 days and older: Carbon dioxide. Each animal were subsequently exsanguinated; Animals less than 14 days of age: subcutaneous injection of sodium pentobarbitone; Pups culled on Day 4 of age requiring thyroid hormone sampling: Decapitation.

MACROSCOPIC PATHOLOGY
A complete gross necropsy were performed on ten male and ten females per group; one male or one female from each litter to ensure that all litters are represented.

ORGAN WEIGHTS
For bilateral organs, left and right organs were weighed together, unless specified in the relevant pathology procedures table. Requisite organs were weighed for animals killed at scheduled intervals. For unselected F1 offspring on Day 22 of age, organs were weighed from ten males and ten females per sex per group from as many litters as possible.

FIXATION
For F0 animals, unselected F1 animals (including decedent offspring), Cohorts 1A, 1B and F2 offspring (including decedents).
- Standard: 10% Neutral Buffered Formalin. Where possible, carcass retained for decedent offspring ≤ 21 days of age.
- Testes (F0 and F1 adults only): modified Davidson’s fluid.
- Eyes: Davidson’s fluid.

HISTOLOGY
Processing to slide:
- Full List: All adult animals killedor dying prematurely. F0/Cohort 1A: All terminal adult animals of Groups 1 and 4 killed at a scheduled interval.
- Reproductive Organs: from F0 animals in Groups 2 and 3 that showed reduced fertility. This included males that failed to sire a pregnancy and females that were not pregnant or females with a litter death.
- Abnormalities only: F0/Cohort 1A: All terminal adult animals of Groups 2 and 3 killed at a scheduled interval.Cohort 1B: All animals.

Processing to block:
- Reproductive Organs: Cohort 1B: All animals.

Staining:
- Routine staining: 4-5 µm sections stained with haematoxylin and eosin.
- Special staining: None scheduled.

LIGHT MICROSCOPY
Tissues preserved for examination were examined as follows:
- Premature deaths: All animals from all groups for all specified tissues.
- Scheduled kill: F0/F1A: All animals from groups 1 and 4 for all specified tissues; All animals from groups 2 and 3 for abnormalities only and reproductive organs for F0 groups 2 and 3 with suspect fertility.
F1B: All animals from all groups for abnormalities only.

- Right testis: A detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle. Any cell- or stage-specificity of testicular findings was noted.
- Ovaries: F0 - Qualitative evaluation of one section from each ovary.
F1 Cohort 1A - Qualitative evaluation of five sections from each ovarywith quantitative assessment of primordial follicle and small growing follicle populations as well as evaluation of corpora lutea numbers in one section from each ovary.
- Vagina: The stage of vaginal estrus was evaluated based on vaginal epithelial morphology (and appearance of the uterus and endometrial glands).

The pathology procedures was detailed in the Table 5 below.

Statistics:

DATA-TYPES
The following data types were analyzed at each timepoint separately, where required, in support of interpretation:
-Body weight, using absolute weights and gains over appropriate study periods
- Food consumption, over appropriate study periods
- Hematology
- Blood chemistry
- Urinalysis
- Mating performance and fertility
- Gestation length
- Estrous cycles
- Litter (implantations, litter size, sex ratio - percentage male, post implantation survival index, live birth index and viability index), for before cull study periods
- Ano-genital distance, adjusted for Day 1 pup body weight
- Sexual maturation, age and body weight at completion
- Thyroid hormone analyses
- Organ weights, absolute and relative to body weight
- Corpora lutea and ovarian primordial follicle count

The following comparisons were performed:
Group 1 vs 2, 3 and 4
Group 1 vs 4 for ovarian follicle counts and corpora lutea data

A sequence of statistical tests was used for body weight, food consumption, implantations, litter size, sex ratio - percentage male, post implantation survival index,ano-genital distance, corpora lutea and ovarian follicle count, organ weight and clinical pathology data either by a parametric analysis or by a non-parametric analysis.

Significant differences between the groups compared were expressed at the 5%(p<0.05) or 1% (p<0.01) level.

Reproductive indices:

Duration of gestation being the time elapsing between mating and commencement of
parturition was recorded. From Day 20 after mating animals were checked 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 noted.

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 paired) x 100
Gestation index: (Number of live litters born / Number pregnant) x 100

Offspring viability indices:

Survival indices (%):
- Post implantation survival index: (Total number of offspring born / Total number uterine implantation sites) x 100
- Live birth index: (Number live off spring on Day 1 after littering / Total number of offspring born) x 100
- Viability index: (Number live off spring on Day 4 before cull / Number live offspring on Day 1 after littering) x 100
- Lactation index: (Number live offspring on Day 21 after littering / Number live offspring on Day 4 (after cull)) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no signs attributed to treatment in males, or in non-mated females during the 10-week pre-pairing period. One female given 6900 ppm was thin from GD 18; there were no other clinical signs attributed to treatment during gestation.
Two females at 4300 ppm and two females given 6900 ppm were thin during the lactation period. There were no other clinical signs attributed to treatment during lactation.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

No test-item related mortality occurred. There were four unscheduled deaths among the F0 animals that were considered unrelated to treatment.
One male (No. 73) given 4300 ppm had abnormal gait from Day 43 and thin build and piloerection from Day 51 and was killed for welfare reasons on Day 58. Macroscopic examination revealed marked enlargement of the ventricles of the heart. Microscopic examination revealed cardiomyopathy of the heart. The major factor contributing to death was recorded as poor clinical condition.
One female (No. 252) given 4300 ppm had pale eyes following parturition (LD 1), hunched posture, whole body pallor, was a thin build (from LD 9) and was killed for welfare reasons on Lactation Day 13. Macroscopic examination revealed marked enlargement of the heart and spleen with many organs appearing pale. Microscopic examination revealed lymphoma of the hemopoietic system. The major factor contributing to death was recorded as lymphoma.
One female (No. 266) given 4300 ppm was found to be underactive, with pale eyes, piloerection, hunched posture, and red discharge (blood) from the vagina and was killed for welfare reasons on Gestation Day 22. These signs may indicate difficulty with parturition. Macroscopic examination revealed abnormal contents of the stomach. Microscopic examination revealed no significant findings. The major factor contributing to death was recorded as poor clinical condition.
One female (No. 283) given 6900 ppm, that had no previous clinical observations, was found dead on Lactation Day 22. No macroscopic or microscopic findings of significance were seen. Many tissues were missing due to cannibalization. The major factor contributing to death was recorded as undetermined.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall body weight gain was unaffected by treatment at 2000, 4300 or 6900 ppm in males, until scheduled necropsy in Week 18 and during the pre-pairing period in females.
Overall body weight gain during gestation at 6900 ppm was statistically significantly low (86% of Control), with the main decrease during GD 6-14 (-21% of Control) and GD 14-20 ( 13% of Control), leading to a statistically significantly lower mean bodyweight than Control at the end of gestation (94% of Control). Body weight gain was unaffected by treatment at 2000 or 4300 ppm.
Females given 6900 ppm showed a mean body weight loss of -3 g during LD 1-4, compared to a mean body weight gain of +4 g in Control. Body weight gain during LD 4-7 was statistically significantly higher than Control (+14 g vs. +7 g) and overall body weight gain (LD 1-21) was unaffected by treatment at 2000, 4300 or 6900 ppm. Body weight loss following weaning of the litter (LD 21-28) was similar to Control at all dietary concentrations of alpha-pinene multiconstituent.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was unaffected by treatment at 2000, 4300 or 6900 ppm in males, until scheduled necropsy in Week 18 and during the pre-pairing period in females.
Overall food consumption (GD 0-20) was statistically lower than Control at 6900 ppm (92% of Control). Food consumption during gestation was unaffected by treatment at 2000 or 4300 ppm.
Overall food consumption (LD 1-21) was not statistically significantly affected by treatment at 2000, 4300 or 6900 ppm, but food consumption at 6900 ppm was statistically significantly low (90% of Control) during LD 1-4 and remained lower than Control throughout lactation (overall mean food consumption: 92% of Control).

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically relevant differences in the cellular composition of the blood in Week 18 of treatment in males and on LD 28 in females.
In males only, mean cell volume was marginally, but statistically significantly low at 6900 ppm (98% of Control) but was within the range of the HCD (90 percentile range from 15 studies (2016-2021): 51.6 to 62.7 fL (n=150)).
Basophil count was statistically significantly low in males at 6900 ppm (57% of Control) and overall white cell count was correspondingly statistically significantly low (77% of Control); however, both were within the range of the HCD (90 percentile ranges from 15 studies (2016 2021): 0.01 to 0.11 x10E9/L and 4.30 to 11.30 x10E9/L (n=150), respectively). In females, basophil count was statistically significantly high, without relationship to dietary concentration at 2000, 4300 or 6900 ppm (2-fold, 2.5 fold or 1.5-fold of Control, respectively), but was within the range of the HCD (90 percentile range from 15 studies (2016-2021): 0.01 to 0.09 x10E9/L (n=146); eosinophil count was statistically significantly low (55% of Control) at 6900 ppm, but was also within the HCD range (90 percentile range from 15 studies (2016-2021): 0.08 to 0.38 x10E9/L (n=146)) and overall white cell count was unaffected.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically relevant differences in the constituents of the blood plasma in Week 18 of treatment in males and on LD 28 in females.
When compared with Control and in males only, cholesterol concentrations at 2000, 4300 or 6900 ppm were high and attained statistical significance at 4300 or 6900 ppm (120%, 129% or 142%, respectively), but was within the range of the HCD (90 percentile range from 15 studies (2016-2021): 0.93 to 2.10 mmol/L (n=150). Total protein and albumin concentrations were marginally high, without relationship to dose, at 2000, 4300 or 6900 ppm (105%, 108% or 107% and 106%, 109% or 109%, respectively), but were within the range of the HCD (90 percentile range from 15 studies (2016-2021): 59 to 68 g/L and 34 to 38 g/L, respectively (n=150)).
In females only, bilirubin concentration was statistically significantly high at 2000, 4300 or 6900 ppm, but was within the range of the HCD (90 percentile range from 15 studies (2016 2021): 0 to 2 µmol/L (n=150)) and creatinine concentration was statistically significantly low at 4300 or 6900 ppm but was within the range of the HCD (90 percentile range from 15 studies (2016-2021): 25 to 46 µmol/L (n=150)). Glucose concentration was marginally, but statistically significantly low at 4300 or 6900 ppm (92% or 89% of Control, respectively), but was within the range of the HCD (90 percentile range from 15 studies (2016-2021): 6.42 to 10.12 mmol/L (n=150). Potassium concentrations were marginally, but statistically significantly high but without relationship to dietary concentration at 4300 or 6900 ppm (109% or 106% of Control, respectively) and within the range of the HCD (90 percentile range from 15 studies (2016-2021): 3.25 to 4.15 mmol/L (n=150)).
Phosphorous concentration was statistically significantly low (87% of Control) in males at 6900 ppm and was high, without relationship to dietary concentration, in females at 2000, 4300 or 6900 ppm (125%, 122% or 122% of Control, respectively); however, both were within the range of the HCD (90 percentile ranges from 15 studies (2016 2021): 1.64 to 2.47 mmol/L and 1.19 to 2.12 mmol/L (n=150), respectively).

