Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

In a reproduction toxicity study conducted by diet on Sprague Dawley rats, the No-Observed-Adverse-Effect-Level (NOAEL) for parental systemic toxicity and reproductive/developmental toxicity is 12000 ppm (males: 749 mg/kg bw/day; females before pairing: 677 mg/kg bw/day; during gestation: 740 mg/kg bw/day; after 13 days of lactation: 1613 mg/kg bw/day).

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 July 2017 - 01 May 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Limited
- Females nulliparous and non-pregnant:
- Age at study initiation:Reproductive phase males: 69 to 76 days old & Reproductive phase females: 83 to 90 days old, Toxicity phase males: 69 to 76 days old
- Weight at study initiation: Reproductive phase males: 333 to 389 g, Female: 231 to 290 g and toxicity phase males: 337 to 386 g
- Housing: Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals. Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, treatment, gestation, littering and lactation periods. Grid bottomed cages were used during pairing. These were suspended above absorbent paper which was changed daily during pairing. Pre-paring: up to four males and up to five females 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: Reproductive phase males: six days before commencement the beginning of treatment. Reproductive phase females: 20 days before commencement the beginning of treatment.
Toxicity phase males: six days before commencement 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. Stability was confirmed for one day at ambient temperature and for eight days frozen for formulations between 1000 and 15000 ppm.
- 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:
- 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 diet prepared for administration during the first week of treatment and the final week of the Reproductive phase treatment (Day 10-12 of lactation) were analyzed for achieved concentration of the test item.
Duration of treatment / exposure:
Males : minimum of 6 weeks.
Females: 3 weeks before paring, then throughout mating and gestation until Day 13 of lactation.
Males toxicity phases: 13 weeks
Frequency of treatment:
Continuous
Dose / conc.:
3 000 ppm (nominal)
Remarks:
corresponding to 186 mg/kg bw/d for reproductive phases males, 179 mg/kg bw/d for females during pre-mating period; 192 mg/kg bw/d during gestation and 432 mg/kg bw/d during lactation
Dose / conc.:
6 000 ppm (nominal)
Remarks:
corresponding to 362 mg/kg bw/d for reproductive phases males, 358 mg/kg bw/d for females during pre-mating period; 381 mg/kg bw/d during gestation and 894 mg/kg bw/d during lactation
Dose / conc.:
12 000 ppm (nominal)
Remarks:
corresponding to 749 mg/kg bw/d for reproductive phases males, to 638 mg/kg bw/d for toxicity phase males, 677 mg/kg bw/d for females during pre-mating period; 740 mg/kg bw/d during gestation and 1613 mg/kg bw/d during lactation
No. of animals per sex per dose:
10 of each sex for the F0 generation; and 10 males at 12000 ppm for the toxicity phase
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose levels of 3000, 6000 and 12000 ppm were selected by the sponsor following completion of the preliminary study. In that study there was no effect on clinical condition, estrous cycles, blood chemistry, organ weights or macropathology. The food consumption for males and females receiving the test item at 3000, 6000 and 12000 ppm was lower than that of Control on Day 1 of treatment, with a dose-dependent decrease. Food consumption remained slightly low in all treated males thereafter. Overall body weight gains for males was low at all treatment levels (range 77 to 84 % of Control), however no similar effect was observed in the females.
Therefore, the high dose level for this study was 12000 ppm with the intermediate and low dose levels chosen to allow the determination of a dose response.

Positive control:
no
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly for all animals. Main phase females: Days 0, 7, 14 and 20 after mating and Days 1 and 7 and 13 of lactation.

BODY WEIGHT: Yes
- Time schedule for examinations: Males: Weekly during acclimation. Day that treatment commences and twice weekly thereafter. Before necropsy.
Main phase females:Weekly during acclimation. Day that treatment commences and twice weekly before pairing. Days 0, 7, 14 and 20 after mating and Days 1, 4 , 7 and 13 of lactation.

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 throughout the study.
Food consumption was not recorded for Reproductive phase males and females during the period when paired for mating (Day 22-25), but recommenced on Day 26.



