Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 September 2015 - 23 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
CAS 85085-41-2
For specific details on test material if available - See in RSS
Name of test material (as cited in study report or in reference): Cedarwood Virginia oil; Cedarwood oil Virginia or Virginia oil
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Obtained from sponsor, Batch Number 1002292672
- Expiration date of the lot/batch: 30 November 2016
- Purity test date: 28 May 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, in a dark place under nitrogen

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:
Solutions of the test item, as received, were prepared immediately before use in DMSO. Solutions were prepared on a weight/volume basis without correction for the displacement due to the volume of the test item. Concentrations were expressed in terms of material as received. All test item solutions were used within 1 hour and 29 minutes from the initial formulation. All dose levels in this report are expressed to three significant figures.

OTHER SPECIFICS: UVCB

Method

Target gene:
Histidine operon
Trypthophan operon
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: All strains: contain the Rfa wall mutation and the uvrB mutation TA98 and TA100 strains: contain the pKM101 plasmid
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: uvrA DNA repair deficiency
Metabolic activation:
with and without
Metabolic activation system:
S9 tissue homogenate
Test concentrations with justification for top dose:
- Toxicity test:
With and without S9:
5000,1580, 500, 158 and 50.0 μg/plate.

- Main Study I (plate incorporation):
Withhout S9
TA1535: 4000, 2000, 1000, 500, 250, 125 and 62.5 μg/plate
TA1537: 4000, 2000, 1000, 500, 250, 125 and 62.5 μg/plate
WP2 uvrA, TA98: 5000, 2500, 1250, 625 and 313 μg/plate
TA100: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate
With S9
TA1535: 4000, 2000, 1000, 500, 250 and 125 μg/plate
TA1537: 4000, 2000, 1000, 500, 250, 125 and 62.5 μg/plate
WP2 uvrA, TA98: 5000, 2500, 1250, 625 and 313 μg/plate
TA100: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate

- Main Study II (preincubation):
Withhout S9
TA1535: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate
TA1537: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate
WP2 uvrA, TA98: 5000, 2500, 1250, 625 and 313 μg/plate
TA100: 500, 250, 125, 62.5, 31.3, 15.6 and 7.81 μg/plate
With S9
TA1535: 2000, 1000, 500, 250, 125, 62,5 and 31.3 μg/plate
TA1537: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate
WP2 uvrA, TA98: 5000, 2500, 1250, 625 and 313 μg/plate
TA100: 1000, 500, 250, 125, 62.5, 31.3 and 15.6 μg/plate

- Main Study II (additional experiment)
Withhout S9
TA1537: 15.6, 7.8, 3.9, 1.95, 0.975, 0.488 μg/plate
TA100: 15.6, 7.8, 3.9, 1.95, 0.975 μg/plate


Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Solubility of the test item was evaluated in a preliminary trial using DMSO. This solvent was used since it is compatible with the survival of the bacteria and the S9 metabolic activity.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
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) if negative or equivocal results are obtained in the first experiment a confirmatory experiment is performed using the pre-incubation method.
- Cell density at seeding (if applicable):

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: ; other: Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation.

Rationale for test conditions:
The study is designed to comply with the experimental methods indicated in the guidelines
Evaluation criteria:
The assay will be considered valid if the following criteria are met:
(i) Mean plate counts for untreated and positive control plates should fall within 2 standard deviations of the current historical mean values.
(ii) The estimated numbers of viable bacteria/plate should fall in the range of 100 – 500 millions for each strain.
(iii) No more than 5%of the plates will be lost through contamination or other unforeseen event The whole assay or only the non-valid treatment series will be repeated in case the acceptance criteria will not be achieved.

Evaluation of data
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels. Evaluation of Ames test data based on a ‘doubling rate’ has been shown to be as effective as statistical techniques in allowing the correct interpretation of test results (Chu et al. 1981). Any increase in revertant numbers should lie outside the historical control range to have biological relevance.
Statistics:
Data are evaluated by performing a regression analysis. This method fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions. The regression equation is
expressed as:
y = a + bx
where:
y = transformed revertant numbers
a = intercept
b = slope value
x = dose level (in the units given).
The regression line does not include the untreated control data, but includes the solvent control data. Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Main experiment I and II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Main experiment I, II and III
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
Main experiment I and II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Main experiment I and II
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Main experiment I, II and III
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: At the end of the incubation period, precipitation of the test item was observed with WP2 uvrA and TA98, at the highest dose level (5000 μg/plate), in the absence and presence of S9 metabolic activation in main experiment I

RANGE-FINDING/SCREENING STUDIES:
Toxicity, as indicated by thinning of the background lawn, was observed with TA100, TA1535 and TA1537 tester strains, at the two or three highest concentrations tested, both in the absence and presence of S9 metabolism. A
more pronounced toxic effect was observed with TA100 tester strain, showinga marked and dose related reduction in revertant numbers. No toxic effect was noticed with WP2 uvrA and TA98, at any dose level tested, in the absence or presence of S9 metabolism.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: see attached
- Negative (solvent/vehicle) historical control data: see attached

Applicant's summary and conclusion

Conclusions:
Based on the results of this study Cedarwood Virginia Oil does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).
Executive summary:

Three experiments were performed to test the gene mutation potential of Cedarwood Oil Virginia according to OECD TG 471. In Main Assay I, using the plate incorporation method, the test item was assayed at dose levels ranging from 16,5-5000µg/plate (concentrations chosen on the basis of the results obtained in a preliminary toxicity test). Toxicity, as indicated by thinning of the background lawn and/or reduction in revertant colonies was observed with TA1535, TA1537 and TA100, starting from 500 μg/plate or 1000 μg/plate, both in the absence and presence of S9 metabolic activation. No toxic effect was noticed with WP2uvrA and TA98. As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at dose levels ranging from 7.81-5000µg/plate. No toxicity was noticed with WP2uvrA and TA98, at any dose level. Toxicity, from severe (TA1537, and TA100) to slight (remaining strains), was observed with the remaining tester strains both in the absence and presence of S9 metabolism. In order to have a sufficient number of analysable concentrations, a further experiment (III), using the pre-incubation method, was performed with TA1537 and TA100 tester strains using dose levels ranging from 0.488-15.6 µg/plate. A slight thinning of the background lawn was observed with both tester strains, at the highest dose level. No relevant increase in the number of revertant colonies was observed, with any tester strain, in the plate incorporation or pre- incubation assay, at any dose level, in the absence or presence of S9 metabolism. The mean plate counts for untreated and positive control plates fell within RTC acceptance criteria based on historical control. It is concluded that the test item Cedarwood Virginia Oil does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism, under the reported experimental conditions. Based on the results of this study Cedarwood Virginia oil is not considered genotoxic.