Endocrine findings:

effects observed, non-treatment-related

Description (incidence and severity):

Serum TSH and T4 concentrations were unaffected by treatment in the F0 (adults). Mean serum TSH concentration in F0 males given 4300 ppm was 2-fold higher than Control and attained statistical significance; however, the concentrations were similar to Control at 6900 ppm, no similar increase was observed in females and values were within the range of the HCD (95 percentile from 13 studies (2019-2021): 24.5 to 3445 pg/mL) and therefore the difference was considered not to be related to treatment.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no toxicologically relevant differences in the composition of the urine in Week 18 of treatment in males and on LD 28 in females. The specific gravity of the urine in males at 6900 ppm was marginally, but statistically significantly low (99% of Control), but was within the range of the HCD (90 percentile range from 7 studies (2016-2021): 1028 to 1059 g/L (n=69)).

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Increased accumulation of hyaline droplets with associated increased incidence and/or severity of basophilic tubules and granular casts was seen in the males given 6900 ppm. The increase in hyaline droplets in the kidneys of males is consistent with the accumulation of alpha-2µ-globulin, a common finding in untreated male rats (Hard et al., 1993). Hyaline droplet accumulation is both sex and species specific (Frazier et al., 2012) and is not generally considered to be significant in man. However, the associated multifocal basophilic tubules and granular casts, known as alpha-2µ-globulin nephropathy are considered to be adverse in the animals affected.
All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including Control), and/or their severity was as expected for this Sprague Dawley rats of this age. Consequently, they were considered not test article related.
The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Two mammary adenocarcinomas were seen in females in the F0 generation, one at 2000 ppm and one at 4300 ppm, with one ameloblastic odontoma seen in the head of a male at 4300 ppm. It is not uncommon to see occasional tumors, particularly mammary adenocarcinomas, in control Sprague Dawley rats of this age. Based on their sporadic incidence and lack of an overall dose relationship, all these tumours are considered incidental.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles were unaffected by treatment during the two-week period before pairing at 2000, 4300 or 6900 ppm.
Estrous cycles were unaffected by treatment following weaning of litters and before termination (LD 25-28) at 2000, 4300 or 6900 ppm.
One Control female and two females at 6900 ppm did not show estrus during LD 25-28; however, it was considered that these animals had a normal 5-day cycle period.

Reproductive performance:

no effects observed

Description (incidence and severity):

At least 96% of pairings in each group showed evidence of successful mating within 4 days of pairing; therefore, pre-coital interval was unaffected by treatment at 2000, 4300 or 6900 ppm.
Mating performance and fertility were unaffected by treatment at 2000, 4300 or 6900 ppm.
One female (No. 249) at 2000 ppm and one female (No. 268) at 4300 ppm were not pregnant.
Macroscopic and microscopic examinations were unremarkable in both animals, with no evidence of implantation sites and with both animals cycling normally. There were no findings in Male No. 50, the mating partner of Animal No. 249 that could account for the suspect fertility. Male No. 68, the mating partner of Animal No. 268, had tubular degeneration of the testes with no sperm in the epididymis, which would have contributed to failure to litter of this pair that was considered to have occurred by chance.
Gestation length was unaffected by treatment at 2000, 4300 or 6900 ppm. All females littered within 22-23 days of mating. Gestation index unaffected by treatment at 2000, 4300 or 6900 ppm.

The overall mean achieved dose during Weeks 1-18 of treatment period was 131, 282 and 469 mg/kg bw/day for males at 2000, 4300 and 6900 ppm, respectively.
The overall mean achieved dose during Weeks 1-10 of the pre-pairing treatment period was 166, 354 and 567 mg/kg bw/day for females at 2000, 4300 and 6900 ppm, respectively.
The overall mean achieved dose during GD 0-20 was 137, 297 and 466 mg/kg bw/day for females at 2000, 4300 and 6900 ppm, respectively.
The overall mean achieved dose during LD 1-14 was 297, 665 and 1047 mg/kg bw/day for females at 2000, 4300 and 6900 ppm, respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

reproductive performance

Effect level:

6 900 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed for F0 parent animals

Remarks on result:

other: Mean achieved doses of 466 mg/kg bw/day and 1047 mg/kg bw/day during F0 gestation and lactation

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

6 900 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed for F0 parent animals

Remarks on result:

other: Mean achieved doses of 469 mg/kg bw/day for F0 males, 567 mg/kg bw/day for F0 females before pairing

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

6 900 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

no effects observed

Description (incidence and severity):

There were no signs attributed to treatment in males, or in non-mated females during the pre pairing period. Two males at 7300 ppm had elevated gait and two females at 7300 ppm had elevated gait and had thin build during Week 1 of the Formal F1 generation. The thin build was attributed to the small size of these animals at this time, when compared with Control.
There were no signs attributed to treatment during gestation and lactation.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

There were no test-item related deaths in the F1 (Cohort 1A or 1B).
One animal (No. 529) given 4600 ppm died following a mishandling incident on Day 24 of the pre-pairing treatment period. No macroscopic findings of significance were seen, and no histopathological investigation was performed on this animal. The major factor contributing to death was recorded as accidental.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Mean body weight gain during PND 21-25 was statistically significantly low at 7300 ppm (males 90% and females 94% of Control, respectively).
Absolute bodyweight on Day 1 of the Formal F1 generation (Day 28 of age (±2 days)) was statistically significantly low at 4600 or 7300 ppm (males 92% or 85%; females 93% or 83% of Control). Subsequent growth improved and overall absolute body weight in in males and females at 4600 ppm was similar to Control and was statistically significantly, but not biologically relevantly, low at 7300 ppm (males 92% and females 89% of Control) and was therefore considered not to be toxicologically adverse. Overall body weight gain in males to termination was similar to Control at all dietary concentrations of alpha pinene multiconstituent.
Absolute body weight at 7300 ppm on GD 0 was statistically significantly low, when compared with Control (93% of Control). Overall body weight gain during gestation (GD 1-20) was marginally statistically significantly low at 4600 or 7300 ppm (89% or 75% of Control, respectively).
Mean body weights at 7300 ppm on LD 1 were statistically significantly low, when compared with Control (89%). Overall body weight gain (LD 1-21) was statistically significantly higher at 7300 ppm (1.7-fold of Control); however, the absolute bodyweight value at LD 21 was 20 g lower than Control (292 g v 312 g) and the Control also had a reduced bodyweight gain compared to the low and mid dose groups (-10 g). Body weight loss following weaning of the litter (LD 21-28) was similar to Control at all dietary concentrations of alpha pinene multiconstituent.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption at 2100, 4600 or 7300 ppm was unaffected in males during the prepairing treatment period and before termination.
Overall food consumption of females during the prepairing treatment period at 2100, 4600 or 7300 ppm was considered to be unaffected by treatment. Overall food intake at 7300 ppm was marginally, but statistically significantly low (93% of Control), but the difference was considered not adverse.
Overall food consumption during GD 0-20 was unaffected by treatment at any dietary concentration.
Overall food consumption during lactation at 2100, 4600 or 7300 ppm was considered to be unaffected by treatment. Overall food intake at 7300 ppm was statistically significantly low (92% of Control), but the difference was considered not adverse.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

COHORT 1A: There were no toxicologically significant differences in the cellular composition of the blood at approximately Week 13 of age. In males only, reticulocyte count was marginally, but statistically significantly low at 4600 or 7300 ppm (80% or 89% of Control, respectively), but failed to show relationship to treatment and was within the range of the HCD (90 percentile range from 6 studies (2019-2021): 0.107 to 0.192 x1012/L (n=60)).
In females only, neutrophil count was statistically significantly low at 7300 ppm (63% of Control) and lymphocyte counts were low at 4600 or 7300 ppm (83% or 69% of Control, respectively); however, both were within the range of the HCD (90 percentile ranges from 15 studies (2016-2021): 0.375 to 1.670 x10E9/L and 2.53 to 9.12 x10E9/L (n=146), respectively) and overall white cell count was correspondingly low (86% or 70% of Control, respectively) that attained statistical significance at 7300 ppm only and was also within the range of the HCD (90 percentile range from 15 studies (2016-2021): 3.18 to 10.65 x10E9/L (n=149)).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

COHORT 1A: There were no toxicologically relevant differences in the constituents of the blood plasma at approximately Week 13 of age. In males only, alkaline phosphatase activities were low, but failed to show dose relationship at 2100, 4600 or 7300 ppm (79%, 91% or 90% of Control, respectively) and lowering of enzyme activity is not toxicologically relevant. Bilirubin concentration was marginally, but statistically significantly low at 2100, 4600 or 7300 ppm, but was within the range of the HCD (90 percentile range from 15 studies (2016-2021): 0 to 2 µmol/L (n=150)).
In females only, glucose concentration was marginally, but statistically significantly low at 7300 ppm (91% of Control), but was within the range of the HCD (90 percentile range from 15 studies (2016-2021): 6.17 to 9.26 mmol/L (n=149)) and cholesterol concentrations were high at 2100, 4600 or 7300 ppm (129%, 137% or 174% of Control, respectively), but were within the range of the HCD (90 percentile range from 15 studies (2016-2021): 1.20 to 2.47 mmol/L (n=149)); the Control value was marginally outside the HCD range. Phosphorus concentration was marginally, but statistically significantly low at 7300 ppm (87% of Control) but was within the HCD range (90 percentile range from 15 studies (2016-2021): 1.71 to 2.65 mmol/L (n=149)).

