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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:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2002

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Version / remarks:
Methodology described in several publications (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

Constituent 1
Chemical structure
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
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Japan SLC Co. (Shizuoka, Japan)
- Age at study initiation: 8 weeks
- Weight at study initiation: no available
- Fasting period before study: not reported
- Diet (e.g. ad libitum): commercial pellets MF (Oriental Yeast Industries Co., Tokyo, Japan) ad l
ibitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 1 week.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24ºC
- Humidity (%): 55-65%
- Photoperiod (hrs dark / hrs light): 12 h light–dark cycle

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: olive oil
- Amount of vehicle (if gavage or dermal): 10 mL/kg
Duration of treatment / exposure:
3, 8 and 24 hours (sampling times after single dose)
Frequency of treatment:
Once
Post exposure period:
No data
Doses / concentrations
Dose / conc.:
2 000 mg/kg bw (total dose)
Remarks:
(for sampling times of 3, 8 and 24 hours)
No. of animals per sex per dose:
- Treatment groups: 4 males per sampling time
- Vehicle control: 12 males per sampling time
- Untreated control group: 12 males
Control animals:
yes, concurrent no treatment
yes, concurrent vehicle
Positive control(s):
None.

Examinations

Tissues and cell types examined:
Stomach, colon, liver, kidney, urinary, bladder, lung, brain and bone marrow.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION:
A single dose was set at 0.5 X LD50 (with a limit of 2000 mg/kg) with the purpose of using a dose at which gross necrosis was not observed in order to avoid false-positive results of the comet assay due to cytotoxicity.

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.
Evaluation criteria:
Migration was calculated as the difference between length and diameter for each of the 50 nuclei. Mean migration of 50 nuclei from each organ was calculated for each individual animal. The differences between the averages of four treated animals and the untreated control animals were compared with the Dunnett test after one-way ANOVA. A P-value less than 0.05 was considered statistically significant.
Statistics:
Dunnett test after one-way ANOVA.

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

Any other information on results incl. tables

Table 1. Migration of nuclear DNA from organs of mice treated with vehicles

Species Treatment Sampling time (h)   Migration (µm, mean of 12 animals)
Stomach Colon Liver Kidney Bladder Lung Brain Bone marrow
Mouse Mean 6.19 5.15 2.55 2.32 4.66 2.70 1.48 0.94
S.E.M. 0.44 0.44 0.24 0.34 0.42 0.24 0.20 1.01
Olive oil 3 Mean 6.46 4.56 2.06 1.90 4.33 2.33 1.56 0.90
S.E.M. 0.57 0.45 0.29 0.32 0.33 0.37 0.38 0.30
8 Mean 7.22 5.09 2.86 2.64 5.63 2.49 1.83 1.08
S.E.M. 0.49 0.40 0.43 0.43 0.49 0.60 0.41 0.41
24 Mean 6.23 4.70 2.49 1.80 4.20 2.27 1.70 1.23
S.E.M. 0.40 0.35 0.20 0.24 0.39 0.27 0.41 0.27

Table 2. Migration of nuclear DNA from organs of mice treated with d-limonene

Chemical Species Dose (mg/kg) Sampling time (h)   Migration (µm, mean of 4 animals)
Stomach Colon Liver Kidney Bladder Lung Brain Bone marrow
d-limonene Mouse 2000 (limit dose) 0 Mean 6.87 4.71 2.76 2.60 4.98 2.58 1.70 1.19
S.E.M. 0.52 0.33 0.65 0.59 1.19 0.37 0.22 0.57
3 Mean 5.74 6.03 2.18 2.88 3.10 2.99 1.50 1.94
S.E.M. 1.15 0.45 0.65 0.75 0.44 1.01 1.50 0.55
8 Mean 6.85 5.34 2.35 1.48 4.88 3.38 1.03 2.05
S.E.M. 0.89 0.36 0.65 0.53 0.47 1.14 1.03 0.80
24 Mean 6.02 5.99 2.15 2.02 4.34 2.15 1.01 1.14
S.E.M. 1.03 1.44 0.56 0.47 0.47 0.37 0.59 1.14

Applicant's summary and conclusion

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