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Description of key information

A 20-week repeated dose toxicity study in rats the NOAEL was >=400 mg/kg bw/day (10000 mg/kg diet) for alpha-Terpinyl Acetate. This NOAEL is supported with information from its main metabolite: Terpineol multi.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Reliability 2 is assigned because study predates current guidelines and GLP but is otherwise adequate for assessment.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
no
Limit test:
no
Species:
rat
Strain:
Osborne-Mendel
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Housing: Individually in wire cages
- Diet: Ad libitum
- Water: Ad libitum

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
7% loss of food flavouring during a 7-day period
Duration of treatment / exposure:
20 weeks
Frequency of treatment:
Fresh diets were made and distributed weekly
Remarks:
Doses / Concentrations:
1000, 2500, 10000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
The rat's weight, food intake and general condition were recorded every week. Haematological examinations were made at termination
Sacrifice and pathology:
At the termination of the experiments the rats were sacrificed and exsanguinated. The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera were removed and the liver, kidneys, spleen, heart, and testes were weighed. These organs, the remaining abdominal and thoracic viscera, and one hind leg, for bone, bone marrow, and muscle, were preserved in 10 % buffered formalin-saline solution for histopathological
examination. For routine histopathology, sections were embedded in paraffin wax and stained with haematoxylin and eosin. Detailed microscopic examinations in the subacute studies were generally done on 6 or 8 rats, evenly divided by sex, from the high dose group and the control group. If changes attributable to the test compound were found in the high dose group, additional animals on lower dosage levels were examined as indicated.
Details on results:
No effect on growth or haematology, and no macroscopic or microscopic change in the tissues in the 10000 ppm exposure group.
No effect on growth or haematology, and no macroscopic change in the tissues in the 2500 and 1000 ppm exposure groups. No microscopic examination performed.
Dose descriptor:
NOAEL
Effect level:
>= 400 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The 400 mg/kg is the derived value based on the 10000 ppm dietary dose)
Critical effects observed:
not specified
Conclusions:
No effects were observed in a 20-weeks repeated dose toxicity study in rats at the tested concentrations (up to 10000 ppm).
Executive summary:

In a 20 -weeks oral exposure study Osborne-Mendel rats (10/dose/sex) were administered alpha-Terpinyl Acetate via diet intake at concentrations of 0 (control), 10000, 2500 and 1000 ppm. Animals were then observed for mortality, weight, food intake and general condition. Haematological examinations were made at termination. At the termination of the experiments the rats were sacrificed and exsanguinated. The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera were removed and the liver, kidneys, spleen, heart, and testes were weighed. Detailed microscopic examinations was done on the animals from the high dose group and the control group. No effect on growth or haematology, and no macroscopic or microscopic change in the tissues in the 10000 ppm exposure group were observed. No effect on growth or haematology, and no macroscopic change in the tissues in the 2500 and 1000 ppm exposure groups. No microscopic examination was performed on rats exposed to 2500 and 1000 ppm. According to the Guidance on information and chemical safety assessment R8 table R 8 -17, rats eat 40 to 50 mg/kg bw/day. 10000 ppm is equivalent to 1% in the diet. Consequently, rats exposed to 10000 ppm in the diet consumed between 400 and 500 mg Terpinyl-Acetate-Alpha per kg bodyweight per day. Therefore, the NOAEL was calculated to be 400 mg/kg bw.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
400 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Two studies were available. One 20-week feeding study with alpha-Terpinyl Acetate published, which is supported with sub-acute toxicity information from Terpineol multi and are sufficiently adequate to cover this endpoint.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

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

Conclusion: The NOAEL of the repeated dose toxicity study of alpha-Terpinyl Acetate has been used as a key study because the study period is 20-weeks and the dosing is via the diet is resembling human health exposure more than via gavage. The supporting study is an OECD TG 422 study conducted with Terpineol multi. This latter substance contains mainly alpha-Terpineol being the main metabolite of alpha-Terpinyl Acetate. The organ effects seen here are considered to be due to gavage and of an adaptive nature, because they were not seen during dietary dosing and/or were reversible in nature. In addition, these effects are not seen in the 20-weeks study.

The repeated dose toxicity from Terpineol multi in the OECD TG 422 test (which is registered in 2010), will be discussed but is not used as a key study because more relevant data are available.

alpha-Terpinyl Acetate

In a 20 -weeks oral exposure study Osborne-Mendel rats (10/dose/sex) were administered alpha-Terpinyl Acetate via diet at concentrations of 0 (control), 10000, 2500 and 1000 ppm. Animals were then observed for mortality, weight, food intake and general condition (Hagan 1967). Haematological examinations were made at termination. At the termination of the experiments the rats were sacrificed and exsanguinated. The tissues of all the rats were examined macroscopically at the time of sacrifice. The viscera were removed and the liver, kidneys, spleen, heart, and testes were weighed. Detailed microscopic examinations were done on the animals from the high dose group and the control group. No effect on growth or haematology, and no macroscopic or microscopic change in the tissues in the 10000 ppm exposure group were observed. No effect on growth or haematology, and no macroscopic change in the tissues in the 2500 and 1000 ppm exposure groups. No microscopic examination was performed on rats exposed to 2500 and 1000 ppm. These values were not converted into mg/kg bw in the publication. Therefore the ECHA guidance was used, according to R8 table R 8 -17, rats eat 40 to 50 mg/kg bw/day. 10000 ppm is equivalent to 1% in the diet. Consequently, rats exposed to 10000 ppm in the diet consumed between 400 and 500 mg alpha-Terpinyl Acetate per kg body weight per day. Therefore, the NOAEL was calculated to be 400 mg/kg bw.