Endocrine findings:

no effects observed

Description (incidence and severity):

Mean serum TSH and T4 concentrations in F1 adults were considered to be unaffected by treatment at 2100, 4600 or 7300 ppm.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

COHORT 1A : There were no toxicologically relevant differences in the composition of the urine at approximately Week 13 of age.
Total potassium concentration was statistically significantly low in females only at 7300 ppm (59% of Control) but was within the HCD range (90 percentile range from 13 studies (2016-2021): 0.321 to 1.001 mmol (n=131)).

Behaviour (functional findings):

not examined

Immunological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Variation was observed in the immunophenotyping parameters within the treatment groups for both male and female splenocytes. The minor increases or decreases in cell percentages and absolute cells/spleen values observed in the treatment groups (Group 2 to 4) when compared to the Group 1 control were likely not the result of the dietary administration of alpha-pinene multiconstituent. This was because the values obtained for T cells (including CD4+ and CD8+ T cell subsets), B cells, NK cells, monocytes and neutrophils in the treated rat spleen leukocytes were either not dose-related, the differences were not statistically significant and/or were within the ranges observed in the Group 1 Control animals and therefore likely due to biological variation.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

COHORT 1A: In the adrenals, when compared to Control, bodyweight relative mean weights were high, attaining statistical significance, for males and females given 4600 or 7300 ppm. A dose response was seen in male relative weights. No histopathological correlate was found to account for this weight change.
In the kidneys, when compared to Control, bodyweight relative weights were high, attaining statistical significance, for males at all dietary concentrations. In females, absolute weights were slightly low, when compared to Control, attaining statistical significance, for females given 4600 or 7300 ppm. No histopathological correlate was found to account for this weight change in females but the increase in hyaline droplets and basophilic tubules seen histologically in males are likely to have contributed to weight increase in males.
In the liver, when compared to Control, relative weights were high, attaining statistical significance, for males given 4600 or 7300 ppm and females given 7300 ppm. No histopathological correlate was found to account for this weight change, but it is common to not be able to detect a microscopic correlate for a small increase in weight in the liver.
In the mesenteric lymph node, when compared to Control, absolute and relative weights were high for males and females, attaining statistical significance for males given 4600 or 7300 ppm, but not dose related. No histopathological correlate was found to account for this weight change.
Terminal body weights were low for males and females, when compared to Control, attaining statistical significance for males given 7300 ppm and females given 4600 or 7300 ppm. This caused statistically significant differences in organ weights of various organs compared to Control. This included brain, epididymis, heart, ovaries, pituitary, spleen, thyroid and parathyroid and seminal vesicle with coagulating gland. These changes were not considered to be a direct effect of treatment with alpha-pinene multiconstituent.
All other differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body weight) ratios but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small. //

COHORT 1B: In the kidneys, when compared to Control, absolute and bodyweight relative weights were high, attaining statistical significance, for males at all dietary concentrations. In females, absolute weights were low, when compared to Control, attaining statistical significance, for females given 7300 ppm. No histopathological correlate was found to account for this weight change in females but the increase in hyaline droplets and basophilic tubules seen histologically in those males examined may have contributed to weight increase in males.
In the liver, when compared to Control, absolute and relative weights were high, attaining statistical significance, for males and females given 4600 or 7300 ppm and for absolute weights in females and also relative weights in males given 2100 ppm. A dose response was seen in male relative weights. No histopathological correlate was found to account for this weight change, but it is common to not be able to detect a microscopic correlate for a small increase in weight in the liver. Terminal body weights were low for females, when compared to Control, attaining statistical significance for females given 7300 ppm. This caused statistically significant low thymus weights in females compared to Control. No macroscopic abnormalities were noted in the thymus and therefore the thymus was not examined histopathologically. This weight change was not considered a direct effect of treatment with alpha-pinene multiconstituent. All other differences in organ weight parameters, statistically significant or not, were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: inconsistency between sexes; presence only in absolute weight or in relative (to body weight) ratios but not both; lack of a dose relationship or correlative findings; and/or the magnitude was considered small.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

COHORT 1A and 1B: All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including Control), and/or were as expected for Sprague Dawley rats of this age; therefore, they were considered not test item related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

COHORT 1A and 1B: Changes related to treatment with Alpha-pinene multiconstituent were seen in the kidneys.
Increased accumulation of hyaline droplets with associated increased incidence and/or severity of basophilic tubules were seen in the males given 7300 ppm. Granular casts were seen in occasional males. Only occasional animals with abnormalities were examined from Cohort 1B.
All other microscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including Control), and/or their severity was as expected for this Sprague Dawley rats of this age. Consequently, they were considered not test article related.
The testes revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Four mammary adenocarcinomas were seen in females, (one in the Control, one at 4600 ppm and two at 7300 ppm). It is not uncommon to see occasional tumours, particularly mammary adenocarcinomas, in Control Sprague Dawley rats of this age. Based on their sporadic incidence and lack of an overall dose relationship, all these tumours are considered incidental.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Estrous cycles from Day 75 of age (Cohort 1A) and at termination (Cohort 1B; LD 25-28) were unaffected by treatment at 2100, 4600 or 7300 ppm.
COHORT 1A : The period of time from vaginal opening to first estrus was unaffected by treatment.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

COHORT 1A: Sperm count, motility and motion parameters and morphology were unaffected by treatment at 2100, 4600 or 7300 ppm.

Reproductive performance:

no effects observed

Description (incidence and severity):

COHORT 1B: Pre-coital interval unaffected by treatment at 2100, 4600 or 7300 ppm. All pairs showed evidence of successful mating within the same period taken by the Control.
Mating performance and fertility were unaffected by treatment at 2100, 4600 or 7300 ppm.

Two Control animals (No. 685 and 697), one animal at 2100 ppm (No. 702) and one animal at 7300 ppm (No. 759) failed to litter. Macroscopic examination for Animal No. 697 revealed marked bilateral fluid distension of the uterus and a cyst. This could be associated with failure to litter in this animal. Macroscopic examinations in the other three animals were unremarkable.
Fertility index was unaffected by treatment at 2100, 4600 or 7300 ppm.
Gestation length was unaffected by treatment at 2100, 4600 or 7300 ppm, as all animals littered within 22-23 days of mating.
Gestation index was unaffected by treatment.

The overall mean achieved dose during the Formal pre-pairing treatment period was 179, 400 and 648 mg/kg bw/day for males at 2100, 4600 or 7300 ppm, respectively.

The overall mean achieved dose during the Formal pre-pairing treatment period was 179, 400 and 648 mg/kg bw/day for females at 2100, 4600 or 7300 ppm, respectively.

The overall mean achieved dose during GD 0-20 was 135, 286 and 494 mg/kg bw/day for females at 2100, 4600 or 7300 ppm, respectively.

The overall mean achieved dose during LD 1-14 was 283, 629 and 1044 mg/kg bw/day for females at 2100, 4600 or 7300 ppm, respectively.

Key result

Dose descriptor:

NOAEL

Remarks:

Reproductive performance

Effect level:

7 300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed for F1B parent animals

Remarks on result:

other: Mean achieved doses of 494 mg/kg bw/day and 1044 mg/kg bw/day during F1B gestation and lactation

Key result

Dose descriptor:

NOAEL

Remarks:

Systemic toxicity

Effect level:

7 300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed for F1 Cohort 1A and 1B

Remarks on result:

other: Mean achieved doses of 648 mg/kg bw/day for F1 males and 659 mg/kg bw/day for F1 females before pairing

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

7 300 ppm

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The clinical condition of the offspring, following parental treatment at 2000, 4300 or 6900 ppm remained good until scheduled termination on PND 22. Clinical signs that are commonly observed in rat litters at this laboratory, including little or no milk in stomach, attached umbilical cord, patchy hair growth and bruising or cuts or encrustations of the skin, were seen at low incidence and showed no relationship to treatment.

Mortality / viability:

no mortality observed

Description (incidence and severity):

The numbers of implantations and total offspring born and subsequent survival of the offspring to Day 21 of age were unaffected by parental treatment at 2000, 4300 or 6900 ppm.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Absolute bodyweights of male and female offspring on PND 1 was similar to Control and therefore unaffected by treatment.

