Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity in vitro (OECD TG 471, based on 3 available Ames studies, WoE): Not mutagenic

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
26 Apr 2018 - 18 Jun 2018
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:
July 21, 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
His, Trp
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 17, 52, 164, 512, 1600 and 5000 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: It has been identified as a suitable solvent
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: ICR-191, 2-aminoanthracene (2AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation) and preincubation
- Cell density at seeding (if applicable): 10^9 cells/mL

DURATION
- Preincubation period: 30 ± 2 minutes by 70 rpm at 37 ± 1°C
- Exposure duration: 48 ± 4 h

SELECTION AGENT (mutation assays): Histidine and tryptophan absence

METHODS OF COLONY COUNTING: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually.

DETERMINATION OF CYTOTOXICITY : Reduction of the bacterial background lawn
Rationale for test conditions:
According to the OECD guideline. Furthermore performing both the pre-incubation and the plate incorporation assay provide more information about the possible mutagenicity of the test item.
Evaluation criteria:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.
Statistics:
No statistical hypothesis testing was done.
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Direct Plate Assay
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Direct Plate Assay
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Direct Plate Assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Direct Plate Assay
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
3.4 fold increases
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Direct Plate Assay
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
2 fold increase
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Direct Plate Assay
Metabolic activation:
without
Genotoxicity:
positive
Remarks:
3.4 fold increase
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Direct Plate Assay
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
1.9 fold increase
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
highest concentration
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
Direct Plate Assay
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Pre-Incubation Assay
Metabolic activation:
without
Genotoxicity:
not determined
Remarks:
Due to the cytotoxicity in the pre-incubation method, the highest dose(s) in the absence of S9-mix in the Salmonella strains resulted in the presence of microcolonies, and therefore the number of revertant colonies could not be determined.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Pre-Incubation Assay
Metabolic activation:
with
Genotoxicity:
not determined
Remarks:
Due to the cytotoxicity in the pre-incubation method, the highest dose(s) in the absence of S9-mix in the Salmonella strains resulted in the presence of microcolonies, and therefore the number of revertant colonies could not be determined.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Pre-Incubation Assay
Metabolic activation:
without
Genotoxicity:
not determined
Remarks:
Due to the cytotoxicity in the pre-incubation method, the highest dose(s) in the absence of S9-mix in the Salmonella strains resulted in the presence of microcolonies, and therefore the number of revertant colonies could not be determined.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Pre-incubation assay
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
2.9 fold increase
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Pre-Incubation Assay
Metabolic activation:
without
Genotoxicity:
not determined
Remarks:
Due to the cytotoxicity in the pre-incubation method, the highest dose(s) in the absence of S9-mix in the Salmonella strains resulted in the presence of microcolonies, and therefore the number of revertant colonies could not be determined.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Pre-Incubation Assay
Metabolic activation:
with
Genotoxicity:
ambiguous
Remarks:
2.1 fold increase
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Pre-Incubation Assay
Metabolic activation:
without
Genotoxicity:
not determined
Remarks:
Due to the cytotoxicity in the pre-incubation method, the highest dose(s) in the absence of S9-mix in the Salmonella strains resulted in the presence of microcolonies, and therefore the number of revertant colonies could not be determined.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Pre-Incubation Assay
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Remarks:
Pre-incubation Assay
Metabolic activation:
without
Genotoxicity:
not determined
Remarks:
Due to the cytotoxicity in the pre-incubation method, the highest dose(s) in the absence of S9-mix in the Salmonella strains resulted in the presence of microcolonies, and therefore the number of revertant colonies could not be determined.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Slight precipitation is distinguished by a noticeable precipitate on the plate, however the precipitate does not influence automated counting of the plates.

RANGE-FINDING/SCREENING STUDIES: Based on the results of the dose-range finding test, the following dose-range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 17, 52, 164, 512,1600 and 5000 μg/plate.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) - refer to any other information

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: reduction in the background lawn
- Due to the cytotoxicity in the pre-incubation method, the highest dose(s) in the absence of S9-mix in the Salmonella strains resulted in the presence of microcolonies, and therefore the number of revertant colonies could not be determined.

