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Toxicological information

Genetic toxicity: in vivo

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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

Reference
Name:
Unnamed
Type:
Constituent

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).