2-ethoxy-4-(hydroxymethyl)phenol

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:

Experimental starting date: 18 June 2013 Experimental completion date: 25 July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-ethoxy-4-(hydroxymethyl)phenol

EC Number:

674-192-5

Cas Number:

4912-58-7

Molecular formula:

C9H12O3

IUPAC Name:

2-ethoxy-4-(hydroxymethyl)phenol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Name: 2-ethoxy-4-(hydroxymethyl)phenol
- CAS number: 4912-58-7

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature (from 1 to 30°C), light shading
- Stability under test conditions: stable at room temperature
- Solubility and stability of the test substance in the solvent/vehicle: hardly soluble in water at 50 mg/mL, soluble in dimethylsulfoxide and acetone

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Time of preparation: prior to use (weighing of the test substance was performed on the day before preparation)
Purity conversion: not converted
Preparation method: In the dose-finding study, 227.80 mg of the test substance was weighed and 4.556 mL of dimethylsulfoxide was added and the test substance was dissolved to prepare a 50 mg/mL test solution. In the main study, 398.91 mg of the test substance was weighed and 7.978 mL of dimehtylsulfoxide was added and the test substance was dissolved to prepare a 50 mg/mL test solution. In the confirmatory study, 369.53 mg of the test substance was weighed and 7.391 mL of dimethylsulfoxide was added and the test substance was dissolved to prepare a 50 mg/mL test solution. Dimethylsulfoxide was previously dehydrated with molecular sieve. In the dose-finding study, total five lower concentrations were prepared by dilution of the 50 mg/mL solution (highest concentration) with dimethylsilfoxide. In the main and confirmatory studies, total four lower concentrations were prepared.

Method

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 specified

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

313, 625, 1250, 2500 and 5000 µg/plate (with and without metabolic activation
In the dose-finding study, six doses including 5000 µg/plate as the highest dose obtained using a common ratio of 4 were tested. The test substance showed neither growth-inhibition nor dose-dependent increase in the number of revertant colonies in any bacterial strains at any dose levels regardless of the presence or absence of metabolic activation. Precipitation of the test substance was not observed at any dose levels in both the presence and absence of metabolic activation. Therefore, in the main and confirmatory studies, total five doses including 5000 µg/plate as the highest dose obtained using a common ratio of 2 were tested.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylsulfoxide
- Justification for choice of solvent/vehicle: Dimethylsulfoxide was selected as a solvent because the test substance was hardly soluble in water at 50 mg/mL but soluble in dimethylsulfoxide, and the stability in dimethylsulfoxide if the test substance was confirmed.

Controls

Untreated negative controls:

yes

Remarks:

diemthylsulfoxide (Lot #: PDM2827)

Negative solvent / vehicle controls:

not specified

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9-aminoacridine

sodium azide

furylfuramide

other: 2-aminoanthracene (2AA)

Remarks:

Dissolved in dimethylsulfoxide.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)
Preparation of bacterial suspension
Each thawed bacterial suspension (50 µL, or 1/5 dilution of bacterial suspension for Eschericia coli WP 2 uvrA) was added on 25 mL of 25 g/L nutrient broth culture medium in a 200 mL Erlenmeyer flask. each flask was shake-cultured for 10 hours at 37°C. The nutrient broth culture medium was kept at 4°C until the start of shale culture. the optical density of bacterial suspension was then measured at 660 nm by spectrophotometer. The number of bacterial cells was confirmed to be 1.0 x 10 9 cells/mL.

DURATION
- Preincubation period: 37°C for 20 minutes
A volume of 0.1 mL of negative control, test substance solution or positive control was put in a small test tube. 0.5 mL of 1/15 mol/L sodium phosphate buffer (pH 7.4) in the absence of metabolic activation or 0.5 mL of S9 mix in the presence of metabolic activation, and 0.1 mL of bacterial suspension were aded into each tube. After mixing, the mixture was shake-cultured by 72 to 78 reciprocations/min. (variation range) at 37°C for 20 minutes. The small test tube was then taken out and 2.0 mL of top agar was added and mixed. The solution was overlaid on a minium glucose agar plate. After the top agar solidified, each minium glucose agar plate was inverted and cultivated for 48 hours at 37°C.
Composition of minium Glucose Agar plate as follows:
magnesium sulfate heptahydrate 0.2 g/L
citric acid monohydrate 2.0 g/L
dipotassium phosphate anhydride 10.0 g/L
monoammonium phosphate 1.92 g/L
sodium hydroxide 0.66 g/L
glucose 20.0 g/L
agar 15.0 g/L

Top agar: soft agar consisting of sodium chloride at 5% and Bacto agar at 0.6% was dissolved by heating. A mixture solution of 0.5 mmol/L biotin, o.5 mmol/L histidine and 0.5 mmol/Ltryptophan was added to the soft agar solution in the ratio of 1:10.
- Exposure duration:
Dose-finding study: 3 minutes, room temperature
Main study: 6 minutes, room temperature
Confirmatory study: 1 minute, room temperature

Identification of Test System
The name of each bacterial strain was indicated on the side of each Erlenmeyer flask with a marking ink. Each minimum glucose agar plate was identified by the indication of study number, name of strain, dose, name of test substance (or other name), negative and positive control substances and presence or absence of metabolic activation.