Historical Control Data of the Solvent Control

 

TA1535

TA1537

TA98

TA100

WP2uvrA

S9-mix

-

+

-

+

-

+

-

+

-

+

Range

3 – 29

3 – 27

3 – 20

3 – 23

8 - 41

8 – 55

61 – 176

60 - 160

10 – 59

9 - 67

Mean

10

11

6

6

16

22

110

106

26

33

SD

3

4

2

3

5

7

17

20

6

8

n

2458

2426

2402

2352

2416

2458

2473

2398

2237

2217

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between May 2016 and May 2018. 

Historical Control Data of the Positive Control Items

 

TA1535

TA1537

TA98

S9-mix

-

+

-

+

-

+

Range

128 – 1530

73 – 1206

58 – 1407

54 – 1051

365 – 1978

250 – 1977

Mean

901

239

817

340

1355

903

SD

174

115

354

160

230

357

n

2400

2296

2051

2337

2357

2367

 

 

TA100

WP2uvrA

S9-mix

-

+

-

+

Range

439 – 1993

408 - 2379

93 – 1958

111 - 1359

Mean

905

1249

1059

444

SD

163

371

506

144

n

2402

2354

2153

2232

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between May 2016 and May 2018. 

Conclusions:
Based on the results of this study it is concluded that Gurjun Balsam Oil (Gurjunene) is mutagenic in the Salmonella typhimurium reverse mutation assay in the tester strains TA100 and TA98, in the absence of S9.
The test item is not mutagenic in the tester strains TA1535 and TA1537, in the Salmonella typhimurium reverse mutation assay or in the Escherichia coli reverse mutation assay, with or without S9.
Although the test item induces a significant increase in the tester strain TA98 in the presence of S9, the increase is below three (3) times the concurrent control. It therefore does not fulfil the acceptability criterium, and is thus not considered mutagenic. The 1.9- and 2.1-fold increases observed in the TA100 strain, in the presence of S9 is considered equivocal, as the level is lower or very close to the two (2) times concurrent control acceptability criterium.
Executive summary:

The potential of Gurjun Balsam Oil (Gurjunene) and/or its metabolites to induce reverse mutations at the histidine locus was tested in a OECD TG 471, Ames study. Mutagenicity was determined in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

 

First mutation assay (direct plate assay): based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 17 to 5000 µg/plate in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. 

- In the absence of S9-mix, the test item induced up to 3.4-fold increases in the number of revertant colonies compared to the solvent control in the tester strains TA100 and TA98. T

- In the presence of S9-mix, the test item induced up to 1.9-, 1.7- and 2.0-fold increases in the number of revertant colonies compared to the solvent control in the tester strains TA100, TA1535 and TA98, respectively.

 

In the second mutation experiment (pre-incubation assay): the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. 

- In the absence of S9-mix, no increases in the number of revertant colonies were observed at any of the dose levels tested in all tester strains. However, due to cytotoxicity in the absence of S9-mix,the number of revertant colonies could not be determined.

- In the presence of S9-mix, the test item induced up to 2.9- and 2.1-fold increases in the number of revertant colonies compared to the solvent control in the tester strains TA98 and TA100, respectively. Both increases were above the laboratory historical control data range.

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

Overall assessment of the results:

It is concluded that Gurjun Balsam Oil (Gurjunene) is mutagenic in the Salmonella typhimurium reverse mutation assay in the tester strains TA100 and TA98, in the absence of S9.

The test item is not mutagenic in the tester strains TA1535 and TA1537, in the Salmonella typhimurium reverse mutation assay or in the Escherichia coli reverse mutation assay, with or without S9.

