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Description of key information

In a Preliminary Toxicity Study, the systemic toxic potential of the registered substance was assessed in a 21-day oral study (dietary administration) in Sprague-Dawley rats to aid in the selection of a suitable high dose for a subsequent OECD 421 screening study. Based on the results of this study, it was concluded that the effects observed at the high dose of 12000 ppm do not preclude the use of this dose level as the high dose for the main OECD 421 study.


In a GLP and OECD 413 compliant 90-day repeated dose toxicity study by inhalation with the registered substance, the No Observed Adverse Effect Concentration (NOAEC) was considered to be 0.3 mg/L, based on the two females decedents at the concentration level of 0.9 mg/L due to general poor clinical condition.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
subacute
Species:
rat
Quality of whole database:
Preliminary study

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
300 mg/m³
Study duration:
subchronic
Experimental exposure time per week (hours/week):
30
Species:
rat
Quality of whole database:
GLP and OECD guideline compliant study

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
GLP and OECD guideline compliant study

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The purpose of this study was to assess the systemic toxic potential of the test item in a 21-day oral study (dietary administration) in Sprague-Dawley rats, and to aid in the selection of a suitable high dose for a subsequent OECD 421 screening study. Three groups, each comprising four male and four female Crl:CD(SD) rats received the test item at dietary concentrations of 3000, 6000 and 12000 ppm. A similarly constituted control group received the vehicle, basal diet with added corn oil. During the study, clinical condition, body weight, food consumption, visual water consumption, estrous cycles, blood chemistry, organ weight and macropathology investigations were undertaken.
The overall mean achieved dosages were 188, 363 and 717 mg/kg bw/day in males and 185, 355 and 681 mg/kg bw/day in females receiving 3000, 6000 and 12000 ppm, respectively.
Administration for 21 days at dose levels up to and including 12000 ppm was well tolerated. There were no premature deaths and no test item-related changes in clinical condition, estrous cycles, blood chemistry and macropathology.
 
Following the start of treatment at 12000 ppm, minor mean body weight loss was recorded for males between Days 1-2 of study, while for females mean progressive weight loss of 8 grams was recorded over Days 1-3 of study. For both sexes at 12000 ppm weight gain was recorded between Days 2-3 for males and Days 3-4 for females. Despite a second period of minor weight loss recorded between Days 5-7 of the study in females, the bodyweight gain between Day 4 and Day 22 was higher than Control (13 g vs. 17 g for Control and 12000 ppm, respectively) and mean bodyweights were similar at the end of treatment. For males at this dose level, mean bodyweight was only slightly lower than Controls (-0.5%) at the end of treatment and bodyweight gain during Days 4-22 was only 9% lower than Control, mainly due to one animal. Following the start of treatment at 6000 or 3000 ppm, mean bodyweight gain in males was unaffected by treatment but mean weight gain in females at 3000 or 6000 ppm were lower than Controls. However, bodyweight gain between Days 4-22 in females was similar to Control at 3000 ppm and lower at 6000 ppm (while higher than Control at 12000 ppm).
Following the start of treatment at 12000 ppm, food consumption in males was slightly lower on Days 1-2 while intake in females was markedly low on Days 1-2 and 2-3 of treatment. Thereafter, food consumption of both sexes improved but females showed a second period of low intake on days 6-9 of study. Food intake in females receiving 6000 ppm was slightly lower on Days 1-2 and 2-3 of the study.
Liver, thymus (males only), spleen (females only), uterus/cervix/oviducts and vagina weights were higher than that of Control for animals that received 3000, 6000 or 12000 ppm, with the exception of liver weight in females receiving 3000 ppm. Thymus weight was considered lower in females that received 12000 ppm.
 
It was therefore concluded that the effects observed at the high dose of 12000 ppm do not preclude the use of this dose level as the high dose for the main OECD 421 study.


 


In a repeated dose toxicity study conducted according to OECD Guideline 413 and in compliance with GLP, (-)-alpha-pinene was administered by inhalation-aerosol to groups of Sprague Dawley rats (10 rats/sex/group) by whole-body inhalation exposure at target exposure levels of 0.15, 0.3 and 0.9 mg/L for 6 hours per day, 5 days per week for 13 weeks. Control animals received air only. Recovery animals were similarly treated for 13 weeks followed by a 4 week off dose period. During the study, clinical condition, body weight, food consumption, ophthalmoscopy, haematology (peripheral blood), blood chemistry, organ weight, broncho-alveolar lavage examinations, macropathology and histopathology investigations were undertaken.


