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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro bacterial reverse mutation (with and without S-9 activation): Negative; OECD 471; Schultz, M. (2018)

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:
15 - 28 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The study was conducted according to GLP following the most recent guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 21 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Version / remarks:
The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labour and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 31 March 2011
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: Fully miscible in DMSO (dimethyl sulfoxide) and assumed stable.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: no

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment.
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: Concentration of stock solution = 50 mg/mL.
- Final preparation of a solid: n/a

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: No
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Pre- Experiment/Experiment I: Salmonella strains & E. coli (presence and absence of S-9 mix) - 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Experiment II: Salmonella strains & E. coli (presence and absence of S-9 mix) - 33; 100; 333; 1000; 2500; and 5000 μg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: guideline recommended
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION:plate incorporation and preincubation

DURATION
- Preincubation period: 20 mins
- Exposure duration: 48 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): n/a
- Fixation time (start of exposure up to fixation or harvest of cells): n/a

SELECTION AGENT (mutation assays): n/a

SPINDLE INHIBITOR (cytogenetic assays): n/a

STAIN (for cytogenetic assays): n/a

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: n/a

NUMBER OF CELLS EVALUATED: n/a

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): n/a

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: n/a

DETERMINATION OF CYTOTOXICITY
- Method: presence of bacterial lawn
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:n/a
- Determination of endoreplication: n/a
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): n/a

- OTHER:
Rationale for test conditions:
The study was based on the in vitro technique described by Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item. These strains have a deleted excision repair mechanism which makes them more sensitive to various mutagens and they will not grow on media which does not contain histidine. When large numbers of these organisms are exposed to a mutagen, reverse mutation to the original histidine independent form takes place. These are readily detectable due to their ability to grow on a histidine deficient medium. Using these strains of Salmonella typhimurium revertants may be produced after exposure to a chemical mutagen, which have arisen as a result of a base-pair substitution in the genetic material (miscoding) or as a frameshift mutation in which genetic material is either added or deleted. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base substitution to tryptophan independence (Green and Muriel (1976)) is used to complement the Salmonella strains.
Evaluation criteria:
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of twofold or above (strains TA 98, TA 100, and WP2 uvrA) or threefold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
SA statistical analysis of the data was not mandatory
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
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:
no cytotoxicity, but tested up to precipitating concentrations
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 examined
- Effects of osmolality: not examined
- Evaporation from medium: not examined
- Water solubility: not examined
- Precipitation: none
- Definition of acceptable cells for analysis: All bacterial strains must have demonstrated the required characteristics as determined by their respective strain checks according to Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000).
- Other confounding effects: no

RANGE-FINDING/SCREENING STUDIES: In the range-finding test (plate incorporation method) the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains, initially at 1500 μg/plate in the absence and presence of S9-mix.

CYTOKINESIS BLOCK (if used)
- Distribution of mono-, bi- and multi-nucleated cells: n/a

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: n/a
- Indication whether binucleate or mononucleate where appropriate: n/a

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: No
- Negative (solvent/vehicle) historical control data: Yes (2009 - 2010 data)

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: presence of bacterial lawn
- Other observations when applicable: no
Conclusions:
The test item was considered to be non-mutagenic under the conditions of the test.
Executive summary:

OECD 471 - 2018:  The test item AD-464 was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA with and without metabolic activation.

Each concentration and the controls were tested in triplicate.;

Pre-Experiment/Experiment I: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II: 33; 100; 333; 1000; 2500; and 5000 μg/plate

Precipitation was observed at 1000 to 5000 μg/plate and 2500 to 5000 μg/plate in pre-incubation and incubation. This did not affect data documentation.

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.

No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation, except in strain TA 100 in the absence of metabolic activation.


In detail, in TA 100 the number of revertants was strongly reduced as indication of bacteriotoxicity in experiment I in the absence of S9 mix at 5000 μg/plate and in experiment II in the absence of S9 mix from 2500 μg/plate onward.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with AD-464 at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix).

There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

Based on the condition of the study, the test item was considered to be non-mutagenic.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

OECD 471 (2018): The test item AD-464 was assessed for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA with and without metabolic activation at concentration at up to 5000 μg/plate.

Precipitation was observed at 1000 to 5000 μg/plate and 2500 to 5000 μg/plate in pre-incubation and incubation. This did not affect data documentation.

In the TA 100  tester strains the number of revertants was strongly reduced as indication of bacteriotoxicity in experiment I in the absence of S9 mix at 5000 μg/plate and in experiment II in the absence of S9 mix from 2500 μg/plate onward.  No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with AD-464 at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix).

There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Based on the condition of the study, the test item was considered to be non-mutagenic.

However, please see below for comment on classification.

For mutagenicity, a single study was carried out (Ames) and found to be negative however, this was considered insufficient to conclude classification in view of a minor constituent in the test item.

According to Regulation (EC) No. 1272/2008 (CLP) principles a classification is made for mutagenicity considering the weight percent of the constituent phenol within the test item that has a harmonised classification for this hazard.

Justification for classification or non-classification

A negative result was obtained for germ cell mutagenicity during an Ames (OECD 471) test on the test item. However, the test item contains phenol. Phenol also resulted in a negative during anAmes test but is classified as a mutagen based on effects seen in a OECD 473 study and OECD 487. Therefore, the negative result seen for the test item here may be a false negative as the content of phenol in the mixture is above the generic concentration limit for mutagenicity. Therefore, further studies would be required exploring other modes of action such as chromosome abberation, in order to conclude on this test item not being a mutagen.

According to Regulation (EC) No. 1272/2008 (CLP) as the weight percent of phenol is > 1 % in the test item and as phenol has a harmonised classification for this hazard of Mut. 2 H341, the test item should considered a Mut. 2, H341.