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

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

15 october- January 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

947-724-1

Molecular formula:

not applicable

IUPAC Name:

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

Test material form:

cream / paste

Details on test material:

Brown solid slightly pasty

Method

Target gene:

Histidine and tryptophan for S. typhimurium and E. coli, respectively.

Species / strain

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:

Obtained from Molecular Toxicology Incorporated, Boone, NC, USA (MolTox™) and prepared according to the method of Ames et al from male Sprague Dawley rats induced with a single dose of Aroclor 1254

Test concentrations with justification for top dose:

In order to determine the toxicity of the test item to the test bacteria, an initial plate incorporation experiment was carried out using TA98 and WP2 uvrA in the presence and absence of S 9 mix. The dose levels used were 1.5; 5; 15; 50; 150; 500; 1500 or 5000 µL/plate. Based on the range finder observations, dose levels for the main experiment were chosen to include the guideline regulatory maximum dose level of 5 µL/plate.

Vehicle / solvent:

Dimethylsulphoxide (DMSO), a commonly used vehicle for which extensive historical control data is available; at concentrations up to 50 mg/mL.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

Salmonella typhimurium and Escherichia coli strains were obtained from Molecular Toxicology Incorporated, Boone, NC, USA (MolTox™) and used in the Bacterial Reverse Mutation test.

The Salmonella typhimurium and Escherichia coli strains are defective in DNA repair (Δuvr B , A- respectively); this confers extra sensitivity to DNA damage. The Salmonella strains have a defective lipopolysaccharide barrier on the cell wall (rfa-) which affords greater permeability to large molecules. The strains TA98 and TA100 also contain a plasmid (pKM101) which enhances error prone repair and confers ampicillin resistance.

The strains are tested routinely for:

1. Auxotrophic control of growth, (histidine or tryptophan dependence).

2. Deep rough mutation (rfa) of the bacterial cell wall (cell membrane permeability, crystal violet sensitivity).

3. Plasmid presence (Ampicillin resistance).

4. Absence of uvrA or uvrB repair enzyme systems (sensitivity to UV irradiation).

TA1535, TA100 and WP2 uvrA are reverted to prototrophy by base substitution mutagens. TA1537 and TA98 are reverted by frame shift mutagens; TA100 can also be affected by these on occasion.

Before the experiment, 25 mL quantities of nutrient broth were inoculated with 100 µL from a freshly thawed vial of the appropriate strain, and incubated overnight at 37 °C to give a pure culture at approximately 10^9 cells per mL.

Rationale for test conditions:

Range finder: As all the Salmonella Escherichia strains show a similar toxic response to most chemicals, only one strain of each species, typically TA98 and WP2 uvrA, was tested by plate incorporation in this experiment. A range of concentrations up to a maximum of 5000 μg/plate was used, with duplicate plates at each concentration, both with and without S-9. The plates examined for integrity of the background lawns after approximately 48 hours ± 1 hour incubation. Experiment I: Mutation experiment, plate incorporation : The mutation experiment was carried out using fresh bacterial cultures for each experiment. The test item was tested at sufficient concentration levels (table1) to provide at least 5 analysable concentrations, with triplicate Petri dishes prepared for each control and experimental point. The following was added to sterile disposable tubes containing 2 mL of L-histidine: D-biotin (Salmonella strains) or L-tryptophan (Escherichia strain) supplemented top agar. The contents of each tube was mixed and added to a Petri dish containing minimal agar. When the top agar has set, the Petri dishes will be inverted and incubated at 37 °C ± 3 °C for approximately 66 hours ± 1 hour. Experiment II: Mutation experiment, preincubation: The mutation experiment was carried out using fresh bacterial cultures for each experiment. The test item was tested at sufficient concentration levels (table2) to provide 5 analysable concentrations, with triplicate Petri dishes prepared for each control and experimental point. Each tube was fitted with a sterile cap and incubated at approximately 37 °C with shaking, for 20 minutes. At the end of this period, 2 mL of top agar (L-histidine: D-biotin for Salmonella strains or L-tryptophan for Escherichia strain) was added to each tube, the contents mixed and added to Petri dishes containing minimal agar. When the top agar has set, the Petri dishes are inverted and incubated at 37 °C ± 3 °C for approximately 66 hours ± 1 hour.

