(1S,5S)-2,6,6-trimethylbicyclo[3.1.1]hept-2-ene

Endpoint summary

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Effects on fertility

Description of key information

In a screening test for reproductive/developmental effects (OECD Guideline 421), the NOAEL for reproductive performance and survival of the offspring is 12000 ppm (709 mg/kg bw/day during pre-pairing period) and the NOAEL for parental toxicity is 12000 ppm for males (728 mg/kg bw/day) and 6000 ppm for females (377 mg/kg bw/day during gestation) due to the lower food consumption and lower mean bodyweights and bodyweight gains observed in females at 12000 ppm, especially during gestation and lactation.

Due to the 31-33% reduction in offspring body weight gain in offspring to mothers treated with 12000 ppm, although almost certainly because of the smaller size of the dams and the lower food consumption during lactation due to the low palatability of the diet at this concentration, and not any specific or intrinsic toxicity of (-)-alpha-pinene, the NOAEL for offspring growth and development is 6000 ppm (807 mg/kg bw/day during lactation).

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 June 2019 to (TBC)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

Adopted on 29 July 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

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 Sprague-Dawley [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
- Females nulliparous and non-pregnant
- Age at study initiation: Males: 69 to 75 days old; Females: 83 to 89 days old
- Weight at study initiation: Males: 340 to 398 g, Females: 228 to 298 g
- Housing:
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. The cages were distributed on the racking to equalize, as far as possible, environmental influences amongst the groups.
For bedding, solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
Pre-paring: up to five animals of one sex per cage; during pairing: one male and one female, after mating: up to four male per cage, during gestation : one female per cage and during lactation: one female and her litter.
- Diet : SDS VRF1 Certified powdered diet ad libitium
- Water: potable water ad libitum
- Acclimation period:
Males: Six days before the beginning of treatment.
Females: 20 days before the beginning of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 40-70%.
- Photoperiod (hrs dark / hrs light): 12 hours light : 12 hours dark.

Route of administration:

oral: feed

Vehicle:

corn oil

Remarks:

test item to corn oil ratio 5:1

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): Weekly. The homogeneity and stability was confirmed at nominal concentrations of 1000 ppm and 15000 ppm for ambient temperature storage (15 to 25ºC) for up to 8 days and frozen storage (-10 to -30ºC) for up to 15 days. Due to the volatile nature of the test item, the low relative mean error and high coefficient of variation results at 1000 ppm were considered acceptable.
- Mixing appropriate amounts with (Type of food): SDS VRF1 Certified powdered diet
- Storage temperature of food: Frozen (nominally -20ºC).

VEHICLE
- Justification for use and choice of vehicle: Corn oil (stabilizer)
- Concentration in vehicle: test item to corn oil ratio 5:1

Details on mating procedure:

- Schedule: Agter three weeks of treatment
- M/F ratio per cage: one female and one male
- Length of cohabitation: Up to two weeks.
- Daily checks for evidence of mating: Ejected copulation plugs in cage tray and sperm in the vaginal smear.
- After successful mating each pregnant female was caged (how): individually in solid bottomed cages

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in Weeks 1-7 and the final week of treatment were taken, samples from Week 1 and the final week of treatment were analyzed for achieved concentration of the test item

Duration of treatment / exposure:

Males : three weeks before pairing, up to necropsy after a minimum of four consecutive weeks.
Females: Three weeks before pairing, then throughout pairing and gestation until Day 12 of lactation [necropsy on Day 13 of lactation (treated diet was available to the animals until the morning of necropsy)].

Frequency of treatment:

Continuous

Dose / conc.:

3 000 ppm (nominal)

Remarks:

corresponding to 181 mg/kg bw/d for reproductive phases males, 180 mg/kg bw/d for females during pre-mating period; 186 mg/kg bw/d during gestation and 418 mg/kg bw/d during lactation

Dose / conc.:

6 000 ppm (nominal)

Remarks:

corresponding to 355 mg/kg bw/d for reproductive phases males, 348 mg/kg bw/d for females during pre-mating period; 377 mg/kg bw/d during gestation and 807 mg/kg bw/d during lactation

Dose / conc.:

12 000 ppm (nominal)

Remarks:

corresponding to 728 mg/kg bw/d for reproductive phases males, 690 mg/kg bw/d for females during pre-mating period; 711 mg/kg bw/d during gestation and 1494 mg/kg bw/d during lactation

No. of animals per sex per dose:

10 of each sex for the F0 generation

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
The doses used in this study (0, 3000, 6000 and 12000 ppm) were selected in conjunction with the Sponsor.
Dietary levels were selected following the completion of the preliminary toxicity study (Covance Study number: HQ82CG). In the preliminary study, four animals/sex were given the test item at dietary inclusion levels of 3000, 6000 and 12000 ppm for three weeks. At the end of treatment there were no unscheduled deaths or clinical signs that could be attributed to the treatment. Body weight gain at the end of the treatment period was lower in males given 6000 or 12000 ppm and all female groups receiving (-)-alpha pinene when compared with controls (corresponding to 93% and 60% of Control at 12000 ppm in males and females, respectively). Liver weights were also increased in all treated animals (corresponding to an increase of 13% and 16% in absolute liver weight compared to Control at 12000 ppm in males and females, respectively). Therefore, the high dose level for this study was chosen to be 12000 ppm. The low and intermediate dose levels were chosen as incremental increases of the high dose and also corresponded to the levels in the preliminary study.

Positive control:

no

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily and daily
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).
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
F0 males: Once each week
F0 females: Once each week until pairing
Gestation phase - Days 0, 7, 14 and 20
Lactation phase - Days 1, 7 and 13

BODY WEIGHT: Yes
- Time schedule for examinations:
Males: Weekly during acclimation. Before feeding of the treated diets on the day that treatment commenced (Day 1) and twice weekly thereafter. On the day of necropsy.
Females: Before feeding of the treated diets on the day that treatment. commenced (Day 1) and twice weekly before pairing. Days 0, 7, 14 and 20 after mating. Day 1, 4, 7, and 13 of lactation.
On the day of necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): YES
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded daily from the day that treatment commenced.
Food consumption was not recorded for males and females during the period when paired for mating, but recommenced for males once pairing of all the animals but one was completed.

For females after mating and during lactation food consumption was performed daily.
From these records the mean daily consumption per animal (g/animal/day) was calculated for each phase

OTHER:
- Parturition Observations and Gestation Length: From Day 20 after mating, females were inspected three times daily for evidence of parturition. The progress and completion of parturition was monitored, numbers of live and dead offspring were recorded and any difficulties observed were recorded.

- Thyroid Hormone Analysis (see Any other information on materials and methods incl. tables): Animals were not fasted and the blood samples (0.1 mL) were performed from the sublingual vein under isoflurane anesthesic (Adults) or by decapitation (Offspring).
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). TSH and T4 parameters were examined.

Oestrous cyclicity (parental animals):

- Dry smears were taken from the beginning of treatment until animals were paired for mating, using cotton swabs
- Wet smears were taken, using pipette lavage, for 14 days before treatment (all females including spares); animals that failed to exhibit regular 4-5 day cycles were not allocated to study, after pairing until mating (for a maximum of 14 days) and for four days before scheduled termination (nominally Days 10-13 of lactation).

Sperm parameters (parental animals):

Immediately after scheduled sacrifice of each male, the left vas deferens, epididymis and testis were removed and the epididymis and testis were weighed. The following tests were performed:
- Sperm motility
- Sperm morphology
- Sperm count
- Homogenisation-resistant spermatid count

Litter observations:

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Clinical observations: Observed approximately 24 hours after birth (Day 1 of age) and then daily for evidence of ill-health or reaction to maternal treatment.
- Litter size: Daily on Days 1-13 of age.
- Sex ratio: Days 1, 4 ,7 and 13 of age.
- Individual offspring bodyweights: Days 1, 4 ,7 and 13 of age.
- Ano-genital distance: Day 1- all F1 offspring
- Nipple/ areolae count: Day 13 of age - male offspring

Postmortem examinations (parental animals):

SACRIFICE
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.
Time of necropsy: F0 males after 4 weeks of treatment, F0 females at the Day 13 of lactation, and F1 offspring: at the Day 13 of age.

