Balsams, gurjun

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:

29-08-2016 to 21-09-2016

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

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

2008

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Version / remarks:

1998

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
- Test item: 207786/A
- Source and lot/batch No.of test material:L4285828
- Expiration date of the lot/batch:30 April 2018 (retest date)

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

OTHER SPECIFICS: UVCB

Target gene:

- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

Direct plate:
- Dose-range finding test: TA 100 and WP2uvrA (without and with S9): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate
Based on the results of experiment 1, the following dose levels were used:
- Experiment 1: TA 1535, TA 1537 and TA 98 (without and with S9): 17, 52, 164, 512, 1600 and 5000 μg/plate
- Experiment 2: Based on the results of experiment 1, the following dose levels were used: TA 1535, TA 1537, TA 98, TA 100 and WP2uvrA (without and with S9): 492, 878, 1568, 2800 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: A solubility test was performed. The test item could not be dissolved in water or dimethyl sulfoxide. The test item was soluble in ethanol. Therefore ethanol was used as solvent in this project.

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: ICR191 (TA1537; without metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: (independent repeat) preincubation

DURATION
- Preincubation period: minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain (in all experiments)

DETERMINATION OF CYTOTOXICITY
- Method: 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:

According to guideline.

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.

Species / strain:

S. typhimurium TA 1535

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

Species / strain:

S. typhimurium TA 1537

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

Species / strain:

S. typhimurium TA 98

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

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In experiment 2

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 2800 and 5000 μg/plate and at 5000 μg/plate at the end of the incubation period at experiment 2

RANGE-FINDING/SCREENING STUDIES: Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA100 (absence and presence of S9-mix). No increase in the number of revertants was observed upon treatment with testing material under any conditions tested.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 78 - 1381 78 - 1058 55 – 1565 55 – 1112 410 – 2057 263 - 1907 549 – 1848 620 - 2651 127 – 1951 85 - 1390
Mean 785 228 653 387 1155 860 892 1404 1263 342
SD 167 105 290 143 370 323 178 327 461 165
n 1684 1662 1448 1536 1646 1686 1650 1677 1370 1410

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between 31 May 2014 and 31 May 2016.

- Negative (solvent/vehicle) historical control data:
TA1535 TA1537 TA98 TA100 WP2uvrA
S9-mix - + - + - + - + - +
Range 4 - 36 3 - 34 3 – 25 3 - 28 9 - 50 9 - 57 63 - 153 60 - 156 12 – 68 12 - 70
Mean 14 13 7 9 17 25 100 103 26 32
SD 6 5 3 4 5 7 16 18 7 8
n 1662 1677 1548 1547 1662 1703 1659 1691 1421 1424

SD = Standard deviation
n = Number of observations
Historical control data from experiments performed between 31 May 2014 and 31 May 2016.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Experiment 1: Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction in the bacterial background lawn was observed in all Salmonella typhimurium bacterial strains. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.
Experiment 2:Cytotoxicity, as evidenced by a decrease in the number of revertants, was only observed in tester strain TA100 in the absence and presence of S9-mix. In all other test strains, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in the absence and presence of
S9-mix.

Conclusions:

Under the conditions of this study, Gurjun balsam oil was determined to be not mutagenic and does not need to be classified for mutagenicity in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay in accordance with the criteria outline in Annex I of CLP (1272/2008/EC).

Executive summary:

The mutagenic activity of Gurjun balsam oil was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent experiments: at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix up to and including 5000 μg/plate. The dose levels were selected based on a dose range finding test with strain TA100 and WP2uvrA. 

In experiment 1, Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction in the bacterial background lawn was observed in all Salmonella typhimurium bacterial strains. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested.

In experiment 2 in all strains, cytotoxicity was only observed in tester strain TA100 in the absence and presence of S9-mix. In all other test strains, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in the absence and presence of S9-mix.

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 four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and E. coli tester strain (WP2uvrA), both in the absence and presence of S9 -metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that Gurjun balsam oil is 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 Gurjun balsam oil was evaluated in accordance with OECD 471 (1997) guideline and according to GLP principles. The test was performed in two independent experiments: at first a direct plate assay was performed and secondly a pre-incubation assay, both in the absence and presence of S9-mix up to and including 5000 μg/plate. The dose levels were selected based on a dose range finding test with strain TA100 and WP2uvrA. In experiment 1, Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction in the bacterial background lawn was observed in all Salmonella typhimurium bacterial strains. In tester strain WP2uvrA, no toxicity was observed at any of the dose levels tested. In experiment 2 in all strains, cytotoxicity was only observed in tester strain TA100 in the absence and presence of S9-mix. In all other test strains, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in the absence and presence of S9-mix. 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 four S. typhimurium tester strains (TA1535, TA1537, TA98 and TA100) and E. coli tester strain (WP2uvrA), both in the absence and presence of S9 -metabolic activation. These results were confirmed in independently repeated experiments. Based on the results of this study it is concluded that Gurjun balsam oil is not mutagenic.

Justification for classification or non-classification

Based on the avilable data, Gurjun Balsam oil - Copaene does not need to be classified for genotoxicity in accordance with the criteria outlined in Annex I of the CLP Regulation (1272/2008/EC).