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.
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 for 14 days before treatment (all females including spares); animals that failed to exhibit 4-5 day cycles were not allocated to study, after pairing until mating 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:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- Clinical observations: Observed approximately 24 hours after birth 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: Toxicity phases males after 13 weeks of treatment, Reproductive phase males after at least 5 weeks of treatment, for Reproductive phase females whose litter died before Day 13: On or after day the last offspring died. Reproductive phase female: Day 13 of lactation. F1 offspring: Selected offspring for thyroid hormone analysis - Day 4 of age and Scheduled kill - 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 of age : blood sampling is uses for Thyroid Hormone analysis (see any other informations on materials and method) and externally normal offspring discarded without examination
- On Day 13 of Age: All animals were subject to an external macroscopic examination; particular attention was paid to the external genitalia.

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.

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 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 = Number of males in litter / Total number of offspring in litter x 100.
Clinical signs:
no effects observed
Description (incidence and severity):
Administration with the tets item at dietary concentrations up to and including 12000 ppm was well tolerated and showed no test-item related changes in clinical condition.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Male animal No.8 (Control), was killed for welfare reasons on Day 31 of treatment due to a physical injury. Clinical signs were limited to abrasion of the skin and included wet abraisons of the head, hindlimb, lower dorsal and the perigenital area. Scabs of the dorsal surface were seen at the macroscopic examination and the histopathological examination identified the injury as a multifocal, epidermal ulceration.
Female animals No. 91 (12000 ppm) was found dead on Day 12 of lactation. Clinical signs were limited to a thin build over days 7-12 of lactation which correlated with slight bodyweight loss and reduced food consumption during the lactation phase. Abnormal contents were observed in the buccal cavity at the macropathology examination.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Overall group mean bodyweight for the reproductive phase males was unaffected by treatment at all dietary concentrations, although bodyweight gain was slightly low for groups 3 and 4 (14% and 12% less than Control, respectively). For females receiving 12000 ppm, mean body weight loss (-9 g) was recorded during Days 1-4 of study followed by high weight gain during days 4-8. Overall bodyweight gain before pairing was low (33% of Control) but the difference did not attain statistical significance.
During gestation, Group 4 females receiving 12000 ppm persistently showed statistically significantly lower bodyweights than Controls from Day 7. The overall weight gain during Days 0-20 was 31% lower than Controls, reflecting low weight gain during Days 0-7, 7-14 and 14 20, leading to statistically significantly lower bodyweight (13% lower than Controls on Day 20).
Bodyweights during gestation were unaffected at 3000 and 6000 ppm.
In the lactation phase, statistically significantly low bodyweights continued in females receiving 12000 ppm (8 to 10% lower than Controls), however, the overall bodyweight gain from Day 1 to 13 was comparable with Control.
Bodyweights during lactation were unaffected at 3000 and 6000 ppm.
Overall group mean bodyweight gain for the toxicity phase males was unaffected by treatment at 12000 ppm.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
The food consumption for reproductive males, and females before pairing receiving 3000, 6000 and 12000 ppm and toxicity phase males at 12000 ppm was lower than that of Control on Day 1 of treatment, with a dose-dependent decrease but was generally similar to that of the Controls intake in males and females thereafter, except for females at 12000 ppm where consumption remained low on Days 2 and 3 of treatment.
There was no significant effect on food consumption during the gestation phase, except slightly low food consumption in females receiving 12000 ppm throughout this period, with statistically significantly low consumptions on Days 17, 18 and 20.
During the lactation phase, females receiving 12000 ppm had statistically significantly low food consumption when compared with Control every day between Days 5 and 11 of lactation. Food consumption at 3000 and 6000 ppm was unaffected during the lactation phase.
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 the test item were seen in the kidneys of males.
Minimal cortical multifocal tubular basophilia was observed in all dose groups, and in four males receiving 6000 ppm and four males receiving 12000 ppm this finding was of slight severity. Cortical tubules with slight hyaline droplets was observed in nearly all animals at all dietary concentrations. Minimal tubular casts was identified in one male receiving 6000 ppm and two males receiving 12000 ppm. These findings, only observed in male rats, were indicative of species- and sex-specific alpha 2μ-globulin nephropathy.
No changes which could be related to treatment with Alpha-pinene multiconstituent were seen in the reproductive tissues examined in reproductive and toxicity males (i.e. up to 90 days of exposure at 12000 ppm).
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.
No adverse effect of treatment was evident on the circulating levels of thyroxine. The microscopic examination of the reproductive organs was unremarkable even after up to 13 weeks of exposure in males.
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):
All females allocated to study showed normal 4/5 day estrous cycles during the acclimatization period.
Nine out of ten females in Control and at 3000 ppm, and eight out of ten females at 6000 and 12000 ppm showed a regular estrous cycle of 4/5 days in length throughout the treatment period. One out of ten animals at 6000 ppm had an irregular estrous cycle. One out of ten females in Control, at 3000 ppm and at 6000 ppm, and two out of ten females at 12000 ppm were considered to be acyclic.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm count and total sperm in the testis for the reproductive phase males were statistically significantly low at 3000, 6000 and 12000 ppm when compared to Control after 5 weeks of treatment. However, these values were within Historical Control Data (HCD). In contrast, particularly high values, even outside maximal values of HCD, were observed in Control (232 vs. 207 million/g for sperm count and 421 vs. 383 million in total count, in Control and maximal HCD value, respectively). Therefore, the statistically significantly low values observed in the treated groups were not due to an effect of the treatment but to particularly high values in Control. This was confirmed by an absence of any dose-related decrease in those values and the absence of effect in epididymal sperm counts. In addition, no similar finding was observed in the toxicity phase males after 13 weeks of treatment at 12000 ppm.
The motility, morphology and concentration of sperm in the epididymis were unaffected by treatment in both the reproductive and toxicity phase males. No effects were observed in the epididymis.
Reproductive performance:
no effects observed
Description (incidence and severity):
Pre-coital interval, gestation length, mating performance, fertility and gestation index were considered unaffected by treatment with the test item. All females that successfully completed the study showed diestrus at termination.
All females that mated were pregnant.
A Group 2 female receiving 3000 ppm (No. 76) and a Group 3 female receiving 6000 ppm (No. 82) had total litter loss pre-day 1 and both were observed to have inactive mammary glands
Key result
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
6 000 ppm
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Offspring bodyweights at 12000 ppm were statistically significantly lower than Control on Day 7 of age (Male offspring: 88%; Female offspring: 87%) reflecting statistically significantly low weight gains from Day 4 to Day 7 of age. Male offspring bodyweights at 12000 ppm were also statistically significantly low compared to Control on Day 13 of age (86%) and female weights were also low (89%). This reflected a lower than Control bodyweight change for offspring over Days 1-13 (83%) which was statistically significant in males.
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
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, Sex Ratio and Survival Indices: Litter size, offspring survival to Day 13 of age and sex ratio were unaffected by parental treatment with the test item.
Key result
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Reproductive effects observed:
no

