Resinoid of Cistus ladaniferus (Cistaceae) obtained from labdanum gum by ethanol extraction

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on a Read across strategy, two extract from the same plant were found to be non mutagenic in Ames test (OECD 471, GLP, K1). Therefore the registred substance is considered to be non mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From July 25 to August 18, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 471 without any deviation.

Reason / purpose for cross-reference:

read-across: supporting information

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:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Economy, Trade and Industry, Japanese Ministry of Health, Labour and Welfare and Japanese Ministry of Agriculture, Forestry and Fisheries.

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected on 05 July 2016 / signed on 28 October 2016

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan gene for Salmonella typhimurium and Escherichia coli, respectively.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The S9 Microsomal fraction was pre-prepared using standardized in-house procedures (outside the confines of this study). The 10% S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.

Test concentrations with justification for top dose:

Experiment 1 (Pre-incubation Method):
1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix.
Experiment 1 (Pre-incubation Method) / repeat:
0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate in all strains with and without S9-mix.
Experiment 2 (Pre-Incubation Method):
Salmonella strains TA100 and TA1535 (absence of S9): 0.05, 0.15, 0.5, 1.5, 5, 15, 50 μg/plate.
Salmonella strains TA98 and TA1537 and E.coli strain WP2uvrA (absence of S9): 0.5, 1.5, 5, 15, 50, 150, 500 μg/plate.
E.coli strain WP2uvrA and Salmonella strains TA100, TA1535 and TA98 (absence of S9): 5, 15, 50, 150, 500, 1500, 5000 μg/plate.
Salmonella strain TA1537 (presence of S9): 1.5, 5, 15, 50, 150, 500, 1500 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: In solubility checks performed in-house the test item was noted as insoluble in dimethyl sulphoxide and dimethyl formamide at 50 mg/mL and acetone at 100 mg/mL but fully soluble in tetrahydrofuran at 200 mg/mL. Tetrahydrofuran was therefore selected as the vehicle. Following solubility information provided by the Sponsor, sterile distilled water was not evaluated as a potential vehicle in this test system.
- Preparation of test materials: The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in tetrahydrofuran by mixing on a vortex mixer and sonication for 10 minutes at 40 °C. No correction was required for purity allowance. Tetrahydrofuran is toxic to the bacterial cells at and above 50 μL (0.05 mL), therefore all of the formulations were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 25 μL (0.025 mL) aliquots. Tetrahydrofuran is considered an acceptable vehicle for use in this test system (Maron et al., 1981). Prior to use, the solvent was dried to remove water using molecular sieves i.e. 2 mm sodium alumino-silicate pellets with a nominal pore diameter of 4 x 10-4 microns.
All formulations were used within four hours of preparation and were assumed to be stable for this period.

Untreated negative controls:

yes

Remarks:

untreated: spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix

Untreated negative controls:

yes

Remarks:

untreated: spontaneous mutation rates

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

SOURCE OF TEST SYSTEM: The bacteria used in the test were obtained from the University of California, Berkeley, and from the British Industrial Biological Research Association.

METHOD OF APPLICATION: preincubation (The test item was confirmed to either have unknown volatility or was suspected to be volatile, therefore all testing was performed using the pre-incubation method (20 minutes at 37 ± 3 °C) except for the untreated controls).

DURATION
- Preincubation period: 20 minutes with shaking
- Exposure duration: approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level in experiment 1 and experiment 2.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning.

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).

Rationale for test conditions:

Experiment 1 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).*
Experiment 1 / repeat - the test item induced excessive toxicity to Salmonella strains TA100 and TA1535 (absence of S9 only) and consequently part of the experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations and employing an amended dose range of 0.05, 0.15, 0.5, 1.5, 5, 15, 50, 150 μg/plate.
Experiment 2 - test item dose levels were selected based on Experiment 1 results. Up to seven test item dose levels per bacterial strain were selected in the second mutation test in order to achieve both a minimum of four non-toxic dose levels and the toxic limit of the test item.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:
- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- Fold increases greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Statistics:

NA

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cf. Table of results

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: immiscible
- Precipitation: A test item precipitate (greasy in appearance with red flecks ) was noted at and abo
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA:
Not needed (no statistical significant increase were noted)
The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the bacterial tester strains initially from 15 μg/plate (TA100), 50 μg/plate (TA1535), 150 μg/plate (TA98) and 500 μg/plate (WP2uvrA and TA1537) in the absence of S9-mix and to three bacterial strains initially from 500 μg/plate (TA1537), 1500 μg/plate (TA100) and 5000 μg/plate (TA1535) in the presence S9-mix. Consequently, the toxic limit or the maximum recommended dose (5000 μg/plate) of the test item was employed as the maximum dose in the second mutation test, depending on bacterial tester strain type and presence or absence of S9-mix. In the second mutation test, the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 15 μg/plate (TA100), 50 μg/plate (TA1535), 150 μg/plate (TA98 and TA1537) and 500 μg/plate (WP2uvrA). In the presence S9-mix weakened bacterial background lawns were noted to three of the bacterial tester strains from 1500 μg/plate (TA1537) and at 5000 μg/plate (TA1535 and TA100). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experiment.

CONCLUSION:
There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2.

Remarks on result:

other:

Remarks:

Table of results are in "Attached background documents"

Cf Tables of results in attached background material

Conclusions:

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item diluted in Tetrahydrofuran both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the Ames pre‑incubation methods in Experiment 1 and 2.

The original dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. However, the test item induced excessive toxicity to Salmonella strains TA100 and TA1535 (absence of S9 only) and consequently part of the experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations and employing an amended dose range of 0.05 to 150 μg/plate. The second experiment was performed at a later date again using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 (and the repeat assay) and ranged between 0.05 and 5000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. Up to seven test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

 

The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the bacterial tester strains initially from 15 μg/plate (TA100), 50 μg/plate (TA1535), 150 μg/plate (TA98) and 500 μg/plate (WP2uvrA and TA1537) in the absence of S9-mix and to three bacterial strains initially from 500 μg/plate (TA1537), 1500 μg/plate (TA100) and 5000 μg/plate (TA1535) in the presence S9-mix. Consequently, the toxic limit or the maximum recommended dose (5000 μg/plate) of the test item was employed as the maximum dose in the second mutation test, depending on bacterial tester strain type and presence or absence of S9-mix. In the second mutation test, the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 15 μg/plate (TA100), 50 μg/plate (TA1535), 150 μg/plate (TA98 and TA1537) and 500 μg/plate (WP2uvrA). In the presence S9-mix weakened bacterial background lawns were noted to three of the bacterial tester strains from 1500 μg/plate (TA1537) and at 5000 μg/plate (TA1535 and TA100). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experiment.

A test item precipitate (greasy in appearance with red flecks ) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2.

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.

 

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

November 07, 2014 to January 12, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD test Guideline No. 471 without any deviation.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine and tryptophan.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

10% S9: S9-mix from the livers of male rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day by oral route).

Test concentrations with justification for top dose:

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate in TA 100, TA 1535 and TA 1537 strains without S9-mix; 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate in TA 98 and WP2uvrA strains without S9-mix
Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA 100, TA 1535 and TA 1537 strains with S9-mix; 15, 50, 150, 500, 1500 and 5000 μg/plate in TA 98 and WP2uvrA strains with S9-mix

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in tetrahydrofuran, therefore, this solvent was selected as the vehicle.
- Preparation of test materials: The test item was accurately weighed and approximate half-log dilutions prepared in tetrahydrofuran by mixing on a vortex mixer and sonication for 30 minutes at 40 °C on the day of each experiment. Tetrahydrofuran is toxic to the bacterial cells at and above 50 μL (0.05 mL), therefore all of the formulations were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 25 μL (0.025 mL) aliquots. Tetrahydrofuran is considered an acceptable vehicle for use in this test system (Maron et al., 1981). All formulations were used within four hours of preparation and were assumed to be stable for this period.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

Without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-Aminoanthracene

Remarks:

With S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Exposure duration: Plates were incubated at 37 °C ± 3 °C for approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning (toxicity).

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using an automated colony counting system. Manual counts were performed at and above 500 μg/plate because of test item precipitation.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- Fold increases greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.