Absolute bodyweight of male and female offspring on PND 1 at 6900 ppm was statistically significantly low (males 93%; females 92% of Control, respectively). Absolute offspring bodyweight at 2000 or 4300 ppm was unaffected by treatment.
Subsequent body weight gain (PND 1-21) was statistically significantly low in offspring at 2000 or 4300 ppm (approximately 92% or 88%, respectively) and was statistically significantly and biologically relevantly low (<85% of Control) at 6900 ppm (approximately 78% of Control, respectively).
Absolute offspring body weight on PND 21 at 6900 ppm was biologically relevantly low (males -10.3 g; 80% and females -10.7 g; 79% of Control). The absolute body weights of both males and females on PND 21 were all statistically significantly lower than Control at all dietary concentrations and showed relationship to dose.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

In males, the absolute mean ano-genital distance on PND 1 was unchanged by treatment, but when adjusted for bodyweight, it was marginally, but statistically significantly longer in males at 6900 ppm. However, this is considered not to be toxicologically relevant because the males in the high dose group had a statistically significantly lower bodyweight than control ( 7.4%). Ano-genital distance of female offspring was unaffected at 2000, 4300 or 6900 ppm.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No male offspring had nipples

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weights of the unselected F1 offspring on PND 22 were unaffected by parental treatment at 2000, 4300 or 6900 ppm.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no macroscopic findings in offspring on Day 22 of age attributable to treatment at 2100, 4600 or 7300 ppm.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Mean serum TSH and T4 concentrations in F1 offspring on Day 22 of age were considered to be unaffected by treatment at 2100, 4600 or 7300 ppm.
The ratio of male to female offspring was unaffected by parental treatment at 2000, 4300 or 6900 ppm.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Growth of the offspring

Generation:

F1

Effect level:

6 900 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Mean achieved doses of 466 mg/kg bw/day and 1047 mg/kg bw/day

Key result

Critical effects observed:

no

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Offspring remained in good clinical condition until scheduled termination on PND 22. Commonly seen signs of cold to touch, little or no milk in stomach, attached umbilical cord, bruising and exfoliation of skin and twisted toes were seen at low incidence and showed no relationship to parental treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

When compared with Control and at 7300 ppm, mean total size (86%), live on PND 1 (85%) and live litter size before culling (PND 4; 90%) were slightly low, with all differences attaining statistical significance; however, this was attributed to the low mean number of implantations (91% of Control). Offspring survival was unaffected by treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Absolute bodyweights of male and female offspring on PND 1 was similar to Control and therefore unaffected by treatment.

Overall body weight gain (PND 1-21) of male and female offspring was statistically significantly low in males at 7300 ppm and females at 4600 or 7300 ppm (87% and 91% or 89% of Control, respectively).

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

When adjusted for body weight, ano-genital distance on Day 1 of age was marginally short in females at 7300 ppm (2.1 mm vs. 2.2 mm in Control); however, this difference was considered not to be toxicologically relevant. Male ano-genital distance was unaffected by parental treatment.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No male offspring had nipples.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Organ weights of the F2 offspring on PND 22 were unaffected by parental treatment at 2100, 4600 or 7300 ppm.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no macroscopic findings in offspring on Day 22 of age attributable to treatment at 2100, 4600 or 7300 ppm.

Histopathological findings:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Sex ratio was unaffected by treatment.

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Key result

Dose descriptor:

NOAEL

Remarks:

Growth of the offspring

Generation:

F2

Effect level:

7 300 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Mean achieved doses of 494 mg/kg bw/day and 1044 mg/kg bw/day

Key result

Critical effects observed:

no

Key result

Reproductive effects observed:

no

Conclusions:

Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity which has the potential to be harmful to human health in the F0 parent animals was 6900 ppm and the F1 Cohort 1A and 1B adult animals was 7300 ppm (Mean achieved doses of 469 mg/kg bw/day for F0 males, 567 mg/kg bw/day for F0 females before pairing, 648 mg/kg bw/day for F1 males and 659 mg/kg bw/day for F1 females before pairing). No NOAEL was established for kidney changes in F0 and F1 males, consistent with the accumulation of alpha-2µ-globulin, which was deemed adverse for the animals, but is generally considered not to be relevant to human health.

The NOAEL for reproductive performance of the F0 and F1B parent animals and survival of the F1 and F2 offspring was concluded to be 6900 ppm or 7300 ppm, respectively (mean achieved doses of 466 mg/kg bw/day and 1047 mg/kg bw/day during F0 gestation and lactation and 494 mg/kg bw/day and 1044 mg/kg bw/day during F1B gestation and lactation). The NOAEL for the growth of the F1 offspring (Days 1-21 of age) was 6900 ppm (mean achieved doses of 466 mg/kg bw/day and 1047 mg/kg bw/day) in F1 offspring and 7300 ppm (mean achieved doses of 494 mg/kg bw/day and 1044 mg/kg bw/day) in F2 offspring, as the reductions in the growth of both generations of offspring was likely caused by reduced palatability of the diet, and the consequent reduced food consumption and bodyweight and/or body weight gain of the females during gestation and lactation, and is therefore not relevant to humans.

Executive summary:

The purpose of this study was to assess of the influence of alpha-pinene multiconstituent on reproductive performance when administered continuously in the diet to Sprague Dawley rats. Cohorts of F1 animals were used to assess the potential for systemic toxicity, and potential effects on sexual maturation and estrous cycles and one cohort was assessed for reproductive performance (OECD 443).

 

In the F0 generation, three groups of 25 male and 25 female rats were to receive alpha-pinene multiconstituent orally, via the diet, at nominal concentrations of 2500, 5500 or 8500 ppm (with corn oil added to the premix at a ratio of 5:1 test item: corn oil). However, the formulation chemistry investigations performed during the course of the study revealed lower than nominal dietary concentrations of alpha-pinene multiconstituent administered in the F0 and F1 generations to all treated groups. To ensure the integrity and validity of the study, the actual dietary concentrations administered (and reported) were calculated, based on the mean dietary concentrations given to each generation, based on the results of the formulation chemistry investigation (performed in Weeks 1, 6, 7 and 10 (F0 generation) and Week 1 and last week of treatment (F1 generation). Therefore, 2000, 4300 or 6900 ppm were administered to the F0 and the F1 to weaning, and 2100, 4600 or 7300 ppm were administered in the F1 after weaning, to Groups 2, 3 and 4, respectively. Males were treated for ten weeks before pairing and up to scheduled termination, after litters had weaned. Females were treated for ten weeks before pairing, throughout pairing and up to scheduled termination on Day 28 of lactation. In the F1 generation, 20 males and 20 females were treated from weaning to their scheduled termination (relevant to each cohort). A similarly constituted Control group in each generation received untreated basal diet with added corn oil for the same duration.

For the F0 generation, data were recorded on clinical observations, body weight, food consumption, estrous cycles, mating performance and fertility, gestation length and parturition observations and reproductive performance. Clinical pathology (hematology, blood chemistry and urinalysis) and thyroid related hormones, organ weight, macroscopic pathology and microscopic pathology investigations were performed.

For F1 offspring, clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, organ weights and macropathology were assessed. Nipple counts were performed on male offspring on Day 13 of age. Serum samples that were collected from selected offspring on Day 22 of age were analyzed for thyroid-related hormones.

At weaning, two cohorts of selected males and females were assigned for further investigations, including sexual maturation, reproductive organ integrity and function and immune functions. The cohorts were as follows:

Cohort	Designation	Animals/Cohort	Sexual maturation assessment	Approximate age at necropsy
1A	Reproductive & general toxicity	20M + 20F	Yes	13 weeks
1B	Reproductive	20M + 20F	Yes	17 weeks

For F1 Cohort 1A, data were recorded on clinical condition, body weight, food consumption, sexual maturation and estrous cycles. Clinical pathology (hematology, blood chemistry and urinalysis) and thyroid-related hormones, sperm assessment, ovarian follicle and corpora lutea counts, organ weight, macroscopic pathology, full microscopic pathology and immunophenotyping investigations were performed.

For F1 Cohort 1B, data was recorded on clinical condition, body weight, food consumption, sexual maturation, estrous cycles, mating performance, fertility, gestation length and gestation index, organ weight and macroscopic and microscopic pathology investigations were performed.

For F2 offspring, clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, organ weights and macropathology were assessed. Nipple counts were performed on male offspring on Day 13 of age.

 

RESULTS

There were four unscheduled deaths in the F0 generation and one unscheduled death in the F1 generation, none were treatment related.

 

There were no clinical signs attributable to treatment in the F0 (adults) or F1 (offspring and adults) or in F2 offspring.

 

Overall mean body weight gain in F0 males and in un-mated females and food intake were unaffected by treatment during the pre-pairing period. In the F0 females, overall mean body weight gain during gestation was statistically significantly low at 6900 ppm (-14%) and mean body weight loss was evident during LD 1-4; both correlated with statistically significantly low food intake (both -10%) in these animals.

 

Absolute mean bodyweight of F1 offspring on Day 1 of age was significantly low (-7% and 8% for males and females, respectively). Subsequent body weight gain (PND 1-21) was statistically significantly low at 2000 or 4300 ppm and statistically significantly and considered to be biologically relevantly low at 6900 ppm (males; -8%, -11% or -12%; females; -8%, -13% or 23% of Control, respectively). Absolute bodyweight of F2 offspring on Day 1 of age was unaffected by treatment. Overall body weight gain (PND 1-21) of male and female offspring was statistically significantly low in males at 7300 ppm and females at 4600 or 7300 ppm (13% and -9% or -11% of Control, respectively). The low PND 1 body weight and subsequent body weight gain to PND 21 seen in the F1 generation at 6900 ppm was not fully replicated in the F2 generation at 7300 ppm and any relationship to treatment for this was not clear although poor palatability of the diet was the most likely cause. 

 

Sexual maturation (vaginal opening) of F1 females at 4600 or 7300 ppm was slightly delayed when compared with Control; however, the values were within the Historical Control Data (HCD) range and probably related to the reduced birthweight and bodyweight gain during Days 1-21 of age, and therefore considered to be not biologically significant. The period of time to first estrus following maturation was unaffected by treatment; maturation in males was unaffected at any dietary concentration. 