Although the test item induces a significant increase in the tester strain TA98 in the presence of S9, the increase is below three (3) times the concurrent control. It therefore does not fulfil the acceptability criterium, and is thus not considered mutagenic. The 1.9- and 2.1-fold increases observed in the TA100 strain, in the presence of S9 is considered equivocal, as the level is lower or very close to the two (2) times concurrent control acceptability criterium.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
22 March 2018 - 14 May 2018
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:
July 1997
Deviations:
yes
Remarks:
1. The test article was distributed in two plates without strains to assist the discrimination between precipitates and colonies 2. The data were statistically analysed using Dunnett’s test
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment 1 (plate incorporation): 1.6, 5, 16, 50, 160, 500, 1600 and 5000 μg/plate
Experiment 2 (plate incorporation method in absence of S9 and the preincubation method in presence of S9): 160, 300, 500, 1000, 2000 and 5000 µg/plate (except for TA100, for which the max dose of 1600µg/plate was chosen due to strain specific toxicity), for TA1535 an additional experiment was performed (no metabolic activation) with an extended narrowed doses range from 50 to 5000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: good solubility, delivering a colorless clear solution
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene (AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding (if applicable): 5.10^8 to 5.10^9 cells/mL

DURATION
- Preincubation period: 20 minutes at 37±1°C
- Exposure duration: 2 to 3 days

SELECTION AGENT (mutation assays): histidine absence

DETERMINATION OF CYTOTOXICITY
- Method: The background lawns of the plates will be examined for signs of toxicity. Other evidence of toxicity may include a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.

Rationale for test conditions:
In accordance with the OECD 471 test guidance
Evaluation criteria:
For valid data, the test article will be considered to be mutagenic in this assay if:
1. A concentration related increase in revertant numbers is ≥2.0-fold (in strains TA98, TA100 or TA102) or ≥3.0-fold (in strains TA1535 or TA1537) the concurrent untreated control values.
2. Any observed response is reproducible under the same treatment conditions The test article will be regarded positive in this assay if both of the above criteria are met.

The test article will be regarded negative in this assay if neither of the above criteria are met.
Results which only partially satisfy the above criteria will be dealt with on a case-bycase basis.
Biological relevance will be taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments. Further experimental work may be deemed necessary to aid evaluation of the data.
Statistics:
The data were statistically analysed using Dunnett’s test to confirm the results.
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Experiment 1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S9 at the maximum tested dose 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Experiment 1
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
In the absence of S9 at the maximum tested dose 5000 μg/plate
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S9 at the maximum tested dose 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Experiment 1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S9 at the maximum tested dose 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Experiment 1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Absence of S9: reduction in background lawn (from 50 to 500 μg/plate) and an absence (1600 – 5000 μg/plate) Presence of S9: reduction in the background bacterial lawn (from 160 to 5000 μg/plate).
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Remarks:
Experiment 1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S9 at the maximum tested dose 5000 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S9 from 300 μg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S9 from 1000µg/plate, in the presence of S9 at 300-1000 µg/plate)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S9 from 200µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In absence of S9 from 100µg/plate, in the prescence of S9 at 800-1600µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
in the absence of S9 from 1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
variability.TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
In Experiment 1, precipitates were observed in the test plates in both conditions with and without metabolic activation at 1600 and 5000 μg/plate in a form of optically clear droplets that ressembled bacterial colonies. A manual scrutiny scoring (double check) was necessary to discriminate transparency droplets from white colonies. This precipitate could have interfered with the counting using an automatic scoring.

In Experiment 2 and the additional Experiment, precipitates were also observed in the test plates in both conditions with and without metabolic activation at the maximum tested dose 1600 μg/plate in the strain TA100 and in the four other strains at 2000 and 5000 μg/plate in a form of optically clear droplets that ressembled bacterial colonies. A manual scrutiny scoring (double check) was necessary to discriminate transparency droplets from white colonies. This precipitate could have interfered with the counting using an automatic scoring.

RANGE-FINDING/SCREENING STUDIES:
Due to the marked toxicity observed in the strain TA1535 without metabolic activation, in the Experiment 2 where only two tested doses were analysable, an additional experiment was performed with an extended narrowed doses range from 50 to 5000 μg/plate. A toxicity was observed from 500 to the maximum tested dose 5000 μg/plate as an absence of the background bacterial lawn.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%) : refer to "attached background information"
- Positive historical control data: available data attached
- Negative (solvent/vehicle) historical control data: available dataattached

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: reduction in background lawn
Conclusions:
Based on the results of this study it is concluded that Gurjun Balsam Oil (Gurjunene) did not induce mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium. These conditions included treatments using both plate incorporation method and pre-incubation method at concentrations up to 5000 µg/plate, in the presence of a rat liver metabolic activation system (S9).
Executive summary:

The mutagenicity of Gurjun balsam oil was assessed in a in vitro gene mutation study in bacteria, according to OECD TG 471, both in the absence and in the presence of metabolic activation.