GROSS 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.

HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 2 were prepared for microscopic examination and weighed, respectively.
In addition for females the number of implantation sites were counted, and the appearance of mammary tissue was examined for female whose litter died before Day 13 of lactation.

Postmortem examinations (offspring):

SACRIFICE
- a part of the F1 is sacrified on Day 4 and 13 of age (by decapitation): blood sampling is uses for Thyroid Hormone analysis (see any other informations on materials and method)
- On Day 13 of Age: Offspring not selected for thyroid hormone sampling were killed by intraperitoneal injection of sodium pentobarbitone.

GROSS NECROPSY
For premature deaths : Where possible, a fresh external macroscopic examination was performed with an assessment of stomach for milk content. Abnormal pups were retained.
For F1 offspring culled on Day 4 of age: Blood samples required. Externally normal offspring were discarded without examination. Externally abnormal offspring identified on dispatch to necropsy were examined externally and retained pending possible future examination.
For F1 offspring culled on Day 13 of age: Blood samples required. All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia. Abnormalities were retained in an appropriate fixative.

HISTOPATHOLOGY / ORGAN WEIGTHS
Thyroid glands were preserved from two offspring - one male and one female in each litter, where possible.

Statistics:

Statistical analyses were performed on the majority of data presented and results of these tests, whether significant or non-significant, are presented on the relevant tables. For some parameters, including estrous cycles before treatment, pre coital interval, mating performance and fertility, gestation index and stage of estrous cycle at termination, the similarity of the data was such that analyses were not considered to be necessary.
All statistical analyses were carried out separately for males and females. Data relating to food consumption were analyzed for males and females before pairing on a cage basis. For all other 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 anomaly and the incidence of the finding within the background control population.

Reproductive indices:

Percentage mating = Number animals mating x 100 / Animals paired.
Conception rate = Number animals achieving pregnancy x 100 / Animals mated.
Fertility index = Number animals achieving pregnancy x 100 / Animals paired.
Gestation index: Calculated for each group as: Number of live litters born x 100 / Number pregnant.

Offspring viability indices:

Post-implantation survival index = Total number offspring born x 100 / Total number uterine implantation sites.
Live birth index = Number live offspring on Day 1 after littering x 100 / Total number of offspring born.
Viability index = Number live offspring on Day 7 x 100 /Number live offspring on Day 1 after littering.
Sex ratio (%males) = Number of males in litter / Total number of offspring in litter x 100.
Lactation index = Number live offspring on Day 13 after littering x 100 /Number live offspring on Day 4 (after blood sampling)

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs that were considered to be related to treatment.
A variety of clinical signs, such as; hair loss, piloerection, thin build and scabs, were noted over the course of the study, however, due to the isolated incidences and low number of animals affected these were considered not to be treatment related.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Female No. 60 (1F) and her litter was euthanized on Day 10 of lactation due to welfare reasons. Macroscopic examination revealed a mass and a depression in the skin of the perigenital region of the dam, which, correlated with a marked focal abscess microscopically. The major factor contributing to death was recorded as skin lesions.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean body weight in males was considered to be unaffected throughout the study.
Group mean body weight change in females showed an initial statistically significant dose related mean weight loss from Days 1-4 in all treated groups when compared with Controls ; then, weight gain during Days 4-8 was statistically significantly higher than controls at 6000 and 12000 ppm. The initial body weight loss resulted in low group mean body weight change before pairing in all female treated groups, when compared with controls, with an apparent dose response.

Group mean body weight gain was statistically significantly low during gestation between
Days 0-7 (50%), 7-14 (58%) and 0-20 (75%) in females receiving 12000 ppm when compared with Controls, leading to statistically significantly lower mean bodyweights from Day 7 of gestation. Females receiving 3000 or 6000 ppm were considered to be unaffected by treatment.

Group mean body weight gain was low during lactation between Days 1-13 (30%) in females receiving 12000 ppm when compared with Controls, leading to statistically significantly lower mean bodyweights throughout lactation and a final mean bodyweight 14% lower than Controls.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Group mean food consumption in males was unaffected by treatment, with the exception of a slightly but statistically significant low food consumption on Day 1 in males receiving 12000 ppm.

Group mean food consumption was statistically significantly low between Days 1-4 of treatment in females receiving 6000 or 12000 ppm, 75% and 20%, respectively, when compared with Controls. Group mean food consumption was observed to be variable (ranging from 10-19 g/animal/day) in females receiving 12000 ppm between Days 4-22 of the treatment.
Group mean food consumption was low between Days 0-3 (47%) and 6-7 (82%) and
8-9 (80%) of gestation in females receiving 12000 ppm when compared with Controls.
Females receiving 3000 or 6000 ppm were considered to have no effect on food consmuption.

Group mean food consumption was statistically significantly low throughout lactation in females receiving 12000 ppm when compared with Controls. At 6000 ppm, mean food consumption was slightly low from Day 3-4 with the difference attaining statistical significance on Day 5-6. At 3000 ppm food intake was slightly low from Day 8-9 but differences did not attain statistical significance.

Food efficiency:

not specified

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

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, treatment-related

Description (incidence and severity):

Changes related to treatment with (-)-alpha-pinene were seen in the kidneys.
Accumulation of hyaline droplets and hyaline droplet nephropathy were seen in males that received 3000, 6000 or 12000 ppm (see Table 3).
The incidence and distribution of all other findings were considered to be unrelated to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

The study design also included an assessment of endocrine disruptor relevant endpoints. This objective was met by including the measurement of the hormone Thyroxine (T4) in adult reproductive males.
All samples taken from Day 13 of age offspring and F0 adult animals in Groups 1 to 4 had mean T4 concentrations comparable with the endogenous levels observed in the control matrix used to prepare the QC samples.
It was therefore concluded that, in the context of this study, the test item showed no evidence of being an endocrine disruptor.

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

There were no differences in the estrous cycle that was considered to be related to treatment with (-)-alpha-pinene.

Reproductive function: sperm measures:

effects observed, non-treatment-related

Description (incidence and severity):

All sperm analysis parameters were considered unaffected by treatment with (-)-alpha-pinene.
There were some statistically significant differences observed, such as straightness and linearity in sperm motion were high in males receiving 12000 ppm and lower numbers of looped tails in morphology in all male treated groups. Due to the relatively small magnitude of difference, the direction of change (i.e. the values were more positive) and the lack of any correlating histopathological findings these were considered incidental to treatment with (-)-alpha-pinene.
Spermatogenic staging:
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and the integrity of the various cell types present within the different stages. No cell or stage specific abnormalities were noted.

Reproductive performance:

no effects observed

Description (incidence and severity):

There were no differences in the estrous cycle, pre-coital interval, mating performance, fertility or gestation length that were considered to be related to treatment with (-)-alpha-pinene. The gestation index was 100% in all groups. All females were in diestrus on Day 13 of lactation.

Key result

Dose descriptor:

NOAEL

Effect level:

12 000 ppm (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

other: The NOAEL for parental toxicity is 12000 ppm for males (728 mg/kg bw/day), i.e. the highest dose tested

Key result

Dose descriptor:

NOAEL

Effect level:

728 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Remarks on result:

other: The NOAEL for parental toxicity is 12000 ppm for males (728 mg/kg bw/day), i.e. the highest dose tested

Key result

Dose descriptor:

NOAEL

Effect level:

709 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: NOAEL for reproductive performance and offspring survival

Key result

Dose descriptor:

NOAEL

Effect level:

377 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Based on reduced food consumption and bodyweight gain observed during gestation and lactation

Key result

Dose descriptor:

NOAEL

Effect level:

12 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: NOAEL for reproductive performance and offspring survival

Key result

Dose descriptor:

NOAEL

Effect level:

6 000 ppm (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Remarks on result:

other: Based on reduced food consumption and bodyweight gain observed during gestation and lactation

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

3 000 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 distribution of signs at physical examination showed no relationship to parental treatment.
There were clinical signs of patchy coat and thin build in separate litters of one maternal female (Animals 82F and 86F, respectively) that received 12000 ppm. Due to these signs only being observed in 1/10 females they were considered not be treatment related.

