Absolute of Cistus ladaniferus (Cistaceae) obtained from labdanum concrete by ethanol extraction

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to limit or cytotoxic concentration in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.

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:

From July 14 to August 17, 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.

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 2 (Pre-Incubation Method):
Salmonella strain TA100 (absence of S9): 5, 15, 50, 150, 500, 1500 µg/plate.
Salmonella strains TA1535 and TA98 (absence of S9): 15, 50, 150, 500, 1500, 5000 µg/plate.
E.coli strain WP2uvrA and Salmonella strain TA1537 (absence of S9): 1.5, 5, 15, 50, 150, 500, 1500 µg/plate.
Salmonella strains TA100, TA98 and TA1537 and E.coli strain WP2uvrA (presence of S9): 15, 50, 150, 500, 1500, 5000 μg/plate.
Salmonella strain TA1535 (presence of S9): 50, 150, 1000, 1500, 3000, 5000 μg/plate.

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.

For Salmonella strain TA1535 (dosed in the presence of S9) intermediate dose levels (1000 and 3000 μg/plate) were included to attain a better dose-response relationship following the observation of small increases in colony frequency in Experiment 1.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Acetone
- Justification for choice of solvent/vehicle: In solubility checks performed in house the test item was noted as immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but fully miscible in acetone at 100 mg/mL. Acetone was therefore selected as the vehicle.
- Preparation of test materials: The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in acetone by mixing on a vortex mixer. No correction was required for purity allowance. Acetone is toxic to the bacterial cells at 0.1 mL (100 µL) after employing the pre-incubation modification; therefore all of the formulations were prepared at concentrations two times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 0.05 mL (50 µL) aliquots (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:

Acetone

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:

Acetone

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 scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity). Several manual counts were required due to revertant colonies spreading slightly, thus distorting the actual plate count.

Rationale for test conditions:

Experiment 1 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
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.
- 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:

Exp1:-S9: 500 µg/plate (WP2uvrA, TA1537), 1500 µg/plate (TA100), 5000 µg/plate (TA 1535, TA98).+S9: 5000 µg/plate (TA1537) Exp2:-S9: 1500 µg/plate (TA1535, TA98, WP2uvrA, TA1537), 5000 µg/plate (TA100).+S9: 3000 µg/plate (TA1535), 5000 µg/plate (TA1537)

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: In solubility checks performed in house the test item was noted as immiscible in sterile distilled water and dimethyl sulphoxide at 50 mg/mL but fully miscible in acetone at 100 mg/mL.
- Precipitation: A test item precipitate (light and globular in appearance) was noted at 5000 g/plate, this observation did not prevent the scoring of revertant colonies.
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA:
Not needed (no statistical significant increase were noted)
The vehicle (acetone) 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 from 150 µg/plate (TA1535, WP2uvrA and TA1537), 500 µg/plate (TA100) and 1500 µg/plate (TA98) in the absence of S9 mix and to three bacterial strains (TA100, TA1537 and TA1535) at 5000 µg/plate in the presence of 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 150 µg/plate (TA1535, WP2uvrA and TA1537), 500 µg/plate (TA100) and 1500 µg/plate (TA98). In the presence of S9-mix weakened bacterial background lawns were noted to two of the bacterial tester strains at 5000 µg/plate (TA1535 and TA1537). 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 biologically relevant 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 biologically relevant 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.

Small increases in TA1535 revertant colony frequency were observed in the presence of S9 mix at 1500 and 5000 µg/plate in Experiment 1. The mean values were in excess of twofold the concurrent vehicle control and some individual revertant counts were just in excess of the maximum in-house untreated/vehicle historical control value for the strain, therefore the results for TA1535 were investigated further in Experiment 2. The small increases in revertant colony frequency observed with TA1535 in Experiment 1 were considered to be of no biological relevance even though some individual counts were marginally above the in-house untreated/vehicle historical control range for the strain. There was no clear dose-response relationship (the increases were very flat over the two dose levels) and the results could not be reproduced in the second experiment which contained extra intermediate test item concentration levels, in an attempt to qualify the response. Furthermore, the test item exhibited variable toxicity under certain circumstances (weakened bacterial background lawns were noted at 3000 and 5000 μg/plate in Experiment 2) and although, weakened lawns weren’t noted to the tester strain in Experiment1, the increases noted at 1500 and 5000 μg/plate may have been an artefact resulting from a modest level of toxicity to the tester strain at the upper test item dose levels i.e. enough weakening of the background lawns (toxicity) to induce a ‘false’ response.

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 acetone 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 dose range for Experiment 1 was predetermined and was 1.5 to 5000 mg/plate. The experiment was repeated on a separate day using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and ranged between 1.5 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. Up to seven test item concentrations 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 (acetone) 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 from 500 μg/plate (WP2uvrA and TA1537), 1500 μg/plate (TA100) and 5000 μg/plate (TA1535 and TA98) in the absence of S9-mix and to one bacterial strain (TA1537) at 5000 μg/plate 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 1500 μg/plate (TA1535, WP2uvrA, TA98 and TA1537) and 5000 μg/plate (TA100). In the presence S9-mix weakened bacterial background lawns were noted to two of the bacterial tester strains from 3000 μg/plate (TA1535) and at 5000 μg/plate (TA1537). 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 (light and globular in appearance) was noted at 5000 µg/plate, this observation did not prevent the scoring of revertant colonies.

There were no biologically relevant 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 biologically relevant 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. Small increases in TA1535 revertant colony frequency were observed in the presence of S9‑mix at 1500 and 5000 µg/plate in Experiment 1. These increases were considered to have no biological relevance because there was no clear dose-response relationship (the increases were very flat over the two dose levels) and the results could not be reproduced in the second experiment which contained extra intermediate test item concentration levels, in an attempt to qualify the response. Furthermore, the test item exhibited variable toxicity under certain circumstances (weakened bacterial background lawns were noted at 3000 and 5000 μg/plate in Experiment 2) and although, weakened lawns weren’t noted to the tester strain in Experiment1, the increases noted at 1500 and 5000 μg/plate may have been an artefact resulting from a modest level of toxicity to the tester strain at the upper test item dose levels i.e. enough weakening of the background lawns (toxicity) to induce a ‘false’ response.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Table 7.6/1: Summary of genotoxicity tests

Test n°	Test / Guideline Reliability	Focus	Strains tested	Metabolic activation	Test concentration	Statement
1   Envigo, 2017	Ames Test (OECD 471) K, rel. 1	Gene mutation	TA 1535, TA 1537, TA 98, TA 100 E. coli WP2uvrA	-S9 +S9	Up to limit or cytotoxic concentration	-S9 : non mutagenic +S9 : non mutagenic

Gene mutation Assays (Tests n° 1):

A Bacterial Reverse mutation Assay (Ames test) was performed according to OECD guideline No. 471 with the substance (Test n°1, see Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies. The substance is therefore considered as non-mutagenic according to the Ames test.

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).