 

In Experiment 1 bacterial strains were exposed to Gurjun balsam oil at 1.6, 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, using the plate incorporation method in the absence and in the presence of S-9. Positive, negative and vehicle controls were also present and the results indicated the validity of the experiment. A reduction in the background bacterial lawn was observed at 5000 µg/plate (absence of S9) in all the strains except in the strain TA100 which showed marked toxicity. In the presence of metabolic activation, no toxicity was observed in the strains except in the strain TA100 with a reduction in the background bacterial lawn from 160 to 5000 µg/plate. No genotoxic effects were observed in any of the tested strains.

 

Experiment 2 was performed in all the tester strains in the absence of S9 using the plate incorporation method and in the presence of S9 using the pre incubation method. The maximum test concentration of 5000 µg/plate was retained for all strains except for the strain TA100 where it was reduced to 1600 µg/plate based on strain specific toxicity. For several strains, cytotoxicity was observed in the absence of metabolic activation (TA100, TA1535, TA1537, TA102, TA98, TA100). No evidence of toxicity was observed in the strains TA98 and TA102, in the presence of metabolic activation. Based on toxicity the doses, an additional experiment was performed on TA1535 with an extended narrowed doses range from 50 to 5000 µg/plate. Toxicity was observed from 500 to the maximum tested dose 5000 µg/plate as an absence of the background bacterial lawn.

 

In both main Experiments and its additional experiment precipitates were observed in the test plates with and without metabolic activation at the maximum tested dose 1600 and 5000 µg/plate (Experiment 1), 1600 µg/plate in the strain TA100 and in the four other strains at 2000 and 5000 µg/plate (Experiment 2 and Additional Experiment) in a form of optically clear droplets that ressembled bacterial colonies. A manual scrutiny scoring (double check) was necessary to discriminate transparency droplets from white colonies. This precipitate could have interfered with the counting using an automatic scoring.

 

Following treatments of all the test strainswith the test material in the absence and presence of metabolic activation, no increases in revertant numbers were observed that were ≥2.0-fold (in strains TA98, TA100 or TA102) or ≥3.0-fold (in strains TA1535 or TA1537) the concurrent vehicle control. This result was confirmed using Dunnett’s test where no increases observed were statistically significant when the data were analysed at the 1% level using Dunnett’s test. This study was considered therefore to have provided no evidence of any mutagenic activity in this assay system.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
20 July 2018 - 10 September 2018
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:
July 1997
Deviations:
yes
Remarks:
1. The test article was distributed in two plates without strains to assist the discrimination between precipitates and colonies 2. The data were statistically analysed using Dunnett’s test
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment 1 (plate incorporation): 1.6, 5, 16, 50, 160, 500, 1600 and 5000 μg/plate, plus 0.5 µg/plate in the strain TA100.
Additional experiment TA1537 (in the absence of S-9) from 0.016, 0.05, 0.16, 0.5, 1.6, 5, 16 and 50 μg/plate.

Experiment 2 (plate incorporation method in absence of S9)
TA98, TA102: 75, 150, 300, 625, 1250, 2500 and 5000 µg/plate
TA100 10, 25, 50, 125, 250, 500, 1250 and 2500 µg/plate
TA1535: 2.5, 5, 10, 25, 50, 125, 250 and 500 µg/plate
TA1537: 0.05, 0.1, 0.5, 1, 2.5, 5, 10 and 25 µg/plate

Experiment 2 (pre-incubation method in presence of S9)
TA98, TA100, TA102: 75, 150, 300, 625, 1250, 2500 and 5000 µg/plate
TA1535: 10, 25, 50, 125, 250, 500, 1250 and 2500 µg/plate
TA1537: 50, 75, 150, 300, 625, 1250 and 2500 µg/plate