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

At 12000 ppm, mean body weights of male and female offspring on Day 1 of age were slightly low. Body weight change was statistically significantly low at Days 4-7 (80%/78%),
7-11 (63%/61%), 11-13 (58%/53%) and 1-13 (69%/67%) of the lactation period in male and female pups that received 12000 ppm when compared with controls.
Body weight change during lactation was slightly low in male (89/88%) and female (88/86%) offspring of maternal females that received 3000 or 6000 ppm when compared with controls, but differences did not attain statistical significance and without dose dependency.

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):

Ano-genital distance was considered unaffected by treatment with (-)-alpha-pinene

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

Male offspring did not develop nipples.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic findings in decedent offspring included, no milk in stomach, cannibalized and autolysis of abdominal contents.
On Day 13 of age (scheduled termination) one litter from a maternal female that received 12000 ppm had pups that were observed to be thin.

Other effects:

no effects observed

Description (incidence and severity):

The study design also included an assessment of endocrine disruptor relevant endpoints. This objective was met by including the measurement of the hormone Thyroxine (T4) in Day 13 offspring, and, because some developmental stages (e.g. gestational and neo-natal) are particularly sensitive to endocrine effects, an external examination of all offspring was conducted, measurement of the ano-genital distance of offspring on Day 1 of age was undertaken and nipple counts for male offspring on Day 13 of age were assessed. No adverse effect of treatment was evident on the circulating levels of thyroxine and therefore there was no requirement to extend the examination to the Day 4 offspring or to the adult females. All offspring were macroscopically ‘normal’, in particular no effects were seen on the external genitalia. Ano-genital distance and male nipple count were not adversely affected by treatment. It was therefore concluded that, in the context of this study, the tgest item showed no evidence of being an endocrine disruptor.

Litter size was unaffected by treatment with (-)-alpha-pinene.
Post implantation survival index (%), live birth index (%) and viability index (%) Day 4 and lactation index (%) Day 13 were all considered to be unaffected by treatment.
The sex ratio of males to females (%M) at Day 4 was slightly high in litters of maternal females that received 12000 ppm; however there was considerable inter-group variation and the initial difference from 50% was similar to that difference in the Control group therefore this is not considered to be an effect of treatment.

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

807 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Probably due to the reduced food consumption and bodyweight gain observed in dams during gestation and lactation

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

6 000 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

Remarks on result:

other: Probably due to the reduced food consumption and bodyweight gain observed in dams during gestation and lactation

Key result

Reproductive effects observed:

no

The homogeneity and stability was confirmed for (-)-alpha-pinene in SDS VRF1 diet with corn oil stabilizer at a ratio of test item to corn oil of 5 to 1 formulations at nominal concentrations of 1000 ppm and 15000 ppm for ambient temperature storage (15 to 25ºC) for up to 8 days and frozen storage (-10 to -30ºC) for up to 15 days. Due to the volatile nature of the test item, the low relative mean error and high coefficient of variation results at 1000 ppm were considered acceptable.

The mean concentrations were within the applied acceptance limits of +10/-15%, confirming the accuracy of formulation, with the exception of Group 2 for Week 1 and the Final week of treatment which has a relative mean error of -17.0% and -17.3% respectively. The difference from mean remained within 1%, confirming precise analysis, with the exception of Group 4 for the Final week of treatment where one sample was excluded due to analytical error so only one value is reported for this occasion.
The procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical procedure

Table 2. Summary of findings in the kidneys for animals killed after 5 weeks of treatment

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (ppm)	0	3000	6000	12000	0	3000	6000	12000
Abnormal Color	1	2	5	5	0	0	0	0
Number of tissues examined	10	10	10	10	10	10	10	10

Table 3.Summary of treatment related findings in the kidneys for animals killed after 5 weeks of treatment

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Dose (ppm)	0	3000	6000	12000	0	3000	6000	12000
Accumulation, Hyaline Droplets
Slight	0	2	3	1	-	-	-	-
Moderate	0	0	2	4	-	-	-	-
Total	0	2	5	5	-	-	-	-
Nephropathy, Hyaline Droplet
Minimal	0	1	2	2	-	-	-	-
Slight	0	0	1	1	-	-	-	-
Moderate	0	0	0	1	-	-	-	-
Total	0	1	3	4	-	-	-	-
Number of tissues examined	1	2	5	5	0	0	0	0

Conclusions:

In conclusion, daily dietary administration of the test item to Sprague-Dawley rats at concentrations of 3000, 6000 or 12000 ppm for three weeks before pairing, up to necropsy after a minimum of four consecutive weeks for males and for three weeks before pairing, throughout pairing, gestation and until Day 12 of lactation for females was well-tolerated in the adult animals, but did elicit histopathological changes in the male kidneys, in a dose dependent manner for all treated groups, indicative of an accumulation of alpha-2 urinary globulin considered as a phenomenon specific to male rats and of no significance to humans.
Following the start of treatment, females were more affected by treatment than males: all groups of treated females showed a dose related initial mean body weight loss on Days 1-4 of study, with females receiving 6000 or 12000 ppm having low food consumption during this period. In contrast, body weight gain of males was unaffected following the start of treatment while food intake was only slightly but statistically significantly low on Day 1 of treatment at 12000 ppm. This initial bodyweight loss associated with reduced food consumption is probably the consequence of the low palatability of the diet, especially at high test item concentrations. This lower food intake was confirmed during gestation and lactation periods in the females exposed to 12000 ppm. As a consequence, mean bodyweights of females at 12000 ppm were statistically significantly lower than Controls from Day 7 of gestation to Day 13 of lactation

There was no effect of treatment on estrus cycles, mating performance, fertility, gestation length or index, litter size or offspring survival.
At 12000 ppm, offspring body weights on Day 1 of age were slightly low and subsequent weight gain was 31-33% lower than in Control, a magnitude which is considered adverse: this almost certainly reflects the smaller size of the dams and the lower food consumption during lactation, and not any specific or intrinsic property of (-)-alpha-pinene. Offspring body weight gain was also slightly low (<15% lower) at 3000 or 6000 ppm but this magnitude of effect is considered not to be adverse and it was not dose related.
There was no effect of treatment on serum T4 levels in F0 males or male and female offspring at Day 13 of age, offspring anogenital distance, nipple counts or external genitalia, or microscopic changes in male and female reproductive organs and thyroids. There is thus no evidence that (-)-alpha-pinene is an endocrine disruptor.

Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive performance and survival of the offspring is 12000 ppm and the NOAEL for parental toxicity is 12000 ppm for males (728 mg/kg bw/day) and 6000 ppm for females (690 mg/kg bw/day) due to the lower food consumption and lower mean bodyweights and bodyweight gains observed in females at 12000 ppm, especially during gestation and lactation.
Due to the 31-33% reduction in offspring body weight gain in offspring to mothers treated with 12000 ppm, although almost certainly because of the smaller size of the dams and the lower food consumption during lactation due to the low palatability of the diet at this concentration, and not any specific or intrinsic property of (-)-alpha-pinene, the NOAEL for offspring growth and development is 6000 ppm (807 mg/kg bw/day during lactation).

Executive summary:

This study was a screening test for reproductive/developmental effects, and assessment of endocrine disruptor relevant endpoints, with dietary administration of the test item for at least five weeks.Three groups of ten male and ten female rats received the test item at dietary concentrations of 3000, 6000 or 12000 ppm. Males were treated daily for three weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for three weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received untreated diet with corn oil for the same duration as treated animals.

During the study, clinical condition, body weight, food consumption, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, sperm analysis (males only), organ weight and macroscopic pathology and histopathology investigations were undertaken. 

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, thyroid hormone analysis and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.

There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age.