 

Pre-pairing estrous cycles in the F0 and F1 females and estrous cycles following weaning of litters and before termination (F0 and F1) were unaffected by treatment. 

 

In the F1 females, mean body weight gain during gestation at 4600 or 7300 ppm was statistically significantly lower than Control (-11 and -25%, respectively); food intake was statistically significantly reduced at 7300 ppm on 7 of 23 days during gestation, particularly in the latter half of gestation. 

 

Mating performance and fertility, gestation length and gestation index in the F0 and F1 generations, litter size, survival and the ratio of male to female offspring and the anogenital distance of the F1 and F2 offspring were unaffected by treatment and no male F1 or F2 offspring had nipples.  

 

Hematology, blood chemistry and urinary parameters were not adversely affected by treatment at any dietary concentration in the F0 or F1 adults. Serum TSH and T4 concentrations were unaffected by treatment in the F0 (adults) or F1 (offspring on PND 22 of age or adults). 

 

In the F2, litters at 7300 ppm, the mean number of implantations and mean total and live litter size before culling were slightly lower than Control; this was considered to be related to the low mean body weight of the females at conception (GD 0). 

 

Sperm count, motility and motion and morphology, splenic immunophenotyping parameters, ovarian follicle and corpora lutea counts were unaffected by treatment in the F1. 

 

There was an increase in the incidence of hyaline droplets and basophilic tubules in F0 males at 6900 ppm and in F1 males at 7300 ppm. The incidence of granular casts in F0 males was increased at 6900 ppm but were seen occasionally in F1 males at 7300 ppm. These findings correlated with an increase in male kidney weights. The increase in hyaline droplets in the kidneys of males is consistent with the accumulation of alpha-2µ-globulin, a common finding in untreated male rats (Hard et al., 1993). Hyaline droplet accumulation is both sex and species specific (Frazier et al., 2012) and is not generally considered to be relevant to man. However, the associated multifocal basophilic tubules and granular casts, known as alpha-2µ-globulin nephropathy was considered adverse in the animals affected.

 

Organ weights of the unselected F1 and the F2 offspring on Day 22 of age were unaffected by parental treatment at 2100, 4300 or 6900 ppm or 2100, 4600 or 7300 ppm, respectively. 

 

There were no macroscopic findings related to treatment in the F0 (adults), F1 (offspring and adults) or F2 generation offspring. 

 

CONCLUSION

Based on the results of this study it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity which has the potential to be harmful to human health in the F0 parent animals was 6900 ppm and the F1 Cohort 1A and 1B adult animals was 7300 ppm (Mean achieved doses of 469 mg/kg bw/day for F0 males, 567 mg/kg bw/day for F0 females before pairing, 648 mg/kg bw/day for F1 males and 659 mg/kg bw/day for F1 females before pairing). No NOAEL was established for kidney changes in F0 and F1 males, consistent with the accumulation of alpha-2µ-globulin, which was deemed adverse for the animals, but is generally considered not to be relevant to human health.

The NOAEL for reproductive performance of the F0 and F1B parent animals and survival of the F1 and F2 offspring was concluded to be 6900 ppm or 7300 ppm, respectively (mean achieved doses of 466 mg/kg bw/day and 1047 mg/kg bw/day during F0 gestation and lactation and 494 mg/kg bw/day and 1044 mg/kg bw/day during F1B gestation and lactation).

The NOAEL for the growth of the F1 offspring (Days 1-21 of age) was 6900 ppm (mean achieved doses of 466 mg/kg bw/day and 1047 mg/kg bw/day) in F1 offspring and 7300 ppm (mean achieved doses of 494 mg/kg bw/day and 1044 mg/kg bw/day) in F2 offspring, as the reductions in the growth of both generations of offspring was likely caused by reduced palatability of the diet, and the consequent reduced food consumption and bodyweight and/or body weight gain of the females during gestation and lactation, and is therefore not relevant to humans. 

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

518 mg/kg bw/day

Study duration:

chronic

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

GLP and OECD guideline compliant study

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

OECD 414 study in rats (Klimisch 1, Key study); maternal NOAEL = 60 mg/kg bw/day; embryo-fetal NOAEL = 110 mg/kg bw/day

OECD 414 study in rabbits (Klimisch 1, Key study); maternal NOAEL = 300 mg/kg bw/day; embryo-fetal NOAEL = 300 mg/kg bw/day

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 September 2020 to [TBC]

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD 414 Guideline without deviations.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

RATIONALE FOR ANIMAL MODEL
The rabbit was chosen as the test species because of the requirement for a non-rodent species by regulatory agencies. The New Zealand White strain was used because of the historical control data available in this laboratory.

TEST ANIMALS
- Source: Envigo RMS (UK)
- Age at study initiation: approximately 21 to 24 weeks old.
- Weight at study initiation: 2.43 to 4.36 kg
- Housing: One animal per cage. Suspended cages fitted with perforated floor panels and mounted in batteries. Undertray lined with absorbent paper which is changed at least 3 times a week. Cages were also fitted with a plastic resting platform.
- Environmental enrichment: A soft white untreated wood block as aspen chew block was provided to each cage throughout the study and replaced when necessary. In addition, a stainless steel key ring was attached to the cage and a cage paper was provided to each cage from Day 20 after mating to allow expression of nesting behavior and was replaced as necessary.
- Diet (e.g. ad libitum): Teklad 2930, pelleted diet, restricted (200 g/animal/day).
In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly. Consumption of hay and vegetables were monitored qualitatively but not quantitatively.
- Water (e.g. ad libitum): Ad libitum, potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.
Water bowls were given to some animals as required.
- Acclimation period: For five days before the start of treatment (Days 1-5 of gestation).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15-21ºC.
- Humidity (%): 45-70%
- Air supply: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod (hrs dark / hrs light):14 hours light : 10 hours dark

IN-LIFE DATES: From: 29 September 2020 to 30 October 2020

Route of administration:

oral: gavage

Vehicle:

other: 1% Methylcellulose

Details on exposure:

DETAILS ON ROUTE OF ADMINISTRATION:
Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.

Rationale for route of administration: The oral (gavage) route of administration was chosen to simulate the conditions of possible human exposure.

PREPARATION OF DOSING SOLUTIONS:
Method of preparation: Starting with the lowest concentration, approximately 50% of the final volume of vehicle (1% methylcellulose) was added to the test item and magnetically stirred until uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was transferred into a beaker and mixed using a high shear homogenizer until visibly homogenous. The formulation was then returned to the container with a lid and mixed using a magnetic stirrer until homogenous for a minimum of 20 minutes.
Formulations were then transferred to their final containers via syringe while being magnetically stirred. Formulations were prepared in ascending order of concentration.
Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Frequency of preparation: Weekly, and divided into 7 daily aliquots.
Storage of formulation: Refrigerated (2-8 °C)

VEHICLE
- Concentration in vehicle: 15, 30 and 60 mg/mL.
- Dose volume: 5 mL/kg bw/day for all groups.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

ANALYSIS of STABILITY
The stability of a homogenous suspension of the test item in the vehicle was demonstrated as part of another study (Covance Study No. CF77KM) which demonstrated that prepared formulations in the concentration range of 5 to 200 mg/mL in the vehicle were stable for up to 15 days following refrigerated storage (2 to 8°C), and for one day following ambient storage (15 to 25°C).

FORMULATION ANALYSIS
Samples of each formulation prepared for administration in the first and last week of treatment were analyzed for achieved concentration of the test item

Details on mating procedure:

Method: Natural mating with New Zealand White bucks of established fertility at the supplier’s facility. Males and females were not closely related
Male/female ratio: 1 : 1 using identified stock New Zealand White bucks.
Checks: natural mating observed.
After mating: each female injected intravenously with 25 i.u. luteinizing hormone.
Day 0 of gestation = Day of mating.
Delivery to Covance: Day 1 after mating.

Duration of treatment / exposure:

Females were treated from Day 6 to Day 28 (inclusive) after mating.

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

75 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

DOSE SELECTION RATIONALE
The dose levels for investigation in this study (0, 75, 150 and 300 mg/kg bw/day) were selected in conjunction with the Sponsor and were based on the results of the preliminary embryo-fetal study in the rabbit, Covance Study No WT37TF.
In the preliminary study, where doses of 87.5, 175 or 350 mg/kg bw/day were tested, there were no unscheduled deaths and no treatment related clinical signs. Two females that received 350 mg/kg bw/day were observed with decreased activity following dose administration (one female on Day 7, the other female on Day 9 after mating), however, this observation was transient in nature. Slight body weight loss was observed following the commencement of treatment, at 350 and 175 mg/kg bw/day (-0.13 and -0.08 kg, respectively). Overall group mean body weight gain for all groups of treated females was lower than control, especially at 350 mg/kg bw/day. Maternal body weight loss adjusted for the contribution of the gravid uterine weight was greater in all treated groups and statistically significant at 350 mg/kg bw/day. Food intake of all groups of treated females was generally lower than control from Day 6 of gestation at 350 mg/kg bw/day or from Day 8 at 175 or 87.5 mg/kg bw/day, with the greatest and statistically significant reduction observed at 350 mg/kg bw/day (Days 6-29: -24% of Control). The lowest food intake was observed at 350 mg/kg bw/day following the commencement of treatment on Days 6 to 9 of gestation when intake was markedly lower than control. Despite a slight increase in liver weights at the high dose, there was no adverse effect of treatment on liver weights at any dose level investigated and no treatment related macroscopic abnormalities were detected among the maternal females or fetuses at scheduled termination. At 350 mg/kg bw/day, male, female and overall fetal weights at 350 mg/kg bw/day were lower than control (about 90% of controls).

Based on these results, the high dose level for investigation in this main embryo-fetal development study was set at 300 mg/kg bw/day with effects on body weight and food intake expected. The intermediate and low dose levels were set at 150 and 75 mg/kg bw/day, respectively, to fulfill the 2-fold to 4-fold dosing internal specified in the test guideline.

