Storax (balsam)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

OECDTG 471: negative with and without metabolic activation

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:

25 Apr 2017 - 12 May 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Identification: Styrax resinoid oil
Appearance: Orange viscous liquid
Batch: K17 031-1
Purity/Composition: 100%
Test item storage: At room temperature
Stable under storage conditions until: 02 February 2019

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

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary test:
TA100 (+S9 and -S9): 0, 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 μg/plate.
WP2uvrA (+S9 (5%, plate incorporation method) and -S9): 0, 1.7, 5.4, 17, 52, 164, 512, 1600, 5000 μg/plate.

Experiment 1:
TA1535, TA 1537, TA98 (+S9 (5%, plate incorporation method) and -S9): 0, 17, 52, 164, 512, 1600, 5000 μg/plate.

Experiment 2:
All Salmonella strains and WP2uvrA (-S9): 0, 164, 275, 492, 878, 1568 μg/plate.
All Salmonella strains and WP2uvrA (+S9(10%, plate incorporation method)): 0, 492, 878, 1568, 2800, 5000 μg/plate.

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: -S9: sodium azide (TA1535), ICR-191 (TA1537), 2-nitrofluorene (TA98), methylmethanesulfonate (TA100), 4-nitroquinoline N-oxide (WP2uvrA). +S9: 2-aminoanthracene (all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION:
With S9 mix: in agar (plate incorporation test)
Without S9 mix: preincubation test

DURATION
- Exposure duration: 48 hr, plate incorporation test

SELECTION AGENT: agar containing Histidine or Tryptophan

NUMBER OF REPLICATIONS: concentrations were tested in triplicate.

DETERMINATION OF CYTOTOXICITY
- Method: Measuring number of revertant colonies and effects on the growth of the bacterial background lawn.

Evaluation criteria:

In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent vehicle 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 the tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three (3) times the concurrent vehicle control.
b) In case a follow up 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:

Not applicable

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Dose range finding/first test: Precipitation of the test item on the plates was observed at the start of the incubation period at concentrations of 1600 and 5000 μg/plate. Precipitation of the test item on the plates was observed at the end of the incubation period at concentrations of 1600 and 5000 μg/plate in the absence of S9-mix and at the dose level of 5000 μg/plate in the presence of S9-mix.

Second experiment: Precipitation of the test item on the plates was observed at the start of the incubation period at the concentration of 1568 μg/plate and above. At the end of the incubation period, precipitation of the test item on the plates was observed at concentrations of 1568 μg/plate and upwards in the absence of S9-mix and at 2800 μg/plate and upwards in the presence of S9-mix.

TOXICITY:
Dose range finding/first test: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. Due to precipitate on the plates at the test item concentration of 5000 μg/plate (absence of S9-mix), the bacterial background lawn and/or the number of revertants of this dose level could not be determined. In strain TA1537 (absence of S9-mix), a fluctuation in the number of revertant colonies below the laboratory historical control data range was observed. However, since no doserelationship was observed, this reduction is not considered to be caused by toxicity of the test item. It is more likely this reduction is caused by an incidental fluctuation in the number of revertant colonies.

Second experiment: In the second mutation assay, 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 all tester strains in the absence and presence of S9-mix. Since in the presence of S9-mix the test item precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the bacterial background lawn and the number of revertants of this dose level could not be determined. In strain TA1537 (absence of S9-mix), fluctuations in the number of revertant colonies below the laboratory historical control data range were observed. However, since no doserelationship was observed, these reductions are not considered to be caused by toxicity of the test item. It is more likely these reductions are caused by incidental fluctuations in the number of revertant colonies.

MUTAGENICITY
Dose range finding/first test: No increase in the number of revertants was observed upon treatment with Styrax resinoid oil under all conditions tested.
Second experiment: In the second mutation assay, no increase in the number of revertants was observed upon treatment with Styrax resinoid oil under all conditions tested.

HISTORICAL CONTROL DATA
The negative control values were within the laboratory historical control data ranges. The 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, except the response for TA1535 in the second experiment. The purpose of the positive control is as a reference for the test system, where a positive response is required to check if the test system functions correctly. Since the value was more than 3 times greater than the concurrent solvent control values, this deviation in the mean plate count of the positive control had no effect on the results of the study.

Conclusions:

In conclusion, based on the results of this study it is concluded that Styrax resinoid oil is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The potential mutagenicity of Styrax resinoid oil and/or its metabolites was investigated with an Ames test according to OECDTG 471. In the dose-range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced and no biologically relevant decrease in the number of revertants was observed. Due to moderate to heavy precipitate on the plates at the test item concentration of 5000 μg/plate (absence of S9-mix), the bacterial background lawn and/or the number of revertants of this dose level could not be determined. 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 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced and no biologically relevant decrease in the number of revertants was observed. Since in the absence of S9-mix the test item precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the bacterial background lawn and the number of revertants of this dose level could not be determined. In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 164 to 1568 μg/plate in the absence and at a concentration range of 492 to 5000 μg/plate in the presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Since in the presence of S9-mix the test item precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the bacterial background lawn and the number of revertants of this dose level could not be determined. Styrax resinoid oil did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrAboth in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In conclusion, based on the results of this study it is concluded that Styrax resinoid oil is not mutagenic in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test:

The potential mutagenicity of Styrax resinoid oil and/or its metabolites was investigated with an Ames test according to OECDTG 471. In the dose-range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced and no biologically relevant decrease in the number of revertants was observed. Due to moderate to heavy precipitate on the plates at the test item concentration of 5000 μg/plate (absence of S9-mix), the bacterial background lawn and/or the number of revertants of this dose level could not be determined. 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 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The test item was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced and no biologically relevant decrease in the number of revertants was observed. Since in the absence of S9-mix the test item precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the bacterial background lawn and the number of revertants of this dose level could not be determined. In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 164 to 1568 μg/plate in the absence and at a concentration range of 492 to 5000 μg/plate in the presence of 10% (v/v) S9-mix in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. The test item was tested up to or beyond a precipitating dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Since in the presence of S9-mix the test item precipitated heavily on the plates at the test item concentration of 5000 μg/plate, the bacterial background lawn and the number of revertants of this dose level could not be determined. Styrax resinoid oil did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrAboth in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. In this study, acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly. In conclusion, based on the results of this study it is concluded that Styrax resinoid oil is not mutagenic in theSalmonella typhimuriumreverse mutation assay and in theEscherichia colireverse mutation assay.

Justification for classification or non-classification

Styrax resinoid oil does not have to be classified and has no obligatory labelling requirement for mutagenicity according to the Globally Harmonized System of Classification and Labelling of Chemicals (GHS) of the United Nations (2015) (including all amendments) and Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).