The achieved dosages for animals during treatment and for reproductive phase females before pairing (F0) were 181, 355 and 728 mg/kg bw/day for males and 180, 348 and 690 mg/kg bw/day for females, at dietary concentrations of 3000, 6000 or 12000 ppm, respectively.
The achieved dosages for reproductive phase females during gestation were 186, 377 and 711 mg/kg bw/day, and during lactation were 418, 807 and 1494 mg/kg bw/day, at dietary concentrations of 3000, 6000 or 12000 ppm, respectively.

 

Parental toxicity

One control female was euthanized on Day 10 of lactation due to welfare reasons not related to treatment. Administration with test item at dietary concentrations up to and including 12000 ppm was well tolerated and there were no clinical signs that were considered to be treatment related.

Body weight in males was unaffected by treatment, while, initial body weight loss resulted in low body weight gain in all treated female groups before pairing and body weight gain was statistically significantly low during the gestation and lactation phases in females receiving 12000 ppm.

Food consumption in males was slightly but statistically significantly low on the first day of treatment at 12000 ppm. Food consumption was low in all female treated groups between Days 1-4, with a variable food consumption (ranging from 10-19 g/animal/day) between Days 4-22 in females receiving 12000 ppm. Food consumption during gestation and lactation was low in females receiving 12000 ppm.

Group mean kidney weight was high in all male treated groups when compared with controls. Group mean liver weight was high in male and females in all treated groups when compared with controls.
Sperm motion, counts and morphology was considered unaffected by treatment.
F0 males at 12000 or 6000 ppm, had a high incidence of pale kidneys. 
Histologically, accumulation of hyaline droplets and hyaline droplet nephropathy was observed in several males that received 3000, 6000 or 12000 ppm, in a dose dependent manner, indicative of the species- and sex-specific alpha 2μ-globulin nephropathy.

Litter Responses (F1)

All reproductive phase females were pregnant and successfully littered down; litter size, post implantation survival index, live birth index, viability index and sex ratio were all considered to be unaffected by treatment.
Ano-genital distance was considered unaffected in male and female offspring, and males did not have nipples at any dose.
Body weight and body weight change was low throughout lactation in male and female offspring of maternal females that received 12000 ppm.

There were no macropathology changes considered to be related to treatment in offspring.

 

In conclusion, daily dietary administration of the test item to Sprague-Dawley rats at concentrations of 3000, 6000 or 12000 ppm for three weeks before pairing, up to necropsy after a minimum of four consecutive weeks for males and for three weeks before pairing, throughout pairing, gestation and until Day 12 of lactation for females was well-tolerated in the adult animals, but did elicit histopathological changes in the males kidneys , in a dose dependent manner for all treated groups, indicative of an accumulation of alpha-2 urinary globulin considered as a phenomenon specific to male rats and of no significance to humans.
Following the start of treatment, females were more affected by treatment than males: all groups of treated females showed a dose related initial mean body weight loss on Days 1-4 of study, with females receiving 6000 or 12000 ppm having low food consumption during this period. In contrast, body weight gain of males was unaffected following the start of treatment while food intake was only slightly but statistically significantly low on Day 1 of treatment at 12000 ppm. This initial bodyweight loss associated with reduced food consumption is probably the consequence of the low palatability of the diet, especially at high test item concentrations. This lower food intake was confirmed during gestation and lactation periods in the females exposed to 12000 ppm. As a consequence, mean bodyweights of females at 12000 ppm were statistically significantly lower than Controls from Day 7 of gestation to Day 13 of lactation

There was no effect of treatment on estrus cycles, mating performance, fertility, gestation length or index, litter size or offspring survival.
At 12000 ppm, offspring body weights on Day 1 of age were slightly low and subsequent weight gain was 31-33% lower than in Control, a magnitude which is considered adverse: this almost certainly reflects the smaller size of the dams and the lower food consumption during lactation, and not any specific or intrinsic property of (-)-alpha-pinene. Offspring body weight gain was also slightly low (<15% lower) at 3000 or 6000 ppm but this magnitude of effect is considered not to be adverse and it was not dose related.

There was no effect of treatment on serum T4 levels in F0 males or male and female offspring at Day 13 of age, offspring anogenital distance, nipple counts or external genitalia, or microscopic changes in male and female reproductive organs and thyroids. There is thus no evidence that (-)-alpha-pinene is an endocrine disruptor.

Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive performance and survival of the offspring is 12000 ppm (709 mg/kg bw/day) and the NOAEL for parental toxicity is 12000 ppm for males (728 mg/kg bw/day) and 6000 ppm for females (377 mg/kg bw/day) due to the lower food consumption and lower mean bodyweights and bodyweight gains observed in females at 12000 ppm, especially during gestation and lactation.
Due to the 31-33% reduction in offspring body weight gain in offspring to mothers treated with 12000 ppm, although almost certainly because of the smaller size of the dams and the lower food consumption during lactation due to the low palatability of the diet at this concentration, and not any specific or intrinsic property of (-)-alpha-pinene, the NOAEL for offspring growth and development is 6000 ppm (807 mg/kg bw/day during lactation).

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

377 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

GLP study conducted acording to OECD guideline 421

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a screening test for reproductive/developmental effects, and assessment of endocrine disruptor relevant endpoints, conducted according to OECD Guideline 421 and in compliance with GLP, the test item was administered to groups of Crl:CD(SD) rats at dietary concentrations for at least five weeks.

Three groups of ten male and ten female rats received the test item at dietary concentrations of 3000, 6000 or 12000 ppm. Males were treated daily for three weeks before pairing, up to necropsy after a minimum of four consecutive weeks. Females were treated daily for three weeks before pairing, throughout pairing, gestation and until Day 12 of lactation. Females were allowed to litter, rear their offspring and were killed on Day 13 of lactation. The F1 generation received no direct administration of the test item; any exposure was in utero or via the milk. A similarly constituted Control group received untreated diet with corn oil for the same duration as treated animals.
During the study, clinical condition, body weight, food consumption, thyroid hormone analysis, estrous cycles, pre-coital interval, mating performance, fertility, gestation length, sperm analysis (males only), organ weight and macroscopic pathology and histopathology investigations were undertaken. 
The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance, thyroid hormone analysis and macropathology for all offspring were also assessed. Nipple counts were performed on male offspring on Day 13 of age.
There was no effect of treatment on the circulating levels of thyroxine (T4) in adult males or in offspring on Day 13 of age.
The achieved dosages for animals during treatment and for reproductive phase females before pairing (F0) were 181, 355 and 728 mg/kg bw/day for males and 180, 348 and 690 mg/kg bw/day for females, at dietary concentrations of 3000, 6000 or 12000 ppm, respectively. The achieved dosages for reproductive phase females during gestation were 186, 377 and 711 mg/kg bw/day, and during lactation were 418, 807 and 1494 mg/kg bw/day, at dietary concentrations of 3000, 6000 or 12000 ppm, respectively.
 
Parental toxicity
One control female was euthanized on Day 10 of lactation due to welfare reasons not related to treatment. Administration with test item at dietary concentrations up to and including 12000 ppm was well tolerated and there were no clinical signs that were considered to be treatment related.
Body weight in males was unaffected by treatment, while, initial body weight loss resulted in low body weight gain in all treated female groups before pairing and body weight gain was statistically significantly low during the gestation and lactation phases in females receiving 12000 ppm.
Food consumption in males was slightly but statistically significantly low on the first day of treatment at 12000 ppm. Food consumption was low in all female treated groups between Days 1-4, with a variable food consumption (ranging from 10-19 g/animal/day) between Days 4-22 in females receiving 12000 ppm. Food consumption during gestation and lactation was low in females receiving 12000 ppm.
Group mean kidney weight was high in all male treated groups when compared with controls. Group mean liver weight was high in male and females in all treated groups when compared with controls. Sperm motion, counts and morphology was considered unaffected by treatment. F0 males at 12000 or 6000 ppm, had a high incidence of pale kidneys.  Histologically, accumulation of hyaline droplets and hyaline droplet nephropathy was observed in several males that received 3000, 6000 or 12000 ppm, in a dose dependent manner, indicative of the species- and sex-specific alpha 2μ-globulin nephropathy.
Litter Responses (F1)
All reproductive phase females were pregnant and successfully littered down; litter size, post implantation survival index, live birth index, viability index and sex ratio were all considered to be unaffected by treatment. Ano-genital distance was considered unaffected in male and female offspring, and males did not have nipples at any dose. Body weight and body weight change was low throughout lactation in male and female offspring of maternal females that received 12000 ppm.
There were no macropathology changes considered to be related to treatment in offspring.
 