 


There were no treatment related clinical signs or effects on food consumption, blood chemistry, ophthalmoscopy, urinalysis, organ weights or broncho-alveolar lavage examinations.


 


There were two unscheduled female deaths during the exposure phase of the study in the group exposed to 0.9 mg/L. Following microscopic examination, no histopathological cause for either death was established. 


In the kidneys of exposure phase males, test item-related accumulation of hyaline droplets in the cortical tubular epithelium and basophilia of the cortical tubular epithelium was seen in all males exposed to (-)-alpha-pinene and demonstrated a relationship to concentration in terms of severity. Immunohistochemical (IHC) staining for alpha‑2µ-globulin in the kidneys of representative exposure phase animals confirmed the presence of this protein in the epithelial hyaline droplets. Tubular granular casts in the outer medulla was seen in the majority of males exposed to 0.3 or 0.9 mg/L and a few males exposed to 0.15 mg/L and cortical tubular degeneration was also seen in two males of each test exposure group. The association of hyaline droplets with basophilic tubules and granular casts, known as alpha‑2µ-globulin nephropathy, is considered to be adverse in the animals affected. These findings accounted for the irregular renal surface seen at necropsy in two males exposed to 0.9 mg/L and for the higher than control mean body weight adjusted kidney weights for males exposed to 0.9 mg/L. These test item-related findings were confined to males.


In the kidneys of recovery phase males, accumulation of hyaline droplets in the cortical tubular epithelium was seen in one male of each group previously exposed to (‑)‑alpha‑pinene. Basophilia of the cortical tubular epithelium was seen in all males previously exposed to (-)-alpha-pinene and in two control males, and tubular granular casts in the outer medulla was seen in a few males previously exposed to 0.15, 0.3 or 0.9 mg/L. Cortical scars, which were considered the result of previous tubular degeneration, were seen in one male and three males previously exposed to 0.3 or 0.9 mg/L, respectively. Therefore, accumulation of hyaline droplets and tubular granular casts both exhibited partial recovery in terms of severity and relative incidence, however, basophilia of the cortical tubular epithelium did not demonstrate recovery.


In the lungs and bronchi of exposure phase animals, findings of an uncertain relationship to the test item were seen in males. Foamy alveolar macrophages were seen at a higher incidence in males exposed to 0.9 mg/L than in control males and alveolar eosinophilic crystals with associated inflammatory cell infiltrate (generally mixed cell) were seen at a clearly higher incidence in males exposed to 0.9 mg/L than in male controls. The foamy macrophages generally accounted for the pale areas seen at necropsy. 


In the lungs and bronchi of recovery phase animals, foamy alveolar macrophages were seen at, or lower than, the control incidence in males and females previously exposed to (‑)‑alpha‑pinene and alveolar eosinophilic crystals with associated inflammatory cell infiltrate (mixed cell) were seen in one male previously exposed to 0.15 mg/L. In consequence, these two findings were considered to be within the background level in these previously exposed male and female animals, and thus the foamy alveolar macrophages was considered to show full recovery.


Clinical signs after exposure noted at 0.9 mg/L tended to be more noticeable on days when the chamber aerosol concentrations were higher than target, and resulted in two deaths in female animals that were considered test item related. These signs were thus considered adverse. A few signs noted at 0.15 or 0.3 mg/L were considered due to the incidence and frequency to be not adverse.


The slight, transient, lower food consumption in females in Week 1 and 2 was not accompanied by any change in body weight and as such the food consumption change is considered not adverse.


There was no pathological correlates seen in the liver to account for the slightly higher weight in animals exposed to 0.9 mg/L; hence this change was considered not adverse and of no toxicological significance.


Therefore, the No Observed Adverse Effect Concentration (NOAEC) was considered to be 0.3 mg/L, based on the two females decedents at the concentration level of 0.9 mg/L due to general poor clinical condition.

Justification for classification or non-classification

Harmonised classification:


The test material has no harmonised classification for repeated dose toxicity according to the Regulation (EC) No. 1272/2008 (CLP).


 


Self-classification:


Based on the available information, no additional self-classification is proposed regarding the specific target organ toxicity after oral dose-repeated exposure according to the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.