The homogeneity and stability was confirmed for the test item in SDS VRF1 Certified diet with a corn oil stabilizer at a ratio of test item to corn oil of 5 to 1 in vehicle formulations at nominal concentrations of 1000 ppm and 15000 ppm during frozen storage (-10 to -30°C) for up to 15 days. 1000 ppm formulations were confirmed stable for 1 day at ambient storage (15 to 25°C). 15000 ppm formulations were confirmed stable for 2 days at ambient storage (15 to 25°C). The accuracy of formulation preparation at 3000 ppm was confirmed.

For Week 1, the mean concentrations for all groups were between -20.7% and -15.8% from nominal which is outside the acceptance limits of +10/-15%. These results were confirmed by redilution and contingency analysis. For Day 10 – 12 of lactation, the mean concentrations were within the acceptance limits of +10/-15%, confirming the accuracy of formulation.

Conclusions:
In conclusion, dietary administration of the test item to Sprague-Dawley rats at concentrations of 3000, 6000 or 12000 ppm for five weeks to reproductive phase males and for three weeks before pairing, throughout gestation and up to Day 13 of lactation in females was well-tolerated in the adult animals but did elicit histopathological changes in the kidneys in the reproductive males, indicative of the species- and sex-specific alpha 2μ-globulin nephropathy.