Additional experiment
TA100: (plate incorporation method in absence of S9) 0.05, 0.125, 0.25, 0.5, 1.25, 2.5, 5, 10, 25, 50, 125, 250, 500, 1250 and 2500 µg/plate
TA100: (pre-incubation method in presence of S9): 0.5, 1.25, 2.5, 5, 12.5, 25, 50, 75, 150, 300, 625, 1250, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: good solubility, delivering a colorless clear solution
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-aminoanthracene (AA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding (if applicable): 5.10^8 to 5.10^9 cells/mL

DURATION
- Preincubation period: 20 minutes at 37±1°C
- Exposure duration: 2 to 3 days

SELECTION AGENT (mutation assays): histidine absence

DETERMINATION OF CYTOTOXICITY
- Method: The background lawns of the plates will be examined for signs of toxicity. Other evidence of toxicity may include a marked reduction in revertants compared to the concurrent vehicle controls and/or a reduction in mutagenic response.

Rationale for test conditions:
In accordance with the OECD 471 test guidance
Evaluation criteria:
For valid data, the test article will be considered to be mutagenic in this assay if:
1. A concentration related increase in revertant numbers is ≥2.0-fold (in strains TA98, TA100 or TA102) or ≥3.0-fold (in strains TA1535 or TA1537) the concurrent untreated control values.
2. Any observed response is reproducible under the same treatment conditions The test article will be regarded positive in this assay if both of the above criteria are met.

The test article will be regarded negative in this assay if neither of the above criteria are met.
Results which only partially satisfy the above criteria will be dealt with on a case-bycase basis.
Biological relevance will be taken into account, for example consistency of response within and between concentrations and (where applicable) between experiments. Further experimental work may be deemed necessary to aid evaluation of the data.
Statistics:
The data were statistically analysed using Dunnett’s test to confirm the results.
Key result
Species / strain:
S. typhimurium TA 1535
Remarks:
Experiment 1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the presence of S9 and in the absence of S-9 at 50 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Experiment 1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In absence and presence of S9 in all dose ranges
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Experiment 1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S9 at 1600-5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Experiment 1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the presence of S9 and in the absence of S-9 at 160 to 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Remarks:
Experiment 1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S9 at 1600-5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Experiment 1 Additional
Metabolic activation:
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 1535
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S-9 from 125 µg/plate up to maximum dose, in the presence of S-9 from 1250 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S-9 from 10 µg/plate up to maximum dose, In the presence of S-9 from 625 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
in the absence of S 9 at 1250 µg/plate
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S-9 from 2500 µg/plate up to maximum dose, In the presence of S-9 from 625 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S-9 from 10 µg/plate up to maximum dose, In the presence of S-9 from 625 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Remarks:
Experiment 2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S-9 from 2500 µg/plate up to maximum dose, In the presence of S-9 at 5000µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Remarks:
Experiment 2 Additional
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the absence of S-9 from 1.25 µg/plate, In the presence of S-9 from 300 µg/plate with up to the maximum dose tested
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
variability.TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation:
In Experiment 1
Precipitates in a form of transparent droplets were observed at 1600 and 5000 µg/plate in both conditions with and without S-9.
In Experiment 2
Precipitates were also observed from 1250 µg/plate (strains TA100 without S-9, TA1537 with S-9) and 2500 µg/plate in the other strains and TA100 with S-9 in a form of transparent droplets in both conditions with or without S-9.

RANGE-FINDING/SCREENING STUDIES:
-Additional experiment strain TA1537 (plate incorporation, absence of S-9) because the number of analyzable doses did not meet the OECD guidelines recommendations (1 out of 5 doses). Following treatments from 0.016 to 50 µg/plate, no evidence of toxicity was observed.
-Additional experiment strain TA100 (plate incorporation method, in the absence of S-9) and (pre-incubation method, presence of S-9) because the number of analyzable doses did not meet the OECD guidelines recommendations (3 out of 5 doses), plus a reduction of the background bacterial lawn was observed in the negative control without S-9. Following treatments from an extended dose range i.e 0.05 to 2500 µg/plate in the absence of S-9 and 0.5 to 5000 µg/plate in the presence of S 9, a marked toxicity was observed from 1.25 µg/plate without S-9 and from 300 µg/plate with S-9 up to the maximum tested dose.