Evaluation criteria:

A test was considered to be positive if the test item induced dose related statistically significant increases in numbers of revertants over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system, compared with negative Controls scored in 2 separate experiments. Biological relevance of the result should be considered. Statistical significance should not be the only determining factor for a positive response. A test was considered to be negative if the test item produced no greater increases in revertants, than may be expected from normal variation in the negative Control number of revertants, for any strain, in either experiment

Statistics:

Mean, standard deviation (s.d.) and Dunnett's t-statistic (t) were calculated for each group and bacterial strain

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium 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

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 5

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

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

BACTERIAL REVERSE MUTATION TEST - EXPERIMENT 1

All negative Controls gave mean counts of spontaneous revertant colonies within historical negative Control ranges obtained in this laboratory.
All positive Controls induced marked increases in the number of revertant colonies and gave mean counts of induced revertant colonies which were within expected historical positive Control ranges. The response to the positive Controls demonstrate that the bacteria were sensitive to the mutagens and that the S-9 mix was able to metabolise the pro-mutagen, 2AA, to a mutagen.
Test item was analysed up to a dose level demonstrating toxicity of 500 μg/plate in TA100 in the absence of S-9 mix, 1500 μg/plate in TA1535, TA1537 and TA98 in the absence of S-9 mix, 3000 μg/plate in TA1537 in the presence of S-9 mix and up to the regulatory maximum dose level of 5000 μg/plate in TA1535, TA98 and TA100 in the presence of S-9 mix and WP2 uvrA in the presence and absence of S-9 mix. Precipitation was noted at 1500 and 3000 μg/plate in TA1537 in the presence of S-9 mix and 5000 μg/plate in TA1535, TA98 and TA100 in the presence of S-9 mix and WP2 uvrA in the presence and absence of S-9 mix.
Toxicity was demonstrated by a slightly reduced background bacterial lawn at 500 μg/plate in TA100 in the absence of S-9 mix, 1500 μg/plate in TA1535, TA1537 and TA98 in the absence of S-9 mix, 3000 μg/plate in TA1537 in the presence of S-9 mix, 5000 μg/plate in TA1535, TA98 and TA100 in the presence of S-9 mix and by a reduced background bacterial lawn at 1500 μg/plate in TA100 and 3000 μg/plate in TA98 in the absence of S-9 mix. There was a reduction (less than 0.5 times that of the negative Control value) in the mean colony counts at
1500 μg/plate in TA1537 and at 5000 μg/plate in WP2 uvrA in the absence of S-9 mix and at 5000 μg/plate in TA100 in the presence of S-9 mix, indicating toxicity of the test item to the bacteria.
There were no increases in revertant numbers, greater than the defined fold-increases (twice the negative Control value for TA98, TA100 or WP2 uvrA or three times the negative Control value for TA1535 or TA1537), compared with the relevant negative Control values in any strain at any dose level of test item, in the presence or absence of S-9 mix, under plate incorporation conditions.

BACTERIAL REVERSE MUTATION TEST - EXPERIMENT 2

All negative Controls gave mean counts of spontaneous revertants within expected ranges obtained in this laboratory.
All positive Controls gave mean counts of induced revertants within historical positive Control ranges obtained in this laboratory. The response to the positive Controls indicated that the bacteria were sensitive to the mutagens and that the S-9 mix was able to metabolise the pro-mutagen, 2AA, to a mutagen.
The test item was tested up to a dose level demonstrating toxicity of 3000 μg/plate in TA1535, TA1537, TA98 and TA100 in the absence of S-9 mix and up to the regulatory maximum dose level of 5000 μg/plate in TA1535, TA1537, TA98 and TA100 in the presence of S-9 mix and WP2 uvrA in the presence and absence of S-9 mix. Precipitation was noted at 3000 μg/plate in TA1537 and TA100 in the absence of S-9 mix and 1500 and 5000 μg/plate in all strains in the presence of S-9 mix and at 5000 μg/plate in WP2 uvrA in the absence of S-9 mix.
Toxicity was demonstrated by a slightly reduced background bacterial lawn at 1500 μg/plate in TA1537 and TA100 in the absence of S-9 mix, at 3000 μg/plate in all Salmonella strains in the absence of S-9 mix and at 5000 μg/plate in TA1537 in the presence of S-9 mix. There was a reduction (less than 0.5 times that of the negative Control value) in the mean colony counts at 3000 μg/plate in TA1537 in the absence of S-9 mix and 5000 μg/plate in TA1535 and
TA1537 in the presence of S-9 mix, indicating toxicity of the test item to the bacteria. There were no increases in revertant numbers, greater than the defined fold-increases (twice the negative Control value for TA98, TA100 or WP2 uvrA or three times the negative Control value for TA1535 or TA1537), compared with the relevant negative Control values in any strain at any dose level of the test item, in the presence or absence of S-9 mix, under pre-incubation conditions.

Applicant's summary and conclusion

Conclusions:

The test item showed no mutagenic potential in the Bacterial Reverse Mutation Assay under the conditions of this test

Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA were exposed to the test item.

Rat liver homogenate (10% liver S9 in standard co-factors) was used as a metabolizing system. Vehicle control, negative (untreated) and positive control groups were also included in mutagenicity tests.

The vehicle (dimethyl sulphoxide) 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 were no increases in revertant numbers greater than twice the negative Control value for TA98, TA100 or WP2uvrAor greater than three times the negative Control value for TA1535 or TA1537 observed at any dose level of, in the presence or absence of S‑9 mix, under either plate incorporation or pre-incubation conditions

Under the test conditions, the test item is not considered as mutagenic in these bacterial systems.