Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Aug 2014 - 16 Sept 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
CAS #
For specific details on test material if available - See in RSS
Name of test material (as cited in study report or in reference): Petitgarin oil, Petitgrain oil (Paraguay), Petitgrain oil (Bigarade) and Petitgrain oil (Terpeneless)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No. of test material: Pettitgrain Oil Paraguay, code number AE02WP (lot no: B-53636)
- Expiration date of the lot/batch:
- Day received: 06 August 2014

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature, protected from light

OTHER SPECIFICS:
Refractive index @20 deg C: 1.4591
Specific gravity @25 deg C: 0.8860

OTHER:
-As of 05/2013 CAS No. 8016-44-2 is no longer valid. An other CAS No. for this material is 8014-17-3

Method

Target gene:
S. typhimurium: Histidine gene
E. coli: Tryptophan gene
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
In the initial test, retests and the confirmatory mutagenicity assay, the following doses were tested 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate both with and without S9-mix.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, (CAS No. 67-68-5; Lot No. SHBD1324V, Purity: 99.98%, Exp. Date: May 2017), obtained from Sigma-Aldrich
- Justification for choice of solvent/vehicle: Based on the solubility of the test substance and compatibility with the target cells. The test substance formed a clear solution in DMSO at approximately 500 mg/mL (the maximum concentration tested in the solubility test)
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DSMO
True negative controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation assay)

DURATION
- Exposure duration: 48-72h

NUMBER OF REPLICATIONS: 2 replications for experiment 1 and 2. 3 replications for confirmation mutagenicity assay.

DETERMINATION OF CYTOTOXICITY
- Method: Count number of revertant colonies
Evaluation criteria:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance.
Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged positive if the increase in mean revertants at the peak of the dose response was greater than or equal to 2.0-times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or greater than the respective threshold cited. A response was evaluated as negative if it was neither positive nor equivocal.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
TA 1537, TA 98 and TA 100 bacteria
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
beginning at 1500 or at 5000 μg per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
beginning at 1500 or at 5000 μg per plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitate was observed at 5000 μg per plate

RANGE-FINDING/SCREENING STUDIES: In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Strain: -S9 (Mean/SD/Min/Max): +S9 (Mean/SD/Min/Max)
TA98: 271/214/34/2274:423/190/47/1711
TA100:608/143/211/1393:730/260/247/2421
TA1535:501/164/20/1593:103/92/20/1472
TA1537:422/386/17/3711:59/56/10/850
WP2 uvrA:380/160/42/1796:245/98/21/969

- Negative (solvent/vehicle) historical control data:
Strain: -S9 (Mean/SD/Min/Max): +S9 (Mean/SD/Min/Max)
TA98:18/8/3/64:24/8/4/60
TA100:98/18/50/251:110/23/55/247
TA1535:11/4/1/43:12/4/1/35
TA1537:7/4/0/28:8/4/0/28
WP2 uvrA:27/10/5/84:30/10/7/80

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, Petitgrain Oil Paraguay (Petitgrain oil citrus aurantium) was determined to be not mutagenic and does not need to be classified for mutagenicity in accordance with the criteria outline in Annex I of CLP (1272/2008/EC).
Executive summary:

The mutagenic activity of Petitgrain Oil Paraguay was evaluated in accordance with OECD 471 guideline and according to GLP principles. The test was performed as a standard plate incorporation assay, both in the absence and presence of S9-mix up to and including 5000 μg/plate. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed from 1500 or at 5000 μg/plate. Precipitation was observed at 5000 μg/plate. Adequate negative and positive controls were included. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100 WP2 uvrA), both in the absence and presence of S9 -metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that Petitgrain oil citrus aurantium was determined to be not mutagenic and does not need to be classified for mutagenicity in accordance with the criteria outline in Annex I of CLP (1272/2008/EC).