4-ethylphenol

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:

July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

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:

p-vinylphenol

EC Number:

220-103-6

EC Name:

p-vinylphenol

Cas Number:

2628-17-3

Molecular formula:

C8H8O

IUPAC Name:

4-vinylphenol

Test material form:

liquid

Details on test material:

- Name as cited in the report: 4-Vinylphenol
- name: p-Vinylphenol
- CAS: 2628-17-3
- Batch No.: STBD7044V
- Storage Conditions: room temperature
- Expiration date: November 2018
- Description: yellow liquid
- Purity: 10.2% in propylene glycol, FG

Specific details on test material used for the study:

The test item was dissolved in propylene glycol (50%) and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity. A correction factor of 9.8 was applied to consider purity of the test item.

Method

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

- Pre-experiment: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
- Main experiment: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Vehicle / solvent:

Propylene glycol

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: Sodium azide (TA100, TA1535), 4-nitro-o-phenylene-diamine (TA98, TA1537)

Remarks:

Without S9-mix

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (all strains)

Remarks:

With S9-mix

Details on test system and experimental conditions:

Samples of each tester strain were grown by culturing for 12 h at 37°C in Nutrient Broth to the late exponential or early stationary phase of growth (approx. 10E9 cells/mL). The nutrient medium consists per litre:
- 8 g Nutrient Broth
- 5 g NaCI
A solution of 125 µL ampicillin (10 mg/mL) (TA 98, TA 100, TA 102) was added in order to retain the phenotypic characteristics of the strain.

The Vogel-Bonner Medium E agar plates with 2 % glucose used in the Ames Test were prepared by Eurofins Munich or provided by an appropriate supplier. Quality controls were performed. Sterilisation was performed for 20 min at 121°C in an autoclave.

The overlay agar contains per litre:
- 7.0 g Agar Agar
- 6.0 g NaCI
- 10.5 mg L-histidine x HCI x H2O
- 12.2 mg biotin
Sterilisation was performed for 20 min at 121°C in an autoclave.

Evaluation criteria:

CYTOTOXICITY:
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately <= 0.5 in relation to the solvent control.

VALIDITY:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA 98, TA 100, TA 102)
- the mean values of the spontaneous reversion frequency of the control plates with and without S9 mix are within the historical control data range
- corresponding background growth on negative control, solvent control and test plates is observed
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.

MUTAGENICITY
The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (exact values).
A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as folIows:
- if in tester strains TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher than the reversion rate of the solvent control.
According to OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

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:

valid

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:

valid

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:

valid

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:

valid

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Any other information on results incl. tables

Precipitation of the test item was observed in all tester strains used in experiment II at a concentration of 5000 µg/plate (with and without metabolic activation).

Toxic effects of the test item were noted in all tester strains evaluated in experiment l and II.

In experiment I toxic effects of the test item were observed in tester strains TA 98 and TA 102 at concentrations of 2500 µg/plate and higher (with and without metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate (with metabolic activation). In tester strains TA 1535 and TA 1537 toxic effects of the test item were observed at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation).

In experiment II toxic effects of the test item were observed in tester strains TA 98 and TA 1535 at concentrations of 1000 µg/plate and higher (with and without metabolic activation). In tester strains TA 100 and TA 102 toxic effects of the test item were noted at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at concentrations of 316 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation). The reduction in the number of revertants down to a mutation faetor of 0.5 found in tester strain TA 1535 at a concentration of 100 µg/plate (without metabolic activation) was regarded as not biologieally relevant due to lack of a dose-response relationship.

No biologially relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 4-Vinylphenol at any concentration level, neither in the presence nor absence of metabolic activation in experiment l and II.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

EXPERIMENT I (Plate-incorporation Test)

Mean revertant colonies per plate

	TA 98	TA98	TA 100	TA 100	TA 1535	TA 1535	TA 1537	TA 1537	TA 102	TA 102
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
neg. control	25	27	102	107	9	5	8	5	248	283
solvent control	25	28	103	115	10	7	7	5	224	262
test item 3.16 µg	30	39	99	102	15	9	8	11	255	391
test item 10.0 µg	20	30	105	108	10	10	7	10	217	303
test item 31.6 µg	28	26	97	115	7	6	6	7	197	304
test item 100 µg	30	41	104	124	8	10	8	6	187	304
test item 316 µg	34	30	96	112	11	14	6	9	217	309
test item 1000 µg	20	31	47	100	14	11	8	8	206	318
test item 2500 µg	12	15	0	0	0	4	0	6	21	84
test item 5000 µg	0	0	0	0	0	0	0	0	0	0
pos. control	277	1368	471	1934	581	94	118	222	1549	770

EXPERIMENT II (Pre-incubation Test)

Mean revertant colonies per plate

	TA 98	TA98	TA 100	TA 100	TA 1535	TA 1535	TA 1537	TA 1537	TA 102	TA 102
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
neg. control	21	27	78	82	9	14	8	8	177	241
solvent control	22	31	80	90	14	12	9	13	188	264
test item 3.16 µg	20	35	95	104	10	11	7	9	196	241
test item 10.0 µg	19	28	84	99	8	14	8	14	181	238
test item 31.6 µg	20	29	84	99	13	11	8	14	206	211
test item 100 µg	24	31	78	83	6	11	6	12	188	229
test item 316 µg	25	32	83	113	14	21	10	14	182	246
test item 1000 µg	0	27	0	72	0	18	0	13	0	237
test item 2500 µg	0	0	0	0	0	0	0	0	0	0
test item 5000 µg	0	0	0	0	0	0	0	0	0	0
pos. control	295	1013	423	1037	554	86	103	137	1657	579

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, 4-Vinylphenol did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, 4-Vinylphenol is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

In order to investigate the potential of 4-Vinylphenol for its ability to induce gene mutations the plate incorporation test (experiment I) and the pre-incubation test (experiment II) were performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102,

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

Precipitation was observed in all tester strains used in experiment II (with and without metabolic activation).

Toxic effects of the test item were noted in all tester strains used in experiment l and II:

- In experiment I toxic effects of the test item were observed at concentrations of 1000 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation), depending on the particular tester strain.

- In experiment II toxic effects of the test item were noted at concentrations of 316 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate and higher (with metabolic activation), depending on the particular tester strain.

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with 4-Vinylphenol at any concentration level, neither in the presence nor absence of metabolic activation in experiment l and II.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments,

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, 4-Vinylphenol did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, 4-Vinylphenol is considered to be non-mutagenic in this bacterial reverse mutation assay.