Terpineol multi

The repeated dose information from Terpineol multi is used as supporting and only to the extent which can be derived from systemic exposure from a reproscreen study. It is, however, presented and discussed in detail because adverse effects were seen.

In the combined repeated dose toxicity study with reproduction/developmental toxicity screening test, conducted following GLP guidelines and OECD guideline 422, three groups, each comprising of ten male and ten female rats for the Main (reproductive) phase and five female rats for the Toxicity phase received Terpineol multi by gavage at doses of 60, 250 or 750 mg/kg bw/day at a dose volume of 5 mL/kg bw/day (Thacker 2010a). Main phase males and Toxicity phase females were dosed daily for a minimum of five consecutive weeks. A similarly constituted Control group received the vehicle, corn oil, at the same volume-dose. An additional ten males and ten females were dosed with the vehicle or at 750 mg/kg/day for five weeks and then given two weeks of recovery before termination. Main phase females were dosed daily for two weeks before pairing, throughout mating, gestation and until Day 6 of lactation. During the study, data was recorded on clinical condition, performance under detailed physical and arena examination, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, water consumption (visual), haematology, blood chemistry, oestrous cycles, mating performance and fertility and gestation length. Organ weight, macroscopic and microscopic pathology investigations were undertaken in the adults. The clinical condition of offspring, litter size and survival, sex ratio and offspring bodyweight were assessed and macroscopic pathology investigations were undertaken. In the 60 mg/kg/day dose group, one male was found dead on Day 31 of study and one female was killed because of parturition difficulties. In the absence of any other death in the intermediate and high dose groups these deaths are not attributed to the test material. No significant findings were recorded for clinical signs, detailed physical examination and arena observations. Underactive behaviour and unsteady reactions, in males and females were observed briefly during Week 1 in animals receiving 750 mg/kg/day and dose‑related increases in post‑dosing salivation and chin rubbing were seen. There were no clear effects on bodyweight in males or unmated females receiving up to 750 mg/kg/day. Males receiving 750 mg/kg/day showed lower overall weight gain (Week 0-5) compared with Control. Bodyweight during the recovery phase was similar to Controls. Bodyweight and bodyweight gain were unaffected during gestation. During lactation females receiving 250 mg/kg/day showed lower weight gain than Controls. There were no adverse effects on food consumption in males, unmated females or females during gestation and lactation but visual assessment of water consumption indicated that males and females receiving 750 mg/kg/day were consuming more water than the Controls during the dosing period.

At 750 mg/kg/day, relative liver weights were significantly higher than Control in males and females and relative kidney weights were significantly higher than Control in males. Testis weight was markedly low in males receiving 750 mg/kg/day and there was also an indication of low epididymal weights at this dose. Liver and kidney weights returned to normal after two weeks when the animals did not receive Terpineol multi but testis and epididymal weights showed no evidence of recovery. Adaptive centrilobular hepatocyte hypertrophy in the liver of females dosed with Terpineol multi at 750 mg/kg/day was not present after 2 weeks recovery and histopathological findings in the kidneys of males receiving 250 and 750 mg/kg/day also resolved after the end of dosing. At 750 mg/kg/day, reduced numbers or complete absence of spermatozoa, accompanied by the presence of degenerate spermatogenic cells in duct(s) were observed in the epididymides and were still present following the 2‑week recovery period. Spermatocele granuloma(ta) that were seen in two males receiving 750 mg/kg/day and one receiving 60 mg/kg/day were not seen at the end of the recovery period. The significance of this change in the single male receiving 60 mg/kg/day is uncertain as spermatocele granuloma(ta) can occur spontaneously in rats of this age and there was no other degenerative change in the testes or epididymides of this animal. Moderate to severe seminiferous tubular atrophy/degeneration was seen in the testes of all animals dosed with Terpineol multi at 750 mg/kg/day, accompanied by minimal to moderate spermatid giant cells and minimal to slight seminiferous tubular vacuolation.

Similar findings were still evident following the 2‑week recovery period but at a lower incidence and severity suggesting a degree of recovery. However, gavage is considered to be a worst case for this effect (confirmed by diet administration). The No-Observed-Adverse–Effect-Level (NOAEL) for males is considered to be >=750 mg/kg bw because the testicular effects are considered to be caused by the type of dosing and no adverse other organ effects were seen (see fertility section for a description of the 14 -dietary dosing testing (Supporting study 1B). The NOAEL of the females is also considered to be >=750 mg/k/g/day because the organ effects were of an adaptive nature and reversible.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
This 20-wk study is selected because the repeated dose exposure is longer than in the other repeat dose tests. In addition, the testing is via the diet, which presents a more regular dosing regime similar to human exposure and thus more appropriate to use for hazard assessment, classification and labelling and risk characterisation.

Justification for classification or non-classification

Repeated dose toxicity information from alpha-Terpinyl Acetate shows that at the highest dose tested of 400 mg/kg bw no effects were seen and therefore classification is not warranted according to Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.