Reproductive performance, fertility and offspring survival were unaffected by parental treatment. There was no effect of treatment on the number of implantations or litter size. From Day 7 of age, growth was slightly impaired for male and female offspring in the groups receiving 12000 ppm but this was not of sufficient magnitude to be considered adverse. No effects were observed in the epididymis.

In the context of this study, the test item showed no evidence of being an endocrine disruptor.
When excluding the species- and sex-specific histopathological findings observed in male kidneys, the No-Observed-Adverse-Effect-Level (NOAEL) for parental systemic toxicity and reproductive/developmental toxicity is 12000 ppm (males: 749 mg/kg bw/day; females before pairing: 677 mg/kg bw/day; during gestation: 740 mg/kg bw/day; after 13 days of lactation: 1613 mg/kg bw/day).
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 four 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 for three weeks before pairing, up to necropsy after a minimum of five consecutive weeks. Females were treated for three weeks before pairing, throughout pairing, gestation and until Day 13 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 the vehicle, basal diet with added corn oil throughout the same period. These animals constituted the Reproductive phase of the study.

An additional ten males were assigned to each of the Control and high dose groups and received either the control or treated diet for 13 consecutive weeks. These animals constituted the Toxicity phase of the study and were used for sperm analysis and histopathology of testes and epididymides at the end of the treatment period.

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

The clinical condition, litter size and survival, sex ratio, body weight, ano-genital distance 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 overall mean achieved dosages for the reproductive phase males were 186, 362 and 749 mg/kg bw/day at 3000, 6000 and 12000 ppm, respectively.

The mean achieved doses for females were 179, 358, and 677 mg/kg bw/day during the pre‑pairing period; 192, 381, and 740 mg/kg bw/day during gestation and 432, 894 and 1613 mg/kg bw/day during lactation at 3000, 6000 and 12000 ppm, respectively.

The overall mean achieved dose for the toxicity phase males was 638 mg/kg bw/day at 12000 ppm.

Two deaths occurred on study (one in Control and one at 12000 ppm), but neither could be attributable to treatment.

Administration with the test item at dietary concentrations up to and including 12000 ppm was well tolerated and showed no test-item related changes in clinical condition.

Overall group mean bodyweight gain for the reproductive phase males was unaffected by treatment at all dietary concentrations. For females before pairing, bodyweight gain was low in those receiving 6000 or 12000 ppm (67 and 33% of Control, respectively), but there was no statistical significance. During gestation, Group 4 females receiving 12000 ppm persistently showed statistically significantly lower bodyweights than Controls. The overall weight gain during Days 0-20 was 31% lower than Controls, reflecting low weight gain throughout gestation. Bodyweights during gestation were unaffected at 3000 and 6000 ppm. In the lactation phase, low bodyweights continued in females receiving 12000 ppm, however, the overall bodyweight gain from Day 1 to 13 was comparable with Control. Bodyweights during lactation were unaffected at 3000 and 6000 ppm. Overall group mean bodyweight gain for the toxicity phase males was unaffected by treatment at 12000 ppm.

The food consumption for reproductive males, and females before pairing receiving 3000, 6000 and 12000 ppm and toxicity phase males at 12000 ppm was lower than that of Control on Day 1 of treatment , with a dose-dependent decrease but was generally similar to that of the Controls intake in males and females thereafter. There was no significant effect on food consumption during the gestation phase, except slightly low food consumption in females receiving 12000 ppm throughout this period, with statistically significant low consumptions on Days 17, 18 and 20. During the lactation phase, females receiving 12000 ppm had statistically significantly low food consumption when compared with Control every day between Days 5 and 11 of lactation. Food consumption at 3000 and 6000 ppm was unaffected during the lactation phase.

All females allocated to study showed normal 4/5 day estrous cycles during the acclimatization period and there was no effect on cycles during treatment.

Pre-coital interval, gestation length, mating performance, fertility and gestation index were considered unaffected by treatment with the test item.

Liver adjusted weights showed a dose dependent increase across both sexes in the reproductive phase animals with a statistically significant difference observed at 6000 and 12000 ppm when compared with Control. However, the magnitude of the weight increase was considered as adaptative.

Reproductive phase male kidney adjusted weights also showed a statistically significant increase however this was not totally dose dependent and no similar observation was noted in the reproductive phase females.