Maternal examinations:

MORTALITY: Yes
- A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.
A complete necropsy was performed in all cases.

CAGE SIDE OBSERVATIONS: Yes
- 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 animal ill-health amongst the occupant. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration: Pre-dose observation; One to two hours after completion of dosing; As late as possible in the working day.
- A detailed physical examination was performed on each animal on Days 1, 6, 12, 18, 24 and 29 after mating to monitor general health.

BODY WEIGHT: Yes
- The weight of each adult was recorded on Days 1, 3 and 6-29 after mating.

FOOD CONSUMPTION : Yes
- The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 2 after mating.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: Animals surviving until the end of the scheduled study period were killed on Day 29 after mating, by intravenous injection of sodium pentobarbitone.
- 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.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
For females surviving to term, the following uterine content was recorded:
Gravid uterine weight (including cervix and ovaries).

For each ovary/uterine horn, the following were recorded for all animals (including those prematurely sacrificed, where possible):
- Number of corpora lutea: Yes
- Number of implantation sites: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of Fetuses (live and dead): Yes

For apparently non-pregnant animals and for apparently empty uterine horns, the absence or number of uterine implantation sites was confirmed.

Blood sampling:

Not examined

Fetal examinations:

Examination of all viable fetuses and placentae: dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded, sampled as appropriate and retained in appropriate fixative. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was also recorded.

Approximately 50% of eviscerated fetuses were decapitated; heads were initially stored in Bouin’s fluid and subject to free-hand serial sectioning. Serial sections were examined for soft tissue abnormalities.
Remaining eviscerated fetuses and torsos were fixed in Industrial Methylated Spirit and processed and stained with Alizarin Red for skeletal development and abnormalities assessment.

Statistics:

For adult parameters, the analyses were carried out using the individual animal as the basic experimental unit. For litter/fetal findings the litter was taken as the treated unit and the basis for statistical analysis and biological significance was assessed with relevance to the severity of the anormaly and the incidence of the finding within the background control population.

The following data types were analyzed at each timepoint separately:
Body weight, using absolute weights and gains over appropriate study periods
Gravid uterine weight and adjusted body weight
Food consumption, over appropriate study periods
Caesarian section litter data (corpora lutea, implantations, pre/post implantation loss, live young and sex ratio - percentage male)
Placental, litter and fetal weights

The following comparisons were performed:
Group 1 vs 2, 3 and 4

A parametric or a non-parametric analysis of statistical tests was used for body weight, gravid uterus weight, food consumption, corpora lutea, implantations, pre/post implantation loss, live young, sex ratio - percentage male, placental, litter and fetal weights data.

Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level given as following:
* p<0.05
** p<0.01

Indices:

Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:
Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] x 100

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).Post-implantation loss was calculated from the formula:
Post-implantation loss (%)= [(Number of implantations – Number of live fetuses) / Number of implantations] x 100

Historical control data:

Historical Control data were routinely supplied for selected observations where this information was considered to assist interpretation of study data, and normally include both fetal and litter incidences.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment related clinical signs observed from Day 6 to Day 29 after mating or post-dose observations observed at any dose level investigated.
Clinical signs of decreased fecal pellets, fecal pellets reduced in size were observed in individual animals on occasion throughout the treatment groups, including Control.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Female 4F 90 that received 300 mg/kg bw/day was euthanized for welfare reasons on Day 21 of gestation due to persistent body weight loss and several days of little/no dry diet intake. There were no findings at macroscopic examination and this female was found to be pregnant with 4 live fetuses (5 early resorptions were noted). This premature death was of uncertain relationship to treatment.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Following the commencement of treatment on Day 6 after mating, slight group mean body weight loss was observed in all groups, including Control, until Day 11-12 of gestation. Thereafter, slight group body weight gain was observed in all groups. Overall (Day 6-29 of gestation) body weight gain was observed in all groups of treated females, however, this gain was slightly lower than Control for females receiving 300 mg/kg bw/day (-33% of Control) but the difference did not attain statistical significance.

Mean gravid uterine weights at termination on Day 29 of gestation was lower than Control (-10%) for females receiving 300 mg/kg bw/day, but without statistical significance. A dose-dependent slight adjusted maternal body weight loss was observed in all groups when taking into account the gravid uterine weight but differences did not attain statistical significance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Following the commencement of treatment and throughout the treatment period, food consumption at 300 mg/kg bw/day was generally lower than controls and differences attained statistical significance on Days 6-8, 14-20, 23 and 25-27 of gestation, leading to a statistically significantly lower mean food consumption between Days 6 and 29 (-20% of Control). Food intake was unaffected by treatment at 150 or 75 mg/kg bw/day.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no treatment related abnormalities detected among the maternal females at macroscopic examination on Day 29 of gestation.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

Administration of alpha-pinene multiconstituent at doses of 75, 150 and 300 mg/kg bw/day was generally well tolerated. At 300 mg/kg bw/day one female which had shown high food consumption prior to the start of treatment was killed for welfare reasons on Day 21 of gestation due to marked body weight loss and a prolonged period of low food intake; however, this was an atypical response (also when considering the absence of mortality at 350 mg/kg bw/day in the preliminary study) and no effect of treatment was inferred. There were no treatment related clinical signs at any dose level investigated. A slight reduction in group mean maternal body weight gain was apparent at 300 mg/kg bw/day (-33% of Control) but the difference did not attain statistical significance. Food consumption at 300 mg/kg bw/day was generally lower than controls and differences attained statistical significance on Days 6-8, 14-20, 23 and 25-27 of gestation, leading to a statistically significantly lower mean food consumption between Days 6 and 29 (-20% of Control). The extent of the reduction in body weight gain and food intake was considered not to be adverse as it was only slight and not associated with any change in maternal clinical condition or adverse effect on pregnancy outcome.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

There was considered to be no effect of treatment on the mean number of implantations, pre or post implantation loss.

Total litter losses by resorption:

effects observed, non-treatment-related

Description (incidence and severity):

One female in each of the 75 or 150 mg/kg bw/day groups had a total litter resorption but no effect of treatment was inferred. It was noted that the mean number of resorptions and thus post implantation loss at 300 mg/kg bw/day was slightly higher than in controls but no effect of treatment was inferred as differences did not attain statistical significance and live litter size was similar to controls.

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

effects observed, non-treatment-related

Description (incidence and severity):

Two females from each group, including controls (1F 4 and 20, 2F 27 and 44, 3F 61 and 69, 4F 80 and 93) were found not to be pregnant at macroscopic examination.

Details on maternal toxic effects:

Litter data was unaffected by maternal treatment at 75 or 150 mg/kg bw/day.

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 300 and 150 mg/kg bw/day, male, female and overall fetal weights were slightly low when compared to controls with statistical significance attained for female and overall fetal weights (90% of Control) at 300 mg/kg bw/day and for overall fetal weights at 150 mg/kg bw/day. Total litter weight was also low at 300 mg/kg bw/day compared to Control.
There was no effect of treatment on group mean placental weights. Total litter, male, female and overall fetal weights were unaffected by maternal treatment at 75 mg/kg bw/day.
At 150 and 300 mg/kg bw/day there was also a decrease in overall mean fetal weight compared to concurrent control. However, all the lower values were within the historical control data.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

Anogenital distance of all rodent fetuses:

no effects observed

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no major abnormalities or minor skeletal or visceral structural abnormalities considered to be related to treatment.
Across all treated groups there was an increase in incidence of incompletely ossified sternebrae compared to concurrent control but within historical control data (HCD) range.
At 150 and 300 mg/kg bw/day there was also an increase in incidence of incomplete ossified epiphyses, and metacarpals compared to concurrent control but also within HCD range. Incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and may be associated with the slight decrease in mean fetal weight seen at these dose levels. It was therefore not considered adverse.

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Embryo-fetal survival and live litter size were unaffected by treatment.

Mean male and female fetal weights, and consequently overall fetal weights, were slightly lower than Control in litters derived from females given 150 or 300 mg/kg bw/day (overall fetal weight was 10% lower than Control at 300 mg/kg bw/day); total litter weights were also slightly low at 300 mg/kg bw/day, but did not show a dose-related response. As differences were slight and not associated with any structural fetal abnormalities they were not deemed to represent an adverse effect of treatment.

There were no major abnormalities or minor skeletal or visceral structural abnormalities considered to be related to treatment.

At 150 and 300 mg/kg bw/day there was also an increased incidence of incomplete ossified epiphyses, and metacarpals compared to concurrent control but within HCD range: incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and may be associated with the slight decrease in mean fetal weight seen at these dose levels. It was therefore not considered adverse. 

Key result

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

FORMULATION ANALYSIS
The mean concentrations were within 7% of the nominal concentration, confirming the accuracy of formulation. The difference from mean and coefficient of variation remained within 4%, confirming precise analysis.

Conclusions:

Oral gavage administration of alpha-pinene multiconstituent at doses up to and including 300 mg/kg bw/day to pregnant New Zealand White rabbits from Day 6-28 of gestation inclusive, was associated with slight reductions in body weight gain and mean food consumption in females given 300 mg/kg bw/day, which were judged as non-adverse. The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was therefore concluded to be 300 mg/kg bw/day. The NOAEL for embryo-fetal survival and development was also concluded to be 300 mg/kg bw/day.

Executive summary:

In a study conducted according to OECD guideline 414 and in GLP conditions, three groups of 24 females received alpha-pinene multiconstituent at doses of 75, 150 or 300 mg/kg bw/day by oral gavage administration at a dose volume of 5 mL/kg bw, from Day 6 to 28 after mating. A similarly constituted Control group received the vehicle, 1% methylcellulose by oral gavage over the same treatment period and at the same volume dose as the treated females. 