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: reduction in background lawn
Remarks on result:
other: A dose-related increase was observed over the dose range from 75 (ratio 1.2) to 625 µg/plate (1.8) and 1250 µg/plate (3.1) without toxicity. The Dunnett’s test is significant at 625 and 1250 µg/plate (p< 0.01), confirming the increase in revertant number
Conclusions:
Based on the results of this study it is concluded that Gurjun Balsam oil (Gurjunene) did not induce mutation in the histidine-requiring strains TA100, TA1535, TA1537 and TA102 of Salmonella typhimurium, These conditions included treatments using both plate incorporation method and pre-incubation method at concentrations up to 5000 µg/plate, in the presence of a rat liver metabolic activation system S9.

Gurjun Balsam Oil (Gurjunene) did induce mutation in the histidine-requiring strain TA98 of Salmonella typhimurium, in the absence of S9 at 1250 µg/plate (the highest dose without toxicity), resulting in an increase in revertant numbers that was ≥2-fold the concurrent vehicle control with a value ratio of 3.1.
Executive summary:

The mutagenicity of Gurjun Balsam oil (Gurjunene) was assessed in an in vitro gene mutation study in bacteria, according to OECD TG 471, both in the absence and in the presence of metabolic activation. Positive, negative and vehicle controls were also present and the results indicated the validity of the experiment.

 

In Experiment 1 bacterial strains were exposed to Gurjun balsam oil at 1.6, 5, 16, 50, 160, 500, 1600 and 5000μg/plate, plus 0.5µg/plate in the strain TA100, using the plate incorporation method in the absence and in the presence of S-9. Additional concentrations were tested for TA1537 (in the absence of S-9) from 0.016, 0.05, 0.16, 0.5, 1.6, 5, 16 and 50μg/plate. A reduction in the background bacterial lawn was observed in all tester strains except in the strains TA98 and TA102 with S‑9 which showed marked toxicity. No significant genotoxic effects were observed in any of the tested strains.

 

Experiment 2 was performed in all the tester strains in the absence of S9 using the plate incorporation method and in the presence of S9 using the pre incubation method. The maximum test concentration of 5000 μg/plate was retained for TA98 and TA102, and was reduced to 2500 μg/plate (TA100), 500 μg/plate (TA1535), and 25 µg/plate (TA1537), for pre-incubation method 2500 µg/plate (TA1535 and TA1537) based on strain specific toxicity. Cytotoxicity, in the form of reduction of background lawn was observed in all strains, both in the presence and absence of S9. In the absence of S-9, from 2500 µg/plate (strains TA98, TA102), 125 µg/plate (strain TA1535), and 10 µg/plate (strain TA100, TA1537) up to the maximum dose tested. In presence of S9, at 5000 µg/plate (strain TA102) and from 1250 µg/plate (strain TA1535 ), 625 µg/plate (strains TA98, TA100, TA1537) up to the maximum dose tested. Precipitates in a form of transparency droplets were observed at 1250 µg/plate (strains TA 100 without S-9, TA1537 with S-9) and at 1600, 2500 and 5000 µg/plate in all the strains both with and without S-9.

 

Following treatments of all the test strains with the test material in the absence and presence of metabolic activation, no increases in revertant numbers were observed that were 2.0-fold (in strains TA100 or TA102) or 3.0-fold (in strains TA1535 or TA1537) the concurrent vehicle control, except for a dose-related increase in strain TA98, in the absence of S9, that was observed over the dose range from 75 (ratio 1.2) to 625 µg/plate (1.8) and 1250 µg/plate (3.1) without toxicity. The Dunnetts test is significant at 625 and 1250 µg/plate (p< 0.01), confirming the increase in revertant number.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Three different in vitro gene mutation studies in bacteria are available for the test substance Gurjun Balsam oil - Gurjunene. These studies were assessed according to a weight-of-evidence (WoE) approach. Here the summaries of the three studies are presented, followed by a WoE evaluation and a conclusion.

