Registration Dossier

Administrative data

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 property 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
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

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

In recent GLP screening test for reproductive/developmental effects, and assessment of endocrine disruptor relevant endpoints (OECD guideline 421):

- 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.

- 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.

Therefore, the registered substance does not need to be classified for reproductive toxicity according to CLP Regulation (EC) No. 1272/2008.

Additional information