In conclusion, daily dietary administration of the test item to Sprague-Dawley rats at concentrations of 3000, 6000 or 12000 ppm for three weeks before pairing, up to necropsy after a minimum of four consecutive weeks for males and for three weeks before pairing, throughout pairing, gestation and until Day 12 of lactation for females was well-tolerated in the adult animals, but did elicit histopathological changes in the males kidneys, in a dose dependent manner for all treated groups, indicative of an accumulation of alpha-2 urinary globulin considered as a phenomenon specific to male rats and of no significance to humans. Following the start of treatment, females were more affected by treatment than males: all groups of treated females showed a dose related initial mean body weight loss on Days 1-4 of study, with females receiving 6000 or 12000 ppm having low food consumption during this period. In contrast, body weight gain of males was unaffected following the start of treatment while food intake was only slightly but statistically significantly low on Day 1 of treatment at 12000 ppm. This initial bodyweight loss associated with reduced food consumption is probably the consequence of the low palatability of the diet, especially at high test item concentrations. This lower food intake was confirmed during gestation and lactation periods in the females exposed to 12000 ppm. As a consequence, mean bodyweights of females at 12000 ppm were statistically significantly lower than Controls from Day 7 of gestation to Day 13 of lactation
There was no effect of treatment on estrus cycles, mating performance, fertility, gestation length or index, litter size or offspring survival. At 12000 ppm, offspring body weights on Day 1 of age were slightly low and subsequent weight gain was 31-33% lower than in Control, a magnitude which is considered adverse: this almost certainly reflects the smaller size of the dams and the lower food consumption during lactation, and not any specific or intrinsic property of (-)-alpha-pinene. Offspring body weight gain was also slightly low (<15% lower) at 3000 or 6000 ppm but this magnitude of effect is considered not to be adverse and it was not dose related. There was no effect of treatment on serum T4 levels in F0 males or male and female offspring at Day 13 of age, offspring anogenital distance, nipple counts or external genitalia, or microscopic changes in male and female reproductive organs and thyroids. There is thus no evidence that (-)-alpha-pinene is an endocrine disruptor.

Based on the results of this study, it is concluded that the No-Observed-Adverse-Effect-Level (NOAEL) for reproductive performance and survival of the offspring is 12000 ppm (709 mg/kg bw/day during pre-pairing period) and the NOAEL for parental toxicity is 12000 ppm for males (728 mg/kg bw/day) and 6000 ppm for females (377 mg/kg bw/day) due to the lower food consumption and lower mean bodyweights and bodyweight gains observed in females at 12000 ppm, especially during gestation and lactation. Due to the 31-33% reduction in offspring body weight gain in offspring to mothers treated with 12000 ppm, although almost certainly because of the smaller size of the dams and the lower food consumption during lactation due to the low palatability of the diet at this concentration, and not any specific or intrinsic property of (-)-alpha-pinene, the NOAEL for offspring growth and development is 6000 ppm (807 mg/kg bw/day during lactation).

Effects on developmental toxicity

Description of key information

OECD 414 study in rats (Klimisch 1, Key study):

• maternal NOAEL = 50 mg/kg bw/day;


• embryo-fetal NOAEL = 100 mg/kg bw/day

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

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

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: DRT / 1000107238
- Appearance: Colorless liquid
- Expiration date of the lot/batch: 30 November 2021

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

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)) strain was used because of the historical control data available at this laboratory.

TEST ANIMALS:
- Source: Charles River (UK) Ltd.
- Sexe: Females
- Age at study initiation (Day 2 after mating): Minimum age 10 weeks.
- Weight at study initiation (Day 3 after mating): 212-326 g on arrival.
- Number of animals per cage: one
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid and solid (polycarbonate) bottom; changed at appropriate intervals. Cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.
- Environmental enrichment: A soft white untreated wood block as aspen chew block was provided to each cage throughout the study and replaced when necessary and a plastic shelter was provided to each cage throughout the study and replaced at the same time as the cages.
- Diet: SDS VRF1 Certified pelleted diet, ad libitum; 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, ad libitum. Bottles were changed at appropriate intervals.
- Acclimation period: 4 days from arrival on Day 2 after mating to commencement of treatment on Day 6 after mating.

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

IN-LIFE DATES: From: 01 December 2021 To: 23 December 2021.

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 possible human exposure.

PREPARATION OF DOSING SOLUTIONS:
Method of preparation: Starting with the lowest concentration the required amount of the test item was weighed out into a beaker and 50% of the final volume of vehicle was added to the test item and magnetically stirred for two minutes to disperse the test item in the vehicle. The beaker was covered with foil during stirring. It was then made up to the required volume with vehicle. The suspension was transferred into a clear glass jar and mixed using a high shear homogenizer until it was visibly homogeneous. A lid was placed on the jar and it was mixed with a magnetic stirrer until homogeneous for a minimum of 20 minutes.
A series of suspensions at the required concentrations were prepared by dilution of individual weighings of the test item.
Frequency of preparation: Weekly, and have been prepared in advance of the first day of dosing within the known stability.
Storage of formulation: Refrigerated (2-8 °C)

VEHICLE
- Concentration in vehicle: 6, 10 and 20 mg/mL
- Dose volume: 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of each formulation prepared for administration in the first week and last week of treatment were analyzed for achieved concentration of the test item.
- Analytical procedure: The analytical method involved extraction and dilution in acetone followed by gas chromatographic analysis with flame ionization detection. Sample concentrations were determined with reference to external standards prepared in the concentration range 10 µg/mL to 100 µg/mL and an internal standard prepared at 0.38 mg/mL.

Details on mating procedure:

Method: Time mated to identified males of the same strain and source at the supplier’s facility.
Day 0 of gestation: When positive evidence of mating was detected.
Delivery to Labcorp: On Day 2 after mating.

Duration of treatment / exposure:

Females were treated from Day 6 to Day 20 (inclusive) after mating.

Frequency of treatment:

Once daily at approximately the same time each day.

Duration of test:

From mating until Day 20 (necropsy).

Dose / conc.:

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

Remarks:

Group 1 - Control

Dose / conc.:

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

Remarks:

Group 2 - Test item

Dose / conc.:

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

Remarks:

Group 3 - Test item

Dose / conc.:

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

Remarks:

Group 4 - Test item

No. of animals per sex per dose:

22 mated females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The doses used in this study (0, 30, 50 and 100 mg/kg bw/day) were selected in conjunction with the Sponsor.
In a preliminary study conducted with gestating females treated at 125 mg/kg bw/day, signs observed in association with the dosing procedures included piloerection (5/6 females), elevated gait (3/6 females), paddling of forepaws (2/6 females) and hunched posture (1/6 females). Two females were also found to be of thin build towards the end of the study (Day 18 or Day 20 of gestation). Mean body weight gain (Day 6-20 of gestation) was markedly low in females treated at 125 mg/kg bw/day when compared with Controls (37% of Control), leading to statistically significantly lower mean bodyweight at Day 20 than Controls (-20%) which correlated with low food consumption (63% of Control) recorded for these females throughout the treatment period. On Day 20 of gestation, mean gravid uterine weight was statistically significantly lower than Controls (-23%). Overall body weight gain (Day 6-20 of gestation) when adjusted for the weight of the gravid uterus revealed mean body weight loss in females treated at 125 mg/kg bw/day of 28 g when compared to a gain of 26 g in Controls. Total litter weight in females treated at 125 mg/kg bw/day was low (-24%) when compared with Controls. As the dose of 125 mg/kg bw/day led to high maternal toxicity, the dose of 100 mg/kg bw/day was considered more suitable as the high dose for this study. The subsequent low and intermediate dose levels selected were 30 and 50 mg/kg bw/day, respectively, in order to assess any dose-related effect.