Genetic toxicity in vitro_OECD471_3015-226_2018_WoE

The mutagenicity of Gurjun balsam oil was assessed in a in vitro gene mutation study in bacteria, according to OECD TG 471, both in the absence and in the presence of metabolic activation.

- In Experiment 1 bacterial strains were exposed to Gurjun balsam oil at 1.6, 5, 16, 50, 160, 500, 1600 and 5000 µg/plate, using the plate incorporation method in the absence and in the presence of S-9. Positive, negative and vehicle controls were also present and the results indicated the validity of the experiment. A reduction in the background bacterial lawn was observed at 5000 µg/plate (absence of S9) in all the strains except in the strain TA100 which showed marked toxicity. In the presence of metabolic activation, no toxicity was observed in the strains except in the strain TA100 with a reduction in the background bacterial lawn from 160 to 5000 µg/plate. No genotoxic effects were observed in any of the tested strains.

- Experiment 2 was performed in all the tester strains in the absence of S9 using the plate incorporation method and in the presence of S9 using the pre incubation method. The maximum test concentration of 5000 µg/plate was retained for all strains except for the strain TA100 where it was reduced to 1600 µg/plate based on strain specific toxicity. For several strains, cytotoxicity was observed in the absence of metabolic activation (TA100, TA1535, TA1537, TA102, TA98, TA100). No evidence of toxicity was observed in the strains TA98 and TA102, in the presence of metabolic activation. Based on toxicity the doses, an additional experiment was performed on TA1535 with an extended narrowed doses range from 50 to 5000 µg/plate. Toxicity was observed from 500 to the maximum tested dose 5000 µg/plate as an absence of the background bacterial lawn.

In both main Experiments and its additional experiment precipitates were observed in the test plates with and without metabolic activation at the maximum tested dose 1600 and 5000 µg/plate (Experiment 1), 1600 µg/plate in the strain TA100 and in the four other strains at 2000 and 5000 µg/plate (Experiment 2 and Additional Experiment) in a form of optically clear droplets that ressembled bacterial colonies. A manual scrutiny scoring (double check) was necessary to discriminate transparency droplets from white colonies. This precipitate could have interfered with the counting using an automatic scoring.

- Conclusion: Following treatments of all the test strains with the test material in the absence and presence of metabolic activation, no increases in revertant numbers were observed that were ≥2.0-fold (in strains TA98, TA100 or TA102) or ≥3.0-fold (in strains TA1535 or TA1537) the concurrent vehicle control. This result was confirmed using Dunnett’s test where no increases observed were statistically significant when the data were analysed at the 1% level using Dunnett’s test. This study was considered therefore to have provided no evidence of any mutagenic activity in this assay system.

Genetic toxicity in vitro_OECD471_20152331_2018_WoE

The potential of Gurjun Balsam Oil (Gurjunene) and/or its metabolites to induce reverse mutations at the histidine locus was tested in a OECD TG 471, Ames study. Mutagenicity was determined in the presence or absence of an exogenous mammalian metabolic activation system (S9). 

 First mutation assay (direct plate assay): based on the results of the dose-range finding test, the test item was tested in the first mutation assay at a concentration range of 17 to 5000 µg/plate in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98. 

- In the absence of S9-mix, the test item induced up to 3.4-fold increases in the number of revertant colonies compared to the solvent control in the tester strains TA100 and TA98. T

- In the presence of S9-mix, the test item induced up to 1.9-, 1.7- and 2.0-fold increases in the number of revertant colonies compared to the solvent control in the tester strains TA100, TA1535 and TA98, respectively.

 In the second mutation experiment (pre-incubation assay): the test item was tested up to concentrations of 5000 µg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation assay. 

- In the absence of S9-mix, no increases in the number of revertant colonies were observed at any of the dose levels tested in all tester strains. However, due to cytotoxicity in the absence of S9-mix,the number of revertant colonies could not be determined.