All other organ weights for the reproductive phase males and females, and the toxicity phase males were unaffected by treatment.

The motility, morphology and concentration of sperm in the epididymis and testis were unaffected by treatment in both the reproductive and toxicity phases (i.e. up to 13 weeks of exposure). No effects were observed in the epididymis.

The macroscopic examination performed after 5 weeks and after 13 weeks of treatment revealed no test item- related lesions. The only histopathological changes related to treatment with the test item were seen in the kidneys of males manifesting as multifocal tubular basophilia in all treated groups and granular casts in 1 or 2 animals at 6000 or 12000 ppm, indicative of the species- and sex-specific alpha 2μ-globulin nephropathy. No changes which could be related to treatment with Alpha-pinene multiconstituent were seen in the reproductive tissues examined in reproductive and toxicity males (i.e. up to 90 days of exposure at 12000 ppm).

F1 Litter Responses

No signs were recorded that were considered to be related to parental treatment with the test item.

All females that mated were pregnant.

A Group 2 female receiving 3000 ppm (No. 76) and a Group 3 female receiving 6000 ppm (No. 82) had total litter loss pre-day 1 and both were observed to have inactive mammary glands.

Litter size, offspring survival to Day 13 of age and sex ratio were unaffected by parental treatment with test item.

Offspring ano-genital distances and nipple counts in male offspring were considered unaffected by treatment.

Offspring bodyweights at 12000 ppm were statistically significantly lower than Control on Day 7 of age (Male offspring: 88%; Female offspring: 87%). Male offspring bodyweights at 12000 ppm were also statistically significantly low compared to Control on Day 13 of age (86%) and weights of females were also low (89%). This reflected lower than Control bodyweight change for male offspring over Days 1-13 (83%) which was statistically significant.

Offspring bodyweights at 3000 and 6000 ppm were unaffected by treatment.

There were no macroscopic findings that were considered to be related to parental treatment.

In conclusion, dietary administration of the test item to Sprague-Dawley rats at concentrations of 3000, 6000 or 12000 ppm for five weeks to reproductive phase males and for three weeks before pairing, throughout gestation and up to Day 13 of lactation in females was well-tolerated in the adult animals but did elicit histopathological changes in the kidneys in the reproductive males, indicative of the species- and sex-specific alpha 2μ-globulin nephropathy. 

Reproductive performance, fertility and offspring survival were unaffected by parental treatment. There was no effect of treatment on the number of implantations or litter size. From Day 7 of age growth was slightly impaired for male and female offspring in the groups receiving 12000 ppm but this was not of sufficient magnitude to be considered adverse.

In the context of this study, alpha-pinene multiconstituent showed no evidence of being an endocrine disruptor.

When excluding the species- and sex-specific histopathological findings observed in male kidneys, the No-Observed-Adverse-Effect-Level (NOAEL) for parental systemic toxicity and reproductive/developmental toxicity is 12000 ppm (males: 749 mg/kg bw/day; females before pairing: 677 mg/kg bw/day; during gestation: 740 mg/kg bw/day; after 13 days of lactation: 1613 mg/kg bw/day).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
749 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
566.5 mg/m³
Study duration:
subchronic
Species:
mouse
Quality of whole database:
The relevance of the effects observed in male rats and mice can be questioned: first, the heat fixation at 65°C of caudae samples for sperm counts may have altered the integrity of the samples; secondly, these changes in sperm levels were not corroborated by other findings such as histopathological changes in other reproductive organs/tissues or other sperm parameters (motility, spermatid counts, etc.). In addition animals were exposed whole body by inhalation, which likely resulted in systemic exposure much higher than intended exposure from target doses (animals likely exposed by oral route through grooming).
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a 90-day inhalation study conducted by NTP similarly to OECD guideline 413, male rats exposed to alpha-pinene showed effects on renal and reproductive systems. The effects on kidneys are not relevant to humans as they are based on sex- and species-specific renal effects linked to alpha 2µ-globulin accumulation. A decrease in sperm count in cauda epididymis at 200 and 400 ppm was observed. Therefore the NOAEC identified for male rats is 100 ppm. A lower body weight gain and death of 6 out of 10 females in the high dose group (400 ppm) were observed. A NOAEC could be defined in female rats at 200 ppm on the basis of mortality and a lower body weight gain.