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 when 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 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 acclimatisation 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 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 3, 5, 12, 18 and 21 after mating to monitor general health.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each adult was recorded on Days 3 and 6 to 21 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 3-5, 6-8, 9-11, 12-14, 15-17 and 18-20 after mating inclusive.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice: Animals surviving until the end of the scheduled study period were killed on Day 21 after mating by Carbon dioxide asphyxiation.
- 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.

ORGAN WEIGHTS: Yes
- The liver, kidneys and thyroid (after partial fixation) were weighed. For bilateral organs, left and right organs were weighed together. Requisite organs were weighed for animals killed at scheduled intervals.
Organ weights were presented both as absolute/unadjusted and adjusted for terminal bodyweight, using the weight recorded on the day of necropsy.

HISTOLOGY: Yes
For all adult female, thyroid were routinely preserved in 10% Neutral Buffered Formalin and then dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. Sections were stained with hematoxylin and eosin.

MICROSCOPY: Yes
Tissues preserved for examination were examined for all animals from all groups for premature deaths and scheduled kill.

The organs weighed, tissue samples fixed and sections examined microscopically are detailed in Table 1.

Ovaries and uterine content:

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

The number of uterine implantation sites were checked after staining with ammonium sulphide.

Blood sampling:

THYROID HORMONE ANALYSIS: Yes
- Blood samples were collected at termination of the study in all surviving adults to scheduled termination on GD 21 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: Subcutaneous injection of sodium pentobarbitone

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. Fetuses examined externally with abnormalities recorded. Particular attention was paid to the external genital organs of male fetuses. The sex and ano-genital distance of each fetus was recorded.

Examination of nominally 50% of fetuses in each litter: Sexed internally, examined for visceral abnormalities by fresh microdissection (Modified Staples technique) and subsequently fixed in Bouin’s solution.
Examination of nominally other 50% of fetuses in each litter: Sexed internally, eviscerated and fixed in Industrial Methylated Spirit (IMS).
Processing: Heads were removed from Bouin’s fixed fetuses and were subject to Wilsons free-hand serial sectioning. Torsos were retained in Bouin’s solution. 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:

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 anomaly 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
- C-section litter data (corpora lutea, implantations, pre/post implantation loss, live young and sex ratio - percentage male)
- Placental, litter and fetal weights
- Ano-genital distance, average for each litter adjusted for litter average fetal body weight
- Organ weights, absolute

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

A parametric or non-parametric analysis sequence 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.

For litter average ano-genital distance data, analysis of covariance was performed using the average fetal weight as covariate, unless non parametric methods were applied. The treatment comparisons were made on adjusted group means in order to allow for differences in fetal weight which might influence the ano-genital distance.

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

Indices:

Pre-implantation loss (%) = [(Number of corpora lutea – Number of implantations) / Number of corpora lutea] x 100
Post-implantation loss (%)= [(Number of implantations – Number of live fetuses) / Number of implantations] x 100

Historical control data:

Historical control data for fetal examinations were available on Rat/Crl:CD(SD)/Charles River UK for a period from november 2017 to April 2022 and included:
- 8 studies (3 preliminary and 5 main studies) for major and minor visceral fetal examinations
- 7 studies (2 preliminary and 5 main studies) for minor skeletal fetal examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Among females surviving until scheduled termination, two females that received 100 mg/kg bw/day were thin between GD 12-14 and 19-21 and one female that received 100 mg/kg bw/day had abnormally pale feces on Day 15-21. Piloerection was observed on GD 11-21 in several females that received 30, 50 or 100 mg/kg bw/day. The number of females observed at 30 or 50 mg/kg bw/day was however similar to Control in the same period, therefore the relationship to treatment is unclear in those groups. For females at 100 mg/kg bw/day between 1 and 13 animals per day were observed with piloerection in relation to dosing in this period.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

Female No. 87 that received 100 mg/kg bw/day was dispatched for reasons of animal welfare on GD 12 due to signs of decreased activity, abnormally cold to touch, piloerection and hunched posture. The animal had shown a weight loss of 22 grams between GD 8 and GD 12, and negligible food consumption from GD 9. Macroscopic examination did not reveal any abnormalities. The animal was pregnant with 12 live embryos.

Female No. 67 that received 100 mg/kg bw/day was dispatched for reasons of animal welfare on Day 17 after mating due to excessive body weight loss. Following commencement of treatment, this animal exhibited an overall weight loss of 40 grams (284 grams on Day 6 after mating; 244 grams on Day 17 after mating). The animal had shown a decrease in food consumption between Days 9 and 12 after mating, however, this increased between Days 12 and 15 after mating. Macroscopic examination did not reveal any abnormalities. This animal was found to be not pregnant.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Group mean bodyweight for females that received 100 mg/kg bw/day was consistently statistically significantly lower than Controls from GD 10 onwards, owing to low body weight gain and body weight losses in these animals on GD 6-15. Mean body weight gain in females receiving 100 mg/kg bw/day was lower than Control from GD 6 to GD 15, with a mean weight loss of 1 gram. Thereafter, on GD 15-17, body weight gain was statistically significantly higher than Control, being 36% higher, and on GD 17-21 mean body weight gain was similar to Control. Overall mean body weight gain (GD 6-21) for females receiving 100 mg/kg bw/day was subsequently statistically significantly low, at 68% of Control.

There was no effect on group mean body weight or mean body weight gain in females receiving 30 or 50 mg/kg bw/day.

When adjusted for the contribution of the gravid uterus, mean adjusted body weight was statistically significantly low in females receiving 100 mg/kg bw/day at 87% of Control, and was associated with an adjusted body weight loss between GD 6 and GD 21 of 6 grams (compared to a gain of 33 grams in Control).

There was no effect of treatment on mean adjusted body weight gain in females receiving 30 or 50 mg/kg bw/day or on the mean gravid uterine weight of female at any dose level.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Compared to Control, females receiving 100 mg/kg bw/day showed a reduction in group mean food consumption throughout the treatment period, resulting in a statistically significantly lower overall group mean food consumption, at 72% of Control.

There was no effect on group mean food consumption in females receiving 30 or 50 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:

effects observed, non-treatment-related

Description (incidence and severity):

There were no statistically significant differences in serum TSH levels in pregnant rats when administered with 30, 50 and 100 mg/kg bw/day of (-)-alpha-pinene when compared to the control group.
Concerning the measurement of T3 and T4 concentrations in rat serum, there was a statistically significant decrease in T3 only (i.e. 323 pg/mL compared to the control data of 456 pg/mL ; p-value < 0.01) in the high dose group of 100 mg/kg bw/day when compared with the control group. However, this decrease is within the laboratory HCD range based on 4 similar studies performed in 2022 (i.e. 272 to 451 pg/mL) and is probably due to a high mean control value. Also, it is not associated to a decrease in T4 or an increase in TSH values. Therefore, this effect is considered to not be attributed to the treatment with no biological significance.
See results in the Table 7.8.1/1.

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):

There was no effect on absolute group mean kidney, liver or thyroid weight at any dose level investigated.

All differences in organ weight parameters were consistent with normal variation and considered incidental. These differences were characterized by one or more of the following: lack of a dose relationship or correlative findings; and/or the magnitude was considered.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no maternal macroscopic abnormalities attributed to treatment detected during necropsy at any dose level investigated.
All macroscopic findings were considered spontaneous and/or incidental because they occurred at a low incidence, were randomly distributed across groups (including concurrent controls), 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:

no effects observed

Description (incidence and severity):

There were no microscopic changes in the thyroid gland at any dose level investigated.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

Daily oral gavage administration of (-)-alpha-pinene to pregnant Sprague Dawley rats at 30 or 50 mg/kg bw/day during the organogenesis phase and fetal phase of gestation (GD 6-20) was maternally well tolerated, with no effects to body weight gain, food consumption, TSH levels, fetal development and survival noted and there were no macro- or microscopic changes found that were attributed to treatment.