The mean concentrations were within 7% of the nominal concentration, confirming the accuracy of formulation. The difference from mean and coefficient of variation remained within 4%, confirming precise analysis.

Animals were killed on Day 29 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterine weight was recorded. All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

Oral gavage administration of alpha-pinene multiconstituent at doses up to and including 300 mg/kg bw/day were generally well tolerated, with no test item-related premature deaths or changes in general clinical condition. Females which received 300 mg/kg bw/day showed a slight reduction in body weight gain throughout the treatment period (-33% of Control) and a statistically significantly lower mean food consumption between Days 6 and 29 (-20% of Control). At scheduled termination on Day 29 of gestation, no test item-related macroscopic abnormalities were detected at any dose level investigated.

Embryo-fetal survival and live litter size were unaffected by treatment. At 300 and 150 mg/kg bw/day, mean fetal weights were lower than Control (10% and 9% respectively) with male, female and total litter weights also slightly lower than control; this was associated with a lower group mean placental weight at 300 mg/kg bw/day. Group mean placental, litter and fetal weights were unaffected at 75 mg/kg bw/day. As differences were slight, within historical control data and not associated with any structural fetal abnormalities they were not deemed to represent an adverse effect of treatment. There were no major fetal abnormalities or minor skeletal or visceral structural abnormalities considered to be related to treatment. At 150 and 300 mg/kg bw/day there was also an increased incidence of incomplete ossified epiphyses, and metacarpals compared to concurrent control but within historical control data  range:  incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and may be associated with the slight decrease in mean fetal weight seen at these dose levels. It was therefore not considered adverse.

Oral gavage administration of alpha-pinene multiconstituent at doses up to and including 300 mg/kg bw/day to pregnant New Zealand White rabbits from Day 6-28 of gestation inclusive, was associated with slight reductions in body weight gain and in mean food consumption in females given 300 mg/kg bw/day, which were judged as non-adverse.

The No Observed Adverse Effect Level (NOAEL) for maternal toxicity was therefore concluded to be 300 mg/kg bw/day. The NOAEL for embryo-fetal survival and development was also concluded to be 300 mg/kg bw/day.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09 September 2020 to 20 November 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD 414 Guideline without deviations.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.31 (Prenatal Developmental Toxicity Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3700 (Prenatal Developmental Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD(SD)

Details on test animals or test system and environmental conditions:

RATIONALE FOR ANIMAL MODEL:
The rat was chosen as the test species because of the requirement for a rodent species by
regulatory agencies. The Sprague Dawley Crl: CD (SD) rat strain was used because of the
historical control data available at this laboratory.

TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 69 to 75 days old.
- Weight at study initiation: 203-289 g
- Housing: Acclimatization - up to four animals; During pairing - one (stock) male and one female; Gestation - one female
Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods. Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing. Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum
- Acclimation period: Five days before commencement of pairing.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod: Artificial lighting, 12 h light : 12 h dark
Environmental Enrichment
- Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing) and replaced when necessary.
- Plastic shelter: Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

IN-LIFE DATES: From: 09 September 2020 To: 08October 2020

Route of administration:

oral: gavage

Vehicle:

other: 1% methylcellulose

Details on exposure:

RATIONALE FOR ROUTE OF ADMINISTRATION:
The oral gavage route of administration was chosen to simulate the conditions of potential
human exposure.

PREPARATION OF DOSING SOLUTIONS:
Method of preparation: The required amount of test item was weighed.
Approximately 50% of the final volume of vehicle was added and magnetically stirred until the test material was uniformly mixed. The remaining vehicle was added to achieve the required volume and the formulation was mixed using a magnetic stirrer until homogeneous for a minimum of 20 minutes. The formulation was transferred to the final containers, via syringe, whilst magnetically
stirring. A series of formulations at the required concentrations were prepared in ascending order.

Frequency of preparation: Weekly.
Storage of formulation: Refrigerated (2-8 °C)

VEHICLE
- Concentration in vehicle: 6, 12 and 22 mg/mL
- Dose volume: 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Achieved concentration: Samples of each formulation were analyzed for the achieved
concentration of the test item.

Stability and homogeneity: Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 5 to 200 mg/mL were analyzed to assess the stability and homogeneity of the test item in the liquid matrix in Covance study CF77KM. Formulations were demonstrated to be homogeneous and stable for up to one day when stored at ambient temperature (15-25 °C) and for 15 days when stored refrigerated (2-8 °C).

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1 with identified stock males
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.
- Day 0 of gestation: When positive evidence of mating was detected.

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.

Duration of treatment / exposure:

Females were treated from Day 6 to Day 19 (inclusive) after mating

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

60 mg/kg bw/day (actual dose received)

Dose / conc.:

110 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

20 mated females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In the preliminary study using dose levels of 100, 125 and 150 mg/kg bw/day, (Covance Study Number KC09YH), 150 mg/kg bw/day was not tolerated with two females found dead at this dose level. Body weight loss was also evident from Day 10 to Day 14 of gestation and mean body weight stasis was recorded at 125 mg/kg bw/day during this period. Body weight gain at 125 mg/kg bw/day was statistically significantly lower than control throughout treatment (-23% of Control), whilst, bodyweight gain at 100 mg/kg bw/day was unaffected by treatment. Adjusted body weight was reduced for all treatment groups that received alphapinene multiconstituent. In addition, there was a large variation in individual adjusted body weight gain in females receiving 125 mg/kg bw/day, with two animals showing body weight loss over Days 6-20. This also correlated with reduced food consumption in these animals. Also, mean total litter weight, male fetal weight, female fetal weight and overall fetal weight was slightly lower at 125 and 150 mg/kg bw/day when compared with Control. Considering the steep response curve, it was considered that 110 mg/kg bw/day would elicit signs of maternal toxicity without causing death. The subsequent doses of 60 and 30 mg/kg bw/day were selected as mid and low doses, respectively, and were not expected to show any toxicity due to the steep response curve.

- Rationale for animal assignment: On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.
Method: To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups. Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.

Maternal examinations:

MORTALITY: Yes
- A viability check was performed near the start and end of each working day. Animals were
killed for reasons of animal welfare where necessary. A complete necropsy was performed in all cases.

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the
occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Detailed observations were recorded daily at the following times in relation to dose administration: A pre-dose observation; One to two hours after completion of dosing all groups; As late as possible in the working day.
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 0, 3 and and 6-20 after mating.

FOOD CONSUMPTION: Yes
- The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-7, 8-10, 11-13, 14-16 and 17-19 after mating inclusive.

POST-MORTEM EXAMINATIONS: Yes
- Animals surviving until the end of the scheduled study period were killed on Day 20 after mating by Carbon dioxide asphyxiation.
- 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 10% Neutral Buffered Formalin. The organs weighed, tissue samples fixed and sections examined microscopically are detailed in Table 1.
For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.
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.

Ovaries and uterine content:

For females surviving to term, the ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight (including cervix and ovaries): Yes
- Number of corpora lutea: Yes
- Number of implantation sites: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
- Number of Fetuses (live and dead): Yes

Blood sampling:

THYROID HORMONE ANALYSIS: Yes
- Blood samples were collected at termination of the study in all surviving adults in sublingual vein after an isoflurane anaesthesia. Samples were kept at ambient temperature (15 to 25°C) for a minimum of 30 minutes prior to centrifugation (2000 g for ten minutes at 4°C).
- Number of aliquots: Two per animal. Aliquot 1: 0.2 mL serum for T3/T4; Aliquot 2: residual serum for TSH.

Fetal examinations:

Method of kill for fetuses: Chilling on a cool plate (approximately 0 °C)
Examination of all viable fetuses and placentae: Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex and ano-genital distance of each fetus was recorded.
Examination of nominally 50% of fetuses in each litter: Sexed internally and eviscerated.
Fixation: Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS). Remaining fetuses were fixed whole in Bouin’s fluid.
Processing: Bouin’s fixed fetuses were subject to free-hand serial sectioning.
IMS fixed fetuses were processed and stained with Alizarin Red.

Fetal Pathology Examination
Bouin’s fixed fetuses: Serial sections were examined for visceral abnormalities.
Alizarin Red stained fetuses: Assessed for skeletal development and abnormalities.

Statistics:

See " Any other information on materials and methods incl. tables"

Indices:

Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss
was considered to reflect losses due to non-fertilization of ova and failure to implant. It was
calculated from the formula:
Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] x 100

Where the number of implantations exceeded the number of corpora lutea observed,
pre-implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to
have occurred).
Post-implantation loss was calculated from the formula:
Post-implantation loss (%)= [(Number of implantations – Number of live fetuses) / Number of implantations] x 100

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs at 30, 60, or 110 mg/kg bw/day.
There were no signs associated with dose administration at 30, 60, or 110 mg/kg bw/day.

Mortality:

mortality observed, treatment-related

Description (incidence):

One animal (No. 61) receiving 110 mg/kg bw/day was euthanized for welfare reasons on Day 14 of gestation (following 8 days of treatment) due to marked body weight loss that correlated with low food consumption. The death was attributed to an exaggerated individual animal response to treatment because this animal exhibited more severe changes in body weight and food intake than the other animals at this dose.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- At 110 mg/kg bw/day there was modest weight gain, or weight loss, from Day 8 to Day 12. Thereafter there was a modest weight gain but with a rate that was less than the control group. Overall, from Day 6 to 20 this group gained significantly less weight than the control group (76 g vs 117 g). For the 30 and 60 mg/kg bw/day groups, there was no significant effect on bodyweight gain over the same period.
- Gravid uterine weight was unaffected by treatment at 30, 60 or 110 mg/kg bw/day. When adjusted for gravid uterine weight, overall body weight stasis was evident at 110 mg/kg bw/day (overall change of -1 g) and adjusted body weight change was 90% of Control. Adjusted body weight change was unaffected at 30 or 60 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was persistently and statistically significantly low during Days 8-20 of gestation at 110 mg/kg bw/day and was marginally, but statistically significantly low, during Days 8-11 of gestation (86% of Control) at 60 mg/kg bw/day. Food intake at 30 mg/kg bw/day was unaffected by treatment.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Serum concentrations of Triiodothyronine, Thyroxine and Thyroid Stimulating Hormone were unaffected by treatment on Day 20 of gestation at 30, 60, or 110 mg/kg bw/day.

