Essential oil of Petitgrain obtained from the leaves and twigs of Citrus aurantium (Rutaceae) by distillation

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in bacteria (OECD TG 471) : not mutagenic

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

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-As of 05/2013 CAS No. 8016-44-2 is no longer valid.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

Target gene:

S. typhimurium: Histidine gene
E. coli: Tryptophan gene

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)

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.

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

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).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

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 Paraguay 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).

Justification for classification or non-classification

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).