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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An in vitro bacterial reverse mutation assay (Ames Test) was performed using Salmonella typhimurium strains TA 1535,TA1537, TA98 and TA 100 and Escherichia coli stain WP2uvrA using both plate pre-incubation and incorporation techniques. Phenol, ethoxylated is considered to be non-mutagenic under the conditions of this test.

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:
04th September 2019 - 27th September 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
See 'Principles of method if other than guideline'
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
See 'Principles of method if other than guideline'
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
yes
Remarks:
See 'Principles of method if other than guideline'
Qualifier:
according to
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
yes
Remarks:
See 'Principles of method if other than guideline'
Qualifier:
according to
Guideline:
other: ICH S2(R1) guideline
Version / remarks:
June 2012
Principles of method if other than guideline:
The following deviations have been noted as follows: All testing was performed using a test item purity value of 99.36%. As this value exceeded 99%, a dose formulation allowance was not made. Upon completion of the study, a new purity value of 98.31% was provided. Applying this value to the dose concentrations used in Experiments 1 and 2 lowered the maxima values, with the result that the maximum recommended dose has not been achieved. A correction factor has therefore been applied to all of the stated test item concentrations. The corrected values are the reported values.
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine locus in the genome of Salmonella typhimurium and tryptophan locus in the genome of Escherichia coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system: Rat liver homogenate metabolizing system (10% liver S9 in standard co-factors).
- source of S9 : The S9 Microsomal fractions (Sprague-Dawley) were purchased from Moltox; Lot No. 4123
- method of preparation of S9 mix: A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.
Test concentrations with justification for top dose:
Experiment 1: 1.48, 4.9, 14.8, 49, 148, 493, 1,478 and 4,927 μg/plate.
Experiment 2: 14.8, 49, 148, 492, 1475 and 4918 μg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO

- Justification for choice of solvent/vehicle: Test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-Aminoanthracene, Benzo[a]pyrene (BP)
Details on test system and experimental conditions:
The test item was immiscible in sterile distilled water at 50 mg/mL but was fully miscible in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.The test item was accurately weighed and, on the day of each experiment, approximate half-log dilutions prepared in dry dimethyl sulphoxide by mixing on a vortex mixer. No correction for purity was originally required. However, on completion of the study, the Sponsor supplied a new purity value of 98.31%, therefore formulation concentrations have been retrospectively amended. All formulations were used within four hours of preparation and were assumed to be stable for this period. Analysis for concentration, homogeneity and stability of the test item formulations is not a requirement of the test guidelines and was, therefore, not determined. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Test for Mutagenicity: Experiment 1 - Plate Incorporation Method
- Dose selection:
The test item was tested using the following method. The maximum concentration was 4927 μg/plate (the OECD TG 471 maximum recommended dose level was not achieved due to a purity change on completion of the study). Eight concentrations of the test item (1.48, 4.9, 14.8, 49, 148, 493, 1478 and 4927* μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.
-Without Metabolic Activation:
0.1 mL aliquot of the appropriate concentration of test item, solvent vehicle or 0.1 mL of the appropriate positive control was added together with 0.1 mL of the bacterial strain culture, 0.5 mL of phosphate buffer and 2 mL of molten, trace amino-acid supplemented media. These were then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls were also performed on the same day as the mutation test. Each concentration of the test item, appropriate positive, vehicle and negative controls, and each bacterial strain, was assayed using triplicate plates.
-With Metabolic Activation:
The procedure was the same as described previously except that following the addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the molten, trace amino-acid supplemented media instead of phosphate buffer.
-Incubation and Scoring:
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity)

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As the result of Experiment 1 was considered negative, Experiment 2 was performed using the pre-incubation method in the presence and absence of metabolic activation (S9-mix).
- Dose selection:
The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 14.8, 49, 148, 492, 1475 and 4918 μg/plate.Six test item concentrations per bacterial strain were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxicity of the test item following the change in test methodology from plate incorporation to pre-incubation.
-Without Metabolic Activation:
A 0.1 mL aliquot of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of the appropriate concentration of test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten, trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates. Negative (untreated) controls were also performed on the same day as the mutation test employing the plate incorporation method. All testing for this experiment was performed in triplicate.
-With Metabolic Activation:
The procedure was the same as described previously (see 3.3.3.2) except that following the addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with shaking) and addition of molten, trace amino-acid supplemented media. All testing for this experiment was performed in triplicate.
-Incubation and Scoring:
All of the plates were incubated at 37 ± 3 °C for between 48 and 72 hours and scored for the presence of revertant colonies using an automated colony counting system. The plates were viewed microscopically for evidence of thinning (toxicity).
Rationale for test conditions:
In accordance with the OECD Testing Guideline 471.
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:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. A fold increase greater than two times the concurrent solvent control for TA100, TA98 and WP2uvrA or a three-fold increase for TA1535 and TA1537 (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
5. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
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 significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control. Values that are statistically significant but are within the in-house historical vehicle/untreated control range were not reported.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
One statistically significant value was noted (WP2uvrA at 1475* μg/plate in the presence of metabolic activation (S9-mix); not identified as statistically significant on Table 5, as per the requirements of the Study Plan), however, this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.
Conclusions:
No significant increases in the frequency of revertant colonies for any of the bacetrial strains were recorded. Phenol, ethoxylated was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The potential of Phenol, ethoxylated to induce gene mutation in bacteria was assessed using a GLP-compliant study performed in accordance with the OECD Testing Guideline 471. Experiments were performed with and without metabolic activation using S9-mix. The bacterial strains used were Salmonella typhimurium, TA98, TA100, TA1535 and TA1537 and Escherichia coli WP2uvrA. Dimethyl sulphoxide was selected as a vehicle and used for negative control, yielding counts of revertant colonies within the normal range. All the positive controls used induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Therefore, the sensitivity of the assay and efficacy of the S9-mix were considered as valid.

Testing was performed using a test item purity value of 99.36%. As this value exceeded 99%, a dose formulation allowance was not made. Upon completion of the study, the Sponsor supplied a new purity value of 98.31%. Application of this value to the dose concentrations used in Experiments 1 and 2 lowered the maxima values, resulting in the maximum recommended dose not being achieved. A correction factor was applied to all of the stated test item concentrations. The corrected values are reported. Plate incorporation method (experiment 1) and pre-incubation method (experiment 2) were used. The maximum dose level of the test item in experiment 1 was 4,927 μg/plate based on the results of experiment 1 the same maximum dose of 4,918 ug/plate was employed in experiment 2.

In the first experiment (plate incorporation method), no visible reduction in the growth of the bacterial background lawn and no significant increases in the frequency of revertant colonies at any dose level both with or without metabolic activation (S9-mix) were noted. In experiment 2 there was no visible reduction in the growth of the bacterial background lawn or biologically relevant increases in the frequency of revertant colonies noted to any of the tester strains at any dose level, either in the presence or absence of metabolic activation (S9-mix). No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of metabolic activation (S9-mix) in Experiments 1 and 2. One statistically significant value was noted (WP2uvrAat 1475 μg/plate in the presence of metabolic activation (S9-mix), however, this response was within the in-house historical vehicle/untreated control range for the strain and was, therefore considered of no biological relevance.

It is therefore concluded that Phenol, ethoxylated was not mutagenic under the conditions of this test.

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

Additional information

Justification for classification or non-classification

The substance does not meet the criteria for classification as mutagenic based on the available experimental data.