Clinical biochemistry findings:

not examined

Endocrine findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Absolute and adjusted thyroid weight was unaffected by treatment at 30, 60, or 110 mg/kg
bw/day.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Macroscopic examination revealed pale contents in the esophagus, jejunum, colon, and rectum; and bedding material was observed in the esophagus and stomach.
There were no treatment-related macroscopic observations in the adults on Day 20 of gestation at 30, 60, or 110 mg/kg bw/day.
All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including Control), and/or were as expected for Sprague-Dawley rats of this age/sex; therefore, they were considered not test item related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Of all the maternal organs only the thyroid was examined microscopically and no abnormalities were found. There were no treatment-related microscopic observations in the thyroid gland in adults at 30, 60, or 110 mg/kg bw/day.

Other effects:

no effects observed

Details on results:

See table of results in the section "Attached background material".

Number of abortions:

no effects observed

Description (incidence and severity):

All animals surviving to scheduled termination were pregnant.

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

The numbers of implantations and the pre- and post-implantation losses (%) were similar to Control and were therefore unaffected by treatment at 30, 60, or 110 mg/kg bw/day.

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Description (incidence and severity):

The numbers of resorptions (early, late) were similar to Control and were therefore unaffected by treatment at 30, 60, or 110 mg/kg bw/day.

Dead fetuses:

no effects observed

Description (incidence and severity):

The numbers of live young were similar to Control and were therefore unaffected by treatment at
30, 60, or 110 mg/kg bw/day.

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

The female was pregnant with 18 live embryos.

Other effects:

no effects observed

Description (incidence and severity):

All animals surviving to scheduled termination were pregnant. The numbers of implantations,
resorptions (early, late), pre- and post-implantation losses (%), live young and the ratio of
male to female fetuses were similar to Control and were therefore unaffected by treatment at
30, 60, or 110 mg/kg bw/day.

Details on maternal toxic effects:

There was no effect of treatment at 30, 60, or 110 mg/kg bw/day on reproductive performance, as assessed by the numbers of resorptions, implantation losses, and live young, the ratio of male to female fetuses, total litter or overall fetal weights and there was no effect of treatment on fetal
ano-genital distance.
See table of results in the section "Attached background material".

Key result

Dose descriptor:

NOAEL

Effect level:

60 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Description (incidence and severity):

Placental weight was marginally low at 110 mg/kg bw/day (87% of Control); placental weight was unaffected by treatment at 30 or 60 mg/kg bw/day.
Total litter and overall fetal weights were unaffected by treatment at 30, 60, or 110 mg/kg
bw/day.

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Description (incidence and severity):

Sex ratio was unaffected by treatment.

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

The litter weight was unaffected by treatment.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

When compared with Control, anogenital distance was unaffected by maternal treatment at
30, 60, or 110 mg/kg bw/day.

External malformations:

no effects observed

Description (incidence and severity):

There were no major or minor treatment related fetal abnormalities at 30, 60, or 110 mg/kg
bw/day.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

There were no major or minor treatment related fetal abnormalities at 30, 60, or 110 mg/kg
bw/day.
One fetus at 30 mg/kg bw/day had a ventricular septum membranous defect and one fetus at
both 60 and 110 mg/kg bw/day had ventricular septum muscular defects, and at 110 mg/kg
bw/day two fetuses from two litters had cleft palate/lip. As the incidence of these major
abnormalities was low and within the Historical Control Data (HCD), a relationship to treatment is not inferred.
There was an increase in incidence of malpositioned testes and brain haemorrhages, when
compared to Control that exceeded the Historical Control Data (HCD) range at 110 mg/kg
bw/day. At 60 mg/kg bw/day there was an increase in incidence of short 13 ribs and brain
haemorrhages, when compared to Control that exceeded the HCD range.
At 30 mg/kg bw/day there was an increase in incidence of malpositioned testes, when
compared to Control that exceeded the HCD range.
As the incidental findings showed no clear dose response they were considered not to be
treatment related.

Visceral malformations:

no effects observed

Other effects:

no effects observed

Details on embryotoxic / teratogenic effects:

There was no effect of
treatment at 30, 60, or 110 mg/kg/day on reproductive performance, as assessed by the
numbers of resorptions, implantation losses, and live young, the ratio of male to female
fetuses, total litter or overall fetal weights and there was no effect of treatment on fetal
ano-genital distance. Mean placental weight was marginally low at 110 mg/kg bw/day;
however, subsequent pathological examination of the fetuses showed that the low incidence
of major or minor abnormalities showed no relationship to treatment at 30, 60, or 110 mg/kg/day; therefore a relationship to treatment is not inferred.
See table of results in the section "Attached background material".

Key result

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Formulation analysis:

Mean concentrations of Alpha-pinene multiconstituent in the first and last formulations were within the applied limits of +10/-15% of nominal concentration, confirming accuracy of the formulations. The difference from mean remained within 7%, confirming precise analysis.

 

Table 2. Analyzed Concentrations for Alpha-pinene multiconstituent in 1% Methylcellulose

Occasion	Group	Nominal conc. (mg/mL)	Analyzed concentration (mg/mL)			RME (%)	Difference from mean (%)
			Analysis 1	Analysis 2	Mean
First preparation	1	0	ND	ND	-	-	-
	2	6	5.28	5.44	5.36	-10.7	±1.47
	3	12	11.0	11.5	11.2	-6.7	±2.33
	4	22	19.7	22.5	21.1	-4.1	±6.68
Second preparation	1	0	ND	ND	-	-	-
	2	6	5.48	5.56	5.52	-8.0	±0.74
	3	12	12.2	12.2	12.2	+1.7	±0.25
	4	22	21.3	21.5	21.4	-2.7	±0.38
Third preparation	1	0	ND	ND	-	-	-
	2	6	5.51	5.52	5.52	-8.0	±0.05
	3	12	11.1	11.7	11.4	-5.0	±2.84
	4	22	20.6	19.8	20.2	-8.2	±1.86

Conclusions:

Based on the results of this study, the maternal No-Observed-Adverse-Effect-Level (NOAEL) was 60 mg/kg bw/day (based on reduced food intake and reduced bodyweight gain) and the embryo-fetal No-Observed-Effect-Level (NOEL) was 110 mg/kg bw/day.

Executive summary:

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, three groups of 20 females received alpha-pinene multiconstituent at doses of 30, 60, or 110 mg/kg bw/day by oral gavage administration at a volume dose of 5 mL/kg bw, from Day 6 to 19 after mating. A similarly constituted Control group received the vehicle, 1% methylcellulose, at the same volume dose as the treated groups. Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.

Clinical observations, body weight, and food consumption were recorded. Adult females were examined macroscopically at necropsy on Day 20 after mating. Gravid uterine and thyroid weights were recorded, and the thyroid, kidney and liver were retained. A microscopic pathology investigation was performed on the thyroid from all pregnant animals.
The ano-genital distance, individual body weight, and placental weights were recorded for all fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination. Blood samples were taken at necropsy for subsequent thyroid hormone analysis in all pregnant animals.

Mean concentrations of alpha-pinene multiconstituent in the first and last formulations were within the applied limits of +10/-15% of nominal concentration, confirming accuracy of the formulations. The difference from mean remained within 7%, confirming precise analysis.

One animal receiving 110 mg/kg bw/day was euthanized for welfare reasons on Day 14 after 8 days of treatment due to marked body weight loss that correlated with low food consumption. The death of this animal was attributed to an exaggerated individual animal response to treatment because this animal exhibited more severe changes in body weight and food intake than the
other animals at this dose.
There were no treatment related clinical signs and there were no signs associated with dosing. Food intake was persistently low at 110 mg/kg bw/day during Days 8-20 of gestation. At 110 mg/kg bw/day, overall body weight gain (Days 6-20) was markedly low (76 g vs 117 g in control) and adjusted body weight change was -1 g (versus +34 g in Control). The reduced bodyweight gain was considered to be correlated with the reduced food consumption.
Gravid uterine weight was unaffected by treatment at any dose.
On Day 20 of gestation, serum concentrations of Triiodothyronine, Thyroxine, and Thyroid Stimulating Hormone were unaffected by treatment at 30, 60, or 110 mg/kg bw/day. Adult thyroid weight was unaffected by treatment at any dose and the tissue was macroscopically and microscopically normal.

Reproductive performance (numbers of resorptions, implantation losses, and live young, the ratio of male to female fetuses, placental, total litter or overall fetal weights) was considered to be unaffected by treatment at 30, 60, or 110 mg/kg bw/day. Fetal ano-genital distance was unaffected by treatment at 30, 60, or 110 mg/kg bw/day. There were no major or minor fetal abnormalities that showed a relationship to treatment at 30, 60, or 110 mg/kg bw/day.

Based on the results of this study, the maternal No-Observed-Adverse-Effect-Level (NOAEL) was 60 mg/kg bw/day (based on reduced food intake and reduced bodyweight gain) and the embryo-fetal No-Observed-Effect-Level (NOEL) was 110 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

60 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

GLP and OECD guideline compliant studies

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Justification for classification or non-classification

No adverse effects on reproductive function and on pre-natal development have been identified in the extended one generation toxicity study as well as in two pre-natal developmental toxicity studies in rats and rabbits. Therefore, the registered substance does not need to be classified for reproductive toxicity according to CLP regulation.