Two animals at 100 mg/kg bw/day were euthanized early, No. 87 on GD 12 and No. 67 on GD 17. Both were dispatched for reasons of animal welfare after both showed sustained body weight loss and decreased food consumption and No. 87 showed signs of decreased activity, abnormally cold to touch, piloerection and hunched posture. No abnormalities were recorded at necropsy and No. 87 was pregnant with 12 live embryos. No. 67 was not pregnant.

Overall mean body weight gain and overall food consumption were both statistically significantly low for females at 100 mg/kg bw/day, at 68% or 72% of Control, respectively. Mean body weight loss was recorded for these females on GD 6-15 (-1 gram), owing to low mean body weight gain (GD 6-9 and GD 9-13) and mean body weight losses (GD 9-13) in this period. At the individual level, 11 pregnant females showed some degree of weight loss during Day 6-15. Group mean food consumption was consistently statistically significantly low in the same period (46-85% of Control), with a period of marked reduction in food consumption observed in several females in this group, particularly in No. 85.
Group mean body weight gain was however statistically significantly higher than Control on GD 15-17 (136% of Control), after which it was similar to Control (GD17-21). These body weight gains coincided with an increase in group mean food consumption, although on GD 15-18 this was still statistically significantly lower than Control.

Among females surviving until scheduled termination at 100 mg/kg bw/day, two females were thin between GD 12-14 and 19-21, resulting from their low food consumption and body weight loss. One female that received 100 mg/kg bw/day had abnormally pale feces on Day 15-21.

At 100 mg/kg bw/day there was no effect on absolute group mean kidney, liver or thyroid weight, no maternal macroscopic abnormalities attributed to treatment detected during necropsy and no microscopic changes in the thyroid gland at any dose level investigated. TSH levels at 100 mg/kg bw/day were similar to Control.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

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

Details on maternal toxic effects:

At scheduled termination on GD 21, two Control females (Nos. 2 and 3), four females receiving 30 mg/kg bw/day (Nos. 24, 25, 26 and 37) and three females receiving 50 mg/kg bw/day (Nos. 46, 47 and 66) were found not to be pregnant. Two females receiving 100 mg/kg bw/day were euthanised for welfare reasons. Reproductive assessment was therefore made using 20 Controls and 18, 19 or 20 females at 30, 50 or 100 mg/kg bw/day, respectively.

There was no effect of maternal treatment on the mean number of implantations, resorptions (early or late), live young, sex ratio, or the levels of pre- or post-implantation losses at any dose level investigated.

Key result

Dose descriptor:

NOAEL

Effect level:

50 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:

effects observed, non-treatment-related

Description (incidence and severity):

Placental weight, male and female weights were statistically significantly low at 100 mg/kg bw/day, being 92%, 93% and 93% of Control, respectively.

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on the live young at any dose level investigated.

Changes in sex ratio:

no effects observed

Description (incidence and severity):

There was no effect of maternal treatment on the sex ratio at any dose level investigated.

Changes in litter size and weights:

effects observed, non-treatment-related

Description (incidence and severity):

Overall litter weights were statistically significantly low at 100 mg/kg bw/day, being 93% of Control.
There was no effect on overall litter weight at 30 or 50 mg/kg bw/day and no effect of maternal treatment on total litter weight at any dose level investigated.

Anogenital distance of all rodent fetuses:

no effects observed

Description (incidence and severity):

The ano-genital distance of the male and female fetuses was unaffected by maternal treatment at any dose level.

Changes in postnatal survival:

not examined

External malformations:

no effects observed

Description (incidence and severity):

The incidence of major and minor abnormalities showed no relationship to treatment.

Skeletal malformations:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of major and minor abnormalities showed no relationship to treatment.
At 100 mg/kg bw/day there was an increase in fetal incidence of delayed/incomplete ossification/unossified 5th and/or 6th sternebrae compared to concurrent control and outside of fetal HCD range; the incidence of litters affected was within the HCD range.
Incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and may be associated with the statistically significant decrease in mean fetal weight seen at this dose level. It is therefore not considered adverse.

Visceral malformations:

no effects observed

Description (incidence and severity):

The incidence of major and minor abnormalities showed no relationship to treatment.

Details on embryotoxic / teratogenic effects:

The incidence of major and minor fetal abnormalities showed no relationship to treatment and there were not affects to embryo-fetal survival at 30 or 50 mg/kg bw/day.

There was no effect on embryo fetal survival, as the mean number of implantations, resorptions (early or late), live young, sex ratio, or the levels of pre- or post-implantation losses were similar to Control.

Fetal development was, however, slightly affected, as placental weight and male, female, and overall litter weight were marginally statistically significantly low at 100 mg/kg bw/day, being 92%, 93%, 93% and 93% of Control, respectively, and there was an increase in fetal incidence of delayed/incomplete ossification/unossified 5th and/or 6th sternebrae compared to Control and outside of fetal Historical Control Data range. Incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and may be associated with the statistically significant decrease in mean fetal weight seen at this dose level. The marginal reduction in mean fetal weight together with the increase in delayed/incomplete ossification of the 5 th/6th sternebrae are expected observations due to the magnitude and persistence of the reduced maternal food consumption recorded (Fleeman et al 2005). It is therefore not considered adverse.

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:

The mean concentrations were within the applied limits of +10/-15% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 3%, confirming precise analysis. 

 

Table 7.8.2/1. Mean serum T3 and T4 concentrations (pg/mL) within treatment groups

Group	Treatment	Dose (mg/kg bw/day)	Parameters	T3 conc. in Females at termination (pg/mL)	T4 conc. in Females at termination (pg/mL)
1	Control (vehicle^a)	0	Mean	456	16200
			SD	171	6110
			CV%	37.5	37.7
			N	22	22
2	Test item	30	Mean	496	19200
			SD	138	7580
			CV%	27.8	39.5
			N	22	22
3		50	Mean	470	19000
			SD	145	8410
			CV%	30.9	44.3
			N	22	22
4		100	Mean	323**	13400
			SD	100	3760
			CV%	31.0	28.1
			N	20	20

^a: 1% Methylcellulose

**: statistically significant difference (p-value < 0.01) was observed between the treatment group (Group 4) and control group (Group 1) by Williams test (two tailed).

Conclusions:

Due to the magnitude of the treatment related body weight loss and low food consumption of females that received 100 mg/kg bw/day, it is concluded that, based on these data, the maternal No-Observed-Adverse-Effect-Level (NOAEL) is 50 mg/kg bw/day and the NOAEL for embryo-fetal survival, growth and development is 100 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 22 females received (-)-alpha-pinene at doses of 30, 50 or 100 mg/kg bw/day by oral gavage administration, from Day 6 to 20 after mating. A similarly constituted Control group received the vehicle, 1% methylcellulose at the same volume dose as treated groups. Animals were killed on Day 21 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 21 after mating, blood samples were taken for thyroid hormone analysis and the gravid uterus weight and thyroid weight were recorded. Microscopic pathology investigations were also undertaken. Ano-genital distance was measured for fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

 

The mean concentrations of the dose formulations were within the applied limits of +10/-15% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 3%, confirming precise analysis.

 

There were two test item related early deaths in the study at the high dose. Two females that received 100 mg/kg bw/day were euthanised, on GD 12 or Day 17 after mating, after one showed signs of decreased activity, abnormally cold to touch, piloerection and hunched posture and both had weight loss and low food consumption.

