Rectified Hydrocarbons by-products from synthetic process of Turpentine and acid, alcohols fraction

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Comet assay.

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Data source

Reference

Reference Type:

publication

Title:

Unnamed

Year:

2002

Materials and methods

Principles of method if other than guideline:

Method: Comet assay (Sasaki et al., 1997; Sasaki et al., 1999; Sasaki et al., 2000; Tsuda et al., 2000)

GLP compliance:

no

Type of assay:

mammalian comet assay

Test material

Test material information

Constituent 1

Reference substance name:

(R)-p-mentha-1,8-diene

EC Number:

227-813-5

EC Name:

(R)-p-mentha-1,8-diene

Cas Number:

5989-27-5

Molecular formula:

C10H16

IUPAC Name:

(4R)-isopropenyl-1-methylcyclohexene

Test animals

Species:

mouse

Strain:

other: ddY

Sex:

male

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: olive oil

Duration of treatment / exposure:

24 hours

Frequency of treatment:

Once

Post exposure period:

No data

Doses / concentrations

Dose / conc.:

2 000 mg/kg bw (total dose)

No. of animals per sex per dose:

- Treatment groups: 4 males
- Vehicle control and untreated control groups: 12 males

Control animals:

yes, concurrent no treatment

yes, concurrent vehicle

Examinations

Tissues and cell types examined:

Stomach, colon, liver, kidney, urinary, bladder, lung, brain and bone marrow

Details of tissue and slide preparation:

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
A preliminary range-finding test was conducted using 4-5 male mice/dose to determine the LD50 value.
Animals were observed for pharmacotoxic signs and were macroscopically necropsied 3, 8 and 24 hours after treatment.

METHOD OF ANALYSIS:
Stomach, colon, liver, kidney, urinary bladder, lung, brain and bone marrow were isolated and the prepared slides were scanned to determine the length of the whole comet, diameter of the head and mean migration of 50 nuclei per organ per animal.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

not applicable

Applicant's summary and conclusion

Conclusions:

Under the test conditions, d-limonene is not considered as mutagenic in Comet assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In an in vivo comet assay, 4 male ddY mice were administered a single oral dose of d-limonene in olive oil by gavage at dose levels of 2000 mg/kg bw. Animals were then observed for pharmacotoxic signs and were macroscopically necropsied 3, 8 and 24 hours after treatment. Stomach, colon, liver, kidney, urinary bladder, lung, brain and bone marrow were isolated and the prepared slides were scanned to determine the length of the whole comet, diameter of the head and mean migration of 50 nuclei per organ per animal. A preliminary range-finding test was also conducted using 4-5 male mice/dose to determine the LD50 value. No death, morbidity or distinctive clinical and microscopic signs were observed. D-limonene did not induced DNA damage in the studied organs. Under the test conditions, d-limonene is not considered as mutagenic in Comet assay and does not need to be classified according to the criteria of the CLP Regulation (EC) N° (1272-2008).