NUMBER OF REPLICATIONS:
Dose-finding study: Each strain (with and without metabolic activation, negative and positive controls in duplicate.
Main and confirmatory studies: Each strain (with and without metabolic activation, negative and positive controls in triplicate.

Colony Counting and Observation
Colony Counting
The number of revertant colonies treated with the test substance in Salmonella typhimurium TA100 and positive controls in all bacterial strains was counted using a colony analyzer CA-11D and other minimum glucose agar plates were coounted manually. When using a colony analyzer, the observed colony counts were corrected by the following equation:
Corrected colony counts:
Observed value from a colony analyzer x 1.20 (correction value)

Colony Observation
Precipitation was checked macroscopically and the presence or absence for the growth of the background lawn was observed with the steromicroscope when counting colonies. Presence or absence of growth-inhibition was examined by growth of the background lawn. The evidence was recorded when any precipitation or growth in-hibition was observed.
The study was judged as a compatible condition in the following cases:
1. Mean values of revertant colonies in the negative and positive controls are within the range of background data. When the values deviate from the range, the deviation can be judged to be accidental by comparison with the backgound data.
2. the mean number of revertant colonies in positive controls are twice or more than that of the negative controls.
3. When there is no lack of the colony counts value.
4. When no contaminants are found in the sterility study.

- OTHER: Steility Test
The sterility test was conducted to examine for contamination in the test substance or S9 mix. Mixture of 0.1 mL of the test solution of highest concentration or 0.5 mL of S9 mix and 2 mL of top agar was overlaid on a minimum glucose agar plate. After the top agar solidified, each minimum glucose agar plate was inverted and cultivated for 48 hours at 37°C.

Rationale for test conditions:

The positive controls were selected because they have been generally used in bacterial reverse mutation tests.
The bacterial strains were selected as they have a high sensitivity for known mutagens and have been generally used in bacterial mutation tests.

Evaluation criteria:

The test results wee judged positive when biologically meaningful increase in the number of revertant colonies, such as dose-related increases with reproducibility were observed.

Statistics:

The number of revertant colonies oer plate, the mean values and standard deviation per dose of the test substance, and the negative control were tablulated for each bacterial strain. The number of revertant colonies per plate and the mean values were tabulated for each strain as for the positive control. Dose-response curves of each strain were drawn for the test substance group.
Two statistical anlayses of Dunnett's multiple coparison method (one-sided test) and linear regression method were used in this test. The number of revertant colonies of each bacterial strain at each dose in the main study and confirmatory study was compared with that of the negative control in both the presence and absence of metabolic activation, and statistically significant difference in the number of revertant colonies between those two groups were analyzed first by the multile comparison method (p<0.05). The dose-reactivity was analyzed by linear regression method (p<0.05) when the statistically significant difference was detected by the multiple comparision method.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Main Study
The test substance showed neither growth-inhibition nor dose dependently increase in the number of revertant colonies in any bacterial strains at any dose levels regardless of the presence or absence of metabolica activation. Precipitation of the test substance was not observed at any dose levels in both the presence and absence of metabolic activation.
Confirmatory Study
The test substance showed neither growth-inhibition nor dose-dependently increase in the number of revertant colonies compared with the negative control in any bacterial strains at any dose levels regardless of the presence or absence of metabolic activation. Precipitation of the test substance was not observed at any dose levels in both the presence and absence of metabolic activation.

Applicant's summary and conclusion

Conclusions:

Based on the results, the inducibility of gene mutation in the test substance was determined as negative under the test conditions employed.

Executive summary:

The inducibility of gene mutation in 2 -ethoxy-4 -(hydroxymethyl)phenol was evaluated by the reverse mutation test with a pre-incubation method at 37°C for 20 minutes using five bacterial strains, Salmonella typhimurium TA98, TA100, TA1535, TA1537, and Escherichia coli WP2 uvrA. The test was composed of the dose-finding, main and confirmatory studies, and the reproducibility between the results from themain study and confirmatory study was confirmed. All studies were performed in the presence and absence of metabolic activation. As a result, biologically meaningful increase in the number of revertant colonies due to mutagenicity of the test substance was not observed in any biological strains at any dose levels regardless of the presence or absence of metabolica activation. The number of revertant colonies in the negative and positive controls were within the range of calculated reference value from background data in both the main and confirmatory studies. No contaminants were found in the sterility test. These results demonstrated that the test was properly performed. No suspected factor affected the reliability of the studies was confirmed.

Based on the above results, the inducibility of gene mutation in the test substance was determined as negative under the test conditions employed.