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Genetic toxicity: in vivo

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Administrative data

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2007
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study well documented, meets generally accepted scientific principles, acceptable for assessment
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2007

Materials and methods

Principles of method if other than guideline:
Method: Comet assay (Tice et al., 2000)
GLP compliance:
no
Type of assay:
mammalian comet assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid

Test animals

Species:
rat
Strain:
other: OFA Sprague-Dawley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (Saint-Germain-sur-l’Arbresle, France)
- Age at study initiation: 5-6 weeks
- Assigned to test groups randomly: Yes
- Housing: Housed in groups of 2-3 in polypropylene cages
- Diet (e.g. ad libitum): Commercial pellets (SAFE, Augy, France), ad libitum
- Water (e.g. ad libitum): Tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55 ± 15%
- Air changes (per hour): 20/hour
- Photoperiod (hours dark / hours light): 20 hours dark / 20 hours light

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 0.1% CMC (carboxymethyl cellulose)
- Amount of vehicle (if gavage): 10 mL/kg bw
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: Test solutions were prepared with 0.1% CMC.
Duration of treatment / exposure:
3-6 or 22-26 hours
Frequency of treatment:
Once
Post exposure period:
No
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 1000 and 2000 mg/kg bw in 0.5% CMC
Basis:
actual ingested
No. of animals per sex per dose:
- Vehicle control and treatment groups: Four males
- Positive control group: Three males
Control animals:
yes, concurrent vehicle
Positive control(s):
Streptozotocin
- Justification for choice of positive control(s): Known renal epigenetic carcinogen
- Route of administration: Intravenous
- Doses / concentrations: 20 mg/kg bw

Examinations

Tissues and cell types examined:
Kidney cells
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: A preliminary range-finding test was conducted using 4 male rats/dose and animals were observed at least 2 days for mortality and clinical signs of toxicity. Maximum tolerated dose (MTD) determined in the preliminary test was selected as the highest dose for the main study.

TREATMENT AND SAMPLING TIMES: After an exposure period of 3-6 or 22-26 hours, treated animals were sacrificed and kidney cells were isolated by specific enzymatic method (Bruggeman et al., 1989). Cytotoxicity was determined on a small sample of each isolated cell suspension following the trypan blue vital dye-exclusion technique.

DETAILS OF SLIDE PREPARATION: Slides (16/dose/expression period) with the cell suspensions (3 × 10^4 cells), embedded in a layer of 0.5% of low melting-point agarose, were immersed in a lysing solution for at least 1 hour at +4 °C in the dark and then run in a horizontal gel electrophoresis unit for 20 min at 0-4 °C by applying an electric current of 0.7 V/cm (25 V/300 mA). After electrophoresis, the slides were neutralized with 0.4 M Tris (pH 7.5) and the DNA was exposed for 5 min to absolute ethanol in order to preserve all the Comet assay slides.

METHOD OF ANALYSIS: Prepared slides were stained with propidium iodide (20 µg/mL distilled water; 25 µL/slide) and scanned using a fluorescent microscope (Leica Microscopy and Scientific Instruments Group, Switzerland), connected through a gated CCD camera to Comet Image Analysis System version 4.0 software (Kinetic Imaging Ltd., UK), to determine mean Olive Tail Moment (OTM) median value in 150 cells per animal (Tice et al., 2000).
Evaluation criteria:
- Olive Tail Moment (OTM) preconised by Olive (1990) was used to evaluate DNA damage.
- OTM, expressed in arbitrary units, is calculated by multiplying the percent of DNA (fluorescence) in the tail by the length of the tail in µm. The tail length is measured between the edge of Comet head and the end of the Comet tail.
Statistics:
- Kruskall-Wallis test was used to display a possible dose–effect relationship.
- Statistical significance of differences in the median values between each group versus the control was determined with the non-parametric Mann-Whitney U-test.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
- No deaths, morbidity, or distinctive clinical signs were observed after any of the treatments.
- Viability, using the trypan-blue exclusion method, was >70% for each cell suspension in all control and treated groups up to the MTD.
- See table 1

Any other information on results incl. tables

Table 1: DNA damage measured by the Comet assay in isolated rat kidney cells 3–6 or 22–26 hours after a single administration of d-limonene at dose levels of 1000 and 2000 mg/kg bw

 

Sampling time (h) 

 Group 

 Dose (mg/kg) 

 OTM 

3-6

d-limonene

 0 

 1.76 

 1000 

 1.81 

 2000 

 1.35 

Streptozotocin

 20 

 41.1*** 

22-26

d-limonene

 0 

 1.87 

 1000 

 1.91 

 2000 

 2.21 

Streptozotocin

 20 

 40.8*** 

Significant difference (Mann–Whitney U-test) as compared with the vehicle control; ***p < 0.001.

OTM: mean Olive Tail Moment median value

Applicant's summary and conclusion

Conclusions:
d-Limonene is not considered as mutagenic in Comet assay on isolated kidney cells and does not need to be classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.
Executive summary:

In an in vivo comet assay, groups of 4 OFA Sprague-Dawley male rats were administered a single oral dose of d-limonene in 0.5% CMC by gavage at dose levels of 0, 1000 and 2000 mg/kg bw. After an exposure period of 3-6 or 22-26 hours, treated animals were sacrificed and the kidney cells were isolated and the prepared slides were scanned to determine mean Olive Tail Moment (OTM) median value in 150 cells per animal using the method described by Tice et al (2000). A preliminary range-finding test has also been conducted using 4 males rats/dose and animals were observed at least 2 days for any clinical signs of toxicity and any mortalities in order to determine the maximum tolerated dose (MTD).

 

Positive control (streptozotocin, 20 mg/kg bw) caused a clear increase in the mean OTM median value. d-Limonene showed no substantial increase in the mean OTM median value.

 

Therefore, d-limonene is not considered as mutagenic in Comet assay on isolated kidney cells and does not need to be classified according to Directive 67/548/EEC and CLP Regulation (EC) No 1272/2008.