- In the presence of S9-mix, the test item induced up to 2.9- and 2.1-fold increases in the number of revertant colonies compared to the solvent control in the tester strains TA98 and TA100, respectively. 

The negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Overall assessment of the results:

It is concluded that Gurjun Balsam Oil (Gurjunene) is mutagenic in the Salmonella typhimurium reverse mutation assay in the tester strains TA100 and TA98, in the absence of S9.

The test item is not mutagenic in the tester strains TA1535 and TA1537, in the Salmonella typhimurium reverse mutation assay or in the Escherichia coli reverse mutation assay, with or without S9.

Although the test item induces a significant increase in the tester strain TA98 in the presence of S9, the increase is below three (3) times the concurrent control. It therefore does not fulfil the stated acceptability criterium for this study, and is thus not considered mutagenic. The 1.9- and 2.1-fold increases observed in the TA100 strain, in the presence of S9 is considered equivocal, as the level is lower or very close to the two (2) times concurrent control acceptability criterium.

Genetic toxicity in vitro_OECD471_ 3031-222_2018_WoE

The mutagenicity of Gurjun Balsam oil (Gurjunene) was assessed in an in vitro gene mutation study in bacteria, according to OECD TG 471, both in the absence and in the presence of metabolic activation. Positive, negative and vehicle controls were also present and the results indicated the validity of the experiment.

In Experiment 1 bacterial strains were exposed to Gurjun balsam oil at 1.6, 5, 16, 50, 160, 500, 1600 and 5000μg/plate, plus 0.5µg/plate in the strain TA100, using the plate incorporation method in the absence and in the presence of S-9. Additional concentrations were tested for TA1537 (in the absence of S-9) from 0.016, 0.05, 0.16, 0.5, 1.6, 5, 16 and 50μg/plate. A reduction in the background bacterial lawn was observed in all tester strains except in the strains TA98 and TA102 with S‑9 which showed marked toxicity. No significant genotoxic effects were observed in any of the tested strains.

Experiment 2 was performed in all the tester strains in the absence of S9 using the plate incorporation method and in the presence of S9 using the pre incubation method. The maximum test concentration of 5000μg/plate was retained for TA98 and TA102, and was reduced to 2500μg/plate (TA100), 500μg/plate (TA1535), and 25µg/plate (TA1537), for pre-incubation method 2500µg/plate (TA1535 and TA1537) based on strain specific toxicity. Cytotoxicity, in the form of reduction of background lawn was observed in all strains, both in the presence and absence of S9. In the absence of S-9, from 2500µg/plate (strains TA98, TA102), 125µg/plate (strain TA1535), and 10µg/plate (strain TA100, TA1537) up to the maximum dose tested. In presence of S9, at 5000 µg/plate (strain TA102) and from 1250µg/plate (strain TA1535 ), 625µg/plate (strains TA98, TA100, TA1537) up to the maximum dose tested. Precipitates in a form of transparency droplets were observed at 1250µg/plate (strains TA 100 without S-9, TA1537 with S-9) and at 1600, 2500 and 5000µg/plate in all the strains both with and without S-9.

Following treatments of all the test strains with the test material in the absence and presence of metabolic activation, no increases in revertant numbers were observed that were≥2.0-fold (in strains TA100 or TA102) or≥3.0-fold (in strains TA1535 or TA1537) the concurrent vehicle control, except for a dose-related increase in strain TA98, in the absence of S9, that was observed over the dose range from 75 (ratio 1.2) to 625µg/plate (1.8) and 1250 µg/plate (3.1) without toxicity. The Dunnett’s test is significant at 625 and 1250µg/plate (p< 0.01), confirming the increase in revertant number.

WoE evaluation

Based on the WoE of the available studies, the test substance shows no mutagenicity in the presence of bioactivation (S9), and is therefore not considered to be mutagenic in bacteria.

Justification for classification or non-classification

Based on the available information, Gurjun Balsam oil - Gurjunene does not need to be classified for genotoxicity, according to the classification criteria outlined in Annex I of 1272/2008/EC (CLP).