Statistics:

Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: None
- Precipitation: A test item precipitate (greasy and particulate in appearance) was noted at and above 500 μg/plate; this observation did not prevent the scoring of revertant colonies.
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2013 and 2014 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- There was no visible reduction in the growth of the bacterial background lawns at any dose level, either in the presence or absence of S9-mix, in the first mutation test (plate incorporation method) and consequently the same maximum dose level was originally selected for the second mutation test. In the initial second experiment, the toxicity of the test item yielded results that differed from Experiment 1 (due to a change in test methodology from plate incorporation to pre-incubation) and consequently, there were an insufficient number of non-toxic dose levels for the majority strains in both the absence and presence of S9-mix (the data is not given in this report). Therefore, a repeat second experiment was performed employing additional dose levels and an expanded dose range. In the second mutation test (pre-incubation method) the test item induced a stronger toxic response with weakened bacterial background lawns initially noted in the absence of S9-mix from 5 μg/plate (TA1535), 15 μg/plate (TA1537), 50 μg/plate (TA100) and 150 μg/plate (TA98 and WP2uvrA). In the presence of S9-mix weakened lawns were initially noted at 50 μg/plate (TA100 and TA1537) and 500 μg/plate (TA1535). No toxicity was noted to TA98 or WP2uvrA at any test item dose level in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The test material formulation, amino acid supplemented top agar and S9-mix used in this experiment were shown to be sterile.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

See the attached document for information on tables of results

Conclusions:

negative with metabolic activation
negative without metabolic activation

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA- according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA- were exposed to test material both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors).

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate in TA 100, TA 1535 and TA 1537 strains without S9-mix; 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate in TA 98 and WP2uvrA strains without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA 100, TA 1535 and TA 1537 strains with S9-mix; 15, 50, 150, 500, 1500 and 5000 μg/plate in TA 98 and WP2uvrA strains with S9-mix

Negative, vehicle (tetrahydrofuran) and positive control groups were also included in mutagenicity tests.

The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

There was no visible reduction in the growth of the bacterial background lawns at any dose level, either in the presence or absence of S9-mix, in the first mutation test (plate incorporation method) and consequently the same maximum dose level was originally selected for the second mutation test. In the second mutation test (pre-incubation method) the test item induced a stronger toxic response with weakened bacterial background lawns initially noted in the absence of S9-mix from 5 μg/plate (TA1535), 15 μg/plate (TA1537), 50 μg/plate (TA100) and 150 μg/plate (TA98 and WP2uvrA). In the presence of S9-mix weakened lawns were initially noted at 50 μg/plate (TA100 and TA1537) and 500 μg/plate (TA1535). No toxicity was noted to TA98 or WP2uvrA at any test item dose level in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (greasy and particulate in appearance) was noted at and above 500 μg/plate; this observation did not prevent the scoring of revertant colonies.

 

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the second mutation test (pre-incubation method). Small, statistically significant increases in revertant colony frequency were observed in the first Experiment at 15 μg/plate (TA98 dosed in the absence of S9-mix) and in the second experiment at 15 μg/plate (TA100 dosed in the presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.5 times the concurrent vehicle controls.

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA- according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Justification for type of information:

Concrete of Cistus ladaniferus (Cistaceae) obtained from stems and leaves by organic solvents extraction and Resinoid of Cistus ladaniferus (Cistaceae) obtained from labdanum gum by ethanol extraction are extracted from the same botanical plant: Cistus ladaniferus (Cistaceae)

Reason / purpose for cross-reference:

read-across source

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

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
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: None
- Precipitation: A test item precipitate (greasy and particulate in appearance) was noted at and above 500 μg/plate; this observation did not prevent the scoring of revertant colonies.
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2013 and 2014 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- There was no visible reduction in the growth of the bacterial background lawns at any dose level, either in the presence or absence of S9-mix, in the first mutation test (plate incorporation method) and consequently the same maximum dose level was originally selected for the second mutation test. In the initial second experiment, the toxicity of the test item yielded results that differed from Experiment 1 (due to a change in test methodology from plate incorporation to pre-incubation) and consequently, there were an insufficient number of non-toxic dose levels for the majority strains in both the absence and presence of S9-mix (the data is not given in this report). Therefore, a repeat second experiment was performed employing additional dose levels and an expanded dose range. In the second mutation test (pre-incubation method) the test item induced a stronger toxic response with weakened bacterial background lawns initially noted in the absence of S9-mix from 5 μg/plate (TA1535), 15 μg/plate (TA1537), 50 μg/plate (TA100) and 150 μg/plate (TA98 and WP2uvrA). In the presence of S9-mix weakened lawns were initially noted at 50 μg/plate (TA100 and TA1537) and 500 μg/plate (TA1535). No toxicity was noted to TA98 or WP2uvrA at any test item dose level in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The test material formulation, amino acid supplemented top agar and S9-mix used in this experiment were shown to be sterile.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

See the attached document for information on tables of results

Conclusions:

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA- according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP). Therefore the registered substance is considered as non mutagenic

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA- were exposed to test material both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors).