 

In a 90-day inhalation study conducted by NTP similarly to OECD guideline 413, mice were exposed to alpha-pinene. Minimal to moderate hyperplasia have been observed in the transitional epithelium of the urinary bladder from 100 ppm in both sexes.

Decreased numbers of sperm per mg cauda in 200 and 400 ppm males and cauda sperm in 100, 200, and 400 ppm males were observed.

A NOAEC for female mice has been set at 50 ppm based on minimal to moderate hyperplasia observed in the transitional epithelium of the urinary bladder and a LOAEC for males at 100 ppm has been determined based on significantly decreased sperm count per mg cauda .

However, the relevance of the effects observed in male rats and mice can be questioned: first, the heat fixation at 65°C of caudae samples for sperm counts may have altered the integrity of the samples; secondly, these changes in sperm levels were not corroborated by other findings such as histopathological changes in other reproductive organs/tissues or other sperm parameters (motility, spermatid counts, etc.). In addition animals were exposed whole body by inhalation, which likely resulted in systemic exposure much higher than intended exposure from target doses (animals likely exposed by oral route through grooming).

Dietary administration of the test item to Sprague-Dawley rats at concentrations of 3000, 6000 or 12000 ppm for five weeks to reproductive phase males and for three weeks before pairing, throughout gestation and up to Day 13 of lactation in females was well-tolerated in the adult animals but did elicit histopathological changes in the kidneys in the reproductive males, indicative of the species- and sex-specific alpha 2μ-globulin nephropathy.  

Reproductive performance, fertility and offspring survival were unaffected by parental treatment. There was no effect of treatment on the number of implantations or litter size. From Day 7 of age, growth was slightly impaired for male and female offspring in the groups receiving 12000 ppm but this was not of sufficient magnitude to be considered adverse. No effects were observed in the epididymis.

In the context of this study, the test item showed no evidence of being an endocrine disruptor.

When excluding the species- and sex-specific histopathological findings observed in male kidneys, the No-Observed-Adverse-Effect-Level (NOAEL) for parental systemic toxicity and reproductive/developmental toxicity is 12000 ppm (males: 749 mg/kg bw/day; females before pairing: 677 mg/kg bw/day; during gestation: 740 mg/kg bw/day; after 13 days of lactation: 1613 mg/kg bw/day).

Effects on developmental toxicity

Description of key information

A study will be conducted in rats according to OECD guideline 414.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no study available (further information necessary)
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 90-day repeated dose toxicity studies by inhalation performed in rats and mice, a decrease in sperm count in cauda epididymis at 200 and 400 ppm was observed in rats and decreased sperm count per mg cauda epididymis at 200 and 400 ppm and decreased sperm count in cauda epididymis at 100, 200, and 400 ppm were observed in mice.

However, the relevance of these effects can be questioned: first, the heat fixation at 65°C of caudae samples for sperm counts may have altered the integrity of the samples; secondly, these changes in sperm levels were not corroborated by other findings such as histopathological changes in other reproductive organs/tissues or other sperm parameters (motility, spermatid counts, etc.). Also, these decreases might be secondary to stress induced by hyperplasia of bladder epithelium at the 3 highest doses in mice. In addition animals were exposed whole body by inhalation, which likely resulted in systemic exposure much higher than intended exposure from target doses (animals likely exposed by oral route through grooming).

A recent OECD 421 study was conducted by dietary route in rats, including satellite groups exposed up to 90 days at 12000 ppm, corresponding to 638 mg/kg bw/day. No effects in sperm parameters, i.e. motility, morphology and sperm count in the testes and epididymides were observed in reproductive males (exposed at least 5 weeks) and toxicity males (exposed up to 13 weeks). Also, no histopathological lesions were observed in the testes and epididymides of these animals.

These results are in contradiction with the decrease in cauda epididymides sperm count observed in the 90-day study conducted in rats by inhalation (from 200 ppm, corresponding to ~330 mg/kg bw/day).

Therefore, no classification for reproduction toxicity is required for the test item according to Regulation (EU) No 1272/2008 (CLP).