At 100 mg/kg bw/day, two females were thin between GD 12-14 and 19-21, one female had abnormally pale feces on Day 15-21. The group mean body weight at this dose level was consistently statistically significantly lower than Controls from GD 10 onwards, owing to low body weight gain and body weight losses in these animals on GD 6-15. Mean body weight gain in females receiving 100 mg/kg bw/day was lower than Control from GD 6 to GD 15, with a mean weight loss of 1 gram. Thereafter, on GD 15-17, body weight gain was statistically significantly higher than Control, being 36% higher, and on GD 17-21 mean body weight gain was similar to Control. Overall mean body weight gain (GD 6-21) of these animals was subsequently statistically significantly low, at 68% of Control. Group mean food consumption was also consistently lower than Control at 100 mg/kg bw/day throughout the treatment period, resulting in a statistically significantly lower overall group mean food consumption, at 72% of Control. When adjusted for the contribution of the gravid uterus, mean adjusted body weight was also statistically significantly low in females receiving 100 mg/kg bw/day, at 87% of Control, and was associated with body loss between GD 6 and GD 21 of 6 grams (compared to a gain of 33 grams in Control).

There were no statistically significant differences in serum Thyroid Stimulating Hormone (TSH) levels in pregnant rats when administered with 30, 50 and 100 mg/kg bw/day of (-)-alpha-pinene when compared to the control group. Concerning the measurement of T3 and T4 concentrations in rat serum, there is a statistically significant decrease (p-value < 0.01) observed in pregnant rats when administered with 100 mg/kg bw/day of (-)-alpha-pinene when compared to the control group. This effect is not attributed to the treatment as it is within the laboratory HCD range. Adult thyroid weight was unaffected by treatment at any dose and the tissue was macroscopically and microscopically normal.

 

There was no effect of treatment on embryo fetal survival, as the mean number of implantations, resorptions (early or late), live young, sex ratio, or the levels of pre- or post-implantation losses were similar to Control. Fetal development was, however, affected, as placental weight and male, female, and overall litter weight were marginally statistically significantly low at 100 mg/kg bw/day, being 92%, 93%, 93% and 93% of Control, respectively, and there was an increase in fetal incidence of delayed/incomplete ossification/unossified 5th and/or 6th sternebrae compared to Control and outside of fetal Historical Control Data range. Incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and may be associated with the statistically significant decrease in mean fetal weight seen at this dose level. It is therefore not considered adverse.

Maternal treatment at 30 or 50 mg/kg bw/day was well tolerated by the dams and there were no effects to embryo fetal survival or development.

 

Due to the magnitude of the treatment related body weight loss and low food consumption of females that received 100 mg/kg bw/day, it is concluded that, based on these data, the maternal No-Observed-Adverse-Effect-Level (NOAEL) is 50 mg/kg bw/day and the NOAEL for embryo-fetal survival, growth and development is 100 mg/kg bw/day.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subacute

Experimental exposure time per week (hours/week):

168

Species:

rat

Quality of whole database:

GLP study conducted according to OECD guideline 414

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a prenatal developmental toxicity study performed according to OECD Guideline 414 and in compliance with GLP, three groups of 22 females received (-)-alpha-pinene at doses of 30, 50 or 100 mg/kg bw/day by oral gavage administration, from Day 6 to 20 after mating. A similarly constituted Control group received the vehicle, 1% methylcellulose at the same volume dose as treated groups. Animals were killed on Day 21 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 21 after mating, blood samples were taken for thyroid hormone analysis and the gravid uterus weight and thyroid weight were recorded. Microscopic pathology investigations were also undertaken. Ano-genital distance was measured for fetuses and all fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.

 

The mean concentrations of the dose formulations were within the applied limits of +10/-15% of the nominal concentration, confirming the accuracy of formulation. The difference from mean remained within 3%, confirming precise analysis.

 

There were two test item related early deaths in the study at the high dose. Two females that received 100 mg/kg bw/day were euthanised, on GD 12 or Day 17 after mating, after one showed signs of decreased activity, abnormally cold to touch, piloerection and hunched posture and both had weight loss and low food consumption.

At 100 mg/kg bw/day, two females were thin between GD 12-14 and 19-21, one female had abnormally pale feces on Day 15-21. The group mean body weight at this dose level was consistently statistically significantly lower than Controls from GD 10 onwards, owing to low body weight gain and body weight losses in these animals on GD 6-15. Mean body weight gain in females receiving 100 mg/kg bw/day was lower than Control from GD 6 to GD 15, with a mean weight loss of 1 gram. Thereafter, on GD 15-17, body weight gain was statistically significantly higher than Control, being 36% higher, and on GD 17-21 mean body weight gain was similar to Control. Overall mean body weight gain (GD 6-21) of these animals was subsequently statistically significantly low, at 68% of Control. Group mean food consumption was also consistently lower than Control at 100 mg/kg bw/day throughout the treatment period, resulting in a statistically significantly lower overall group mean food consumption, at 72% of Control. When adjusted for the contribution of the gravid uterus, mean adjusted body weight was also statistically significantly low in females receiving 100 mg/kg bw/day, at 87% of Control, and was associated with body loss between GD 6 and GD 21 of 6 grams (compared to a gain of 33 grams in Control).

There were no statistically significant differences in serum Thyroid Stimulating Hormone (TSH) levels in pregnant rats when administered with 30, 50 and 100 mg/kg bw/day of (-)-alpha-pinene when compared to the control group. Concerning the measurement of T3 and T4 concentrations in rat serum, there is a statistically significant decrease (p-value < 0.01) observed in pregnant rats when administered with 100 mg/kg bw/day of (-)-alpha-pinene when compared to the control group. This effect is not attributed to the treatment as it is within the laboratory HCD range. Adult thyroid weight was unaffected by treatment at any dose and the tissue was macroscopically and microscopically normal.

 

There was no effect of treatment on embryo fetal survival, as the mean number of implantations, resorptions (early or late), live young, sex ratio, or the levels of pre- or post-implantation losses were similar to Control. Fetal development was, however, affected, as placental weight and male, female, and overall litter weight were marginally statistically significantly low at 100 mg/kg bw/day, being 92%, 93%, 93% and 93% of Control, respectively, and there was an increase in fetal incidence of delayed/incomplete ossification/unossified 5th and/or 6th sternebrae compared to Control and outside of fetal Historical Control Data range. Incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and may be associated with the statistically significant decrease in mean fetal weight seen at this dose level. It is therefore not considered adverse.

Maternal treatment at 30 or 50 mg/kg bw/day was well tolerated by the dams and there were no effects to embryo fetal survival or development.

 

Due to the magnitude of the treatment related body weight loss and low food consumption of females that received 100 mg/kg bw/day, it is concluded that, based on these data, the maternal No-Observed-Adverse-Effect-Level (NOAEL) is 50 mg/kg bw/day and the NOAEL for embryo-fetal survival, growth and development is 100 mg/kg bw/day.

Justification for classification or non-classification

In recent GLP screening test for reproductive/developmental effects, prenatal developmental toxicity study and assessment of endocrine disruptor relevant endpoints (OECD guidelines 421 and 414 on rats):

• Parental toxicity was observed due to the lower food consumption and lower mean bodyweights and bodyweight gains observed in females at 12000 ppm, especially during gestation and lactation in the OECD 421;


• Maternal toxicity was observed due to the magnitude of the treatment related body weight loss and low food consumption of females that received 100 mg/kg bw/day in the OECD 414;


• No effects on fertility and reproductive parameters were observed up to the highest dose tested. Furthermore, decrease in bodyweight and bodyweight gain of offspring were observed at 12000 ppm, probably due to the decrease in food consumption of dams during the lactation period in the OECD 421


• Fetal development was affected, as placental weight and male, female, and overall litter weight were marginally statistically significantly low at 100 mg/kg bw/day, being 92%, 93%, 93% and 93% of Control, respectively, and there was an increase in fetal incidence of delayed/incomplete ossification/unossified 5th and/or 6th sternebrae compared to Control and outside of fetal Historical Control Data range. Incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and may be associated with the statistically significant decrease in mean fetal weight seen at this dose level. It is not considered adverse and, therefore, no effects on fertility and reproductive parameters were observed up to the highest dose tested of 100 mg/kg bw/day in the OECD 414.

Therefore, the registered substance does not need to be classified for reproductive toxicity according to CLP Regulation (EC) n° 1272/2008.