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate in TA 100, TA 1535 and TA 1537 strains without S9-mix; 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate in TA 98 and WP2uvrA strains without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA 100, TA 1535 and TA 1537 strains with S9-mix; 15, 50, 150, 500, 1500 and 5000 μg/plate in TA 98 and WP2uvrA strains with S9-mix

Negative, vehicle (tetrahydrofuran) and positive control groups were also included in mutagenicity tests.

The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

There was no visible reduction in the growth of the bacterial background lawns at any dose level, either in the presence or absence of S9-mix, in the first mutation test (plate incorporation method) and consequently the same maximum dose level was originally selected for the second mutation test. In the second mutation test (pre-incubation method) the test item induced a stronger toxic response with weakened bacterial background lawns initially noted in the absence of S9-mix from 5 μg/plate (TA1535), 15 μg/plate (TA1537), 50 μg/plate (TA100) and 150 μg/plate (TA98 and WP2uvrA). In the presence of S9-mix weakened lawns were initially noted at 50 μg/plate (TA100 and TA1537) and 500 μg/plate (TA1535). No toxicity was noted to TA98 or WP2uvrA at any test item dose level in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (greasy and particulate in appearance) was noted at and above 500 μg/plate; this observation did not prevent the scoring of revertant colonies.

 

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the second mutation test (pre-incubation method). Small, statistically significant increases in revertant colony frequency were observed in the first Experiment at 15 μg/plate (TA98 dosed in the absence of S9-mix) and in the second experiment at 15 μg/plate (TA100 dosed in the presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.5 times the concurrent vehicle controls.

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA- according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

Therefore the registered substance is considered as non mutagenic

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Justification for type of information:

Resinoid of Cistus ladaniferus (Cistaceae) obtained from labdanum gum by organic solvents extraction and Resinoid of Cistus ladaniferus (Cistaceae) obtained from labdanum gum by ethanol extraction are extracted from the same botanical plant: Cistus ladaniferus (Cistaceae)

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cf. Table of results

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: immiscible
- Precipitation: A test item precipitate (greasy in appearance with red flecks ) was noted at and abo
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA:
Not needed (no statistical significant increase were noted)
The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the bacterial tester strains initially from 15 μg/plate (TA100), 50 μg/plate (TA1535), 150 μg/plate (TA98) and 500 μg/plate (WP2uvrA and TA1537) in the absence of S9-mix and to three bacterial strains initially from 500 μg/plate (TA1537), 1500 μg/plate (TA100) and 5000 μg/plate (TA1535) in the presence S9-mix. Consequently, the toxic limit or the maximum recommended dose (5000 μg/plate) of the test item was employed as the maximum dose in the second mutation test, depending on bacterial tester strain type and presence or absence of S9-mix. In the second mutation test, the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 15 μg/plate (TA100), 50 μg/plate (TA1535), 150 μg/plate (TA98 and TA1537) and 500 μg/plate (WP2uvrA). In the presence S9-mix weakened bacterial background lawns were noted to three of the bacterial tester strains from 1500 μg/plate (TA1537) and at 5000 μg/plate (TA1535 and TA100). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experiment.

CONCLUSION:
There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2.

Remarks on result:

other:

Remarks:

Table of results are in "Attached background documents"

Cf Tables of results in attached background material

Conclusions:

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA. Therefore the registered substance is not considered to be mutagenic

Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item diluted in Tetrahydrofuran both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the Ames pre‑incubation methods in Experiment 1 and 2.

The original dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. However, the test item induced excessive toxicity to Salmonella strains TA100 and TA1535 (absence of S9 only) and consequently part of the experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations and employing an amended dose range of 0.05 to 150 μg/plate. The second experiment was performed at a later date again using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 (and the repeat assay) and ranged between 0.05 and 5000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix. Up to seven test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

 

The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the bacterial tester strains initially from 15 μg/plate (TA100), 50 μg/plate (TA1535), 150 μg/plate (TA98) and 500 μg/plate (WP2uvrA and TA1537) in the absence of S9-mix and to three bacterial strains initially from 500 μg/plate (TA1537), 1500 μg/plate (TA100) and 5000 μg/plate (TA1535) in the presence S9-mix. Consequently, the toxic limit or the maximum recommended dose (5000 μg/plate) of the test item was employed as the maximum dose in the second mutation test, depending on bacterial tester strain type and presence or absence of S9-mix. In the second mutation test, the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 15 μg/plate (TA100), 50 μg/plate (TA1535), 150 μg/plate (TA98 and TA1537) and 500 μg/plate (WP2uvrA). In the presence S9-mix weakened bacterial background lawns were noted to three of the bacterial tester strains from 1500 μg/plate (TA1537) and at 5000 μg/plate (TA1535 and TA100). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experiment.

A test item precipitate (greasy in appearance with red flecks ) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2.

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.

  Therefore the registered substance is not considered to be mutagenic

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test on Labdanum resinoid:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item diluted in Tetrahydrofuran both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the Ames pre‑incubation methods in Experiment 1 and 2.

In the first mutation test, the test item caused a visible reduction in the growth of the bacterial background lawns of all of the bacterial tester strains initially from 15 μg/plate (TA100), 50 μg/plate (TA1535), 150 μg/plate (TA98) and 500 μg/plate (WP2uvrA and TA1537) in the absence of S9-mix and to three bacterial strains initially from 500 μg/plate (TA1537), 1500 μg/plate (TA100) and 5000 μg/plate (TA1535) in the presence S9-mix. Consequently, the toxic limit or the maximum recommended dose (5000 μg/plate) of the test item was employed as the maximum dose in the second mutation test, depending on bacterial tester strain type and presence or absence of S9-mix. In the second mutation test, the test item again induced a toxic response with weakened bacterial background lawns noted in the absence of S9-mix from 15 μg/plate (TA100), 50 μg/plate (TA1535), 150 μg/plate (TA98 and TA1537) and 500 μg/plate (WP2uvrA). In the presence S9-mix weakened bacterial background lawns were noted to three of the bacterial tester strains from 1500 μg/plate (TA1537) and at 5000 μg/plate (TA1535 and TA100). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experiment.

A test item precipitate (greasy in appearance with red flecks ) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.

There were no increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1. Similarly, no increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2.

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.

 

Ames test on cistus concrete

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP,Salmonella typhimuriumstrains TA 1535, TA 1537, TA 98 and TA 100 andEscherichia colistrain WP2 uvrA- were exposed to test material both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors).

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate in TA 100, TA 1535 and TA 1537 strains without S9-mix; 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate in TA 98 and WP2uvrA strains without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA 100, TA 1535 and TA 1537 strains with S9-mix; 15, 50, 150, 500, 1500 and 5000 μg/plate in TA 98 and WP2uvrA strains with S9-mix

Negative, vehicle (tetrahydrofuran) and positive control groups were also included in mutagenicity tests.

The vehicle control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

There was no visible reduction in the growth of the bacterial background lawns at any dose level, either in the presence or absence of S9-mix, in the first mutation test (plate incorporation method) and consequently the same maximum dose level was originally selected for the second mutation test. In the second mutation test (pre-incubation method) the test item induced a stronger toxic response with weakened bacterial background lawns initially noted in the absence of S9-mix from 5 μg/plate (TA1535), 15 μg/plate (TA1537), 50 μg/plate (TA100) and 150 μg/plate (TA98 and WP2uvrA). In the presence of S9-mix weakened lawns were initially noted at 50 μg/plate (TA100 and TA1537) and 500 μg/plate (TA1535). No toxicity was noted to TA98 or WP2uvrA at any test item dose level in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (greasy and particulate in appearance) was noted at and above 500 μg/plate; this observation did not prevent the scoring of revertant colonies.

 

There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation in the second mutation test (pre-incubation method). Small, statistically significant increases in revertant colony frequency were observed in the first Experiment at 15 μg/plate (TA98 dosed in the absence of S9-mix) and in the second experiment at 15 μg/plate (TA100 dosed in the presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.5 times the concurrent vehicle controls.

 

Under the test condition, test material is not mutagenic with and without metabolic activation inS. typhimurium(strains TA1535, TA1537, TA98 and TA100) andE.coliWP2 uvrA- according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding germ cell mutagenicity according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonised System of classification and labelling of chemicals (GHS).