4-ethylphenol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

4-Ethylphenol did not induce gene mutations in Salmonella typhimurium and Escherichia coli strains applying pre-incubation or plate incorporation up to 2000 µg/plate in a GLP compliant study according to OECD Technical Guidelines (TG) 471. 4-Ethylphenol was also tested for chromosome aberration in V79 Chinese hamster cells in absence and presence of metabolic activation in a GLP compliant study to OECD 473. The test material induced chromosome aberrations in presence of metabolic activation in a short-term treatment (6 h) and after a 24 h treatment without metabolic activation.

The observations described above are supported by results obtained with the structurally similar 4-vinylphenol. 4-Vinylphenol was tested in three GLP compliant studies according to OECD TG 471, 473 and 476. Like 4-ethylphenol, 4-vinylphenol did not induce gene mutations in Salmonella typhimurium and Escherichia coli strains. Furthermore, it did also not increase the mutant frequency in the HPRT-Assay using V79 cells. In an in vitro chromosome aberration test with human peripheral blood lymphocyte cells, indications were seen for a clastogenic potential.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

July 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Justification for read-across: see attachment

Reason / purpose for cross-reference:

read-across source

Remarks:

Vinylphenol

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

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.

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

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

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.

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

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

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

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

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

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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 (phenobarbital and 5,6-benzoflavone induced)

Test concentrations with justification for top dose:

DOSE RANGE FINDING TEST
50, 150, 500, 1500, 5000 µg/plate - bacterial growth inhibition was observed at 1500 μg/plate and above in all tester strains

MAIN TEST
62.5, 125, 250, 500, 1000, 2000 µg/plate

Vehicle / solvent:

The test substance was dissolved into dimethyl sulfoxide (DMS0, Lot No ACQ2095, Wako Pure Chemical Industries, Ltd.) and prepared into a high-concentration solution which was then
serially diluted with the same solvent and supplied for immediate use in the test.

Untreated negative controls:

no

Remarks:

Solvent control served as negative control

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

furylfuramide

other: 2-aminoanthracene

Details on test system and experimental conditions:

The test was conducted according to the pre-incubation method with and without the S9 mix. 0.1 mL of the tester strain solution was poured into a small test tube and mixed with 0.1 mL of the test substance solution and 0.5 mL of phosphate buffer solution (as well as with 0.5 mL of the S9 mix in tests using the S9 mix), and pre-incubated for 20 min. at 37 °C. After adding 2 mL of top agar, it was poured onto a synthetic media plate and solidified. In testing control groups, the test substance solution was replaced by a test solvent (which serves as a negative control) and by positive substance solutions. The same negative and positive groups were used for tests conducted in one series.

Each plate was incubated at 37°C for 48 hours. The number of revertant colonies produced was determined using a colony analyzer or by visual examination. Precipitation from the test
substance was macroscopically examined. The condition of biofilms formed at the surface of agar was examined with unaided eyes or with a stereoscopic microscope to determine whether the test substance inhibits bacterial growth. The dose-determination was carried out on a single plate and on three plates of negative and positive control groups, resp. Three replicates for each dose of each control group (negative/positive) were used in the main test, from the results of which values and standard deviations were determined.

0.1 mL of the solution containing the test substance at the highest dose and 0.5 mL of the S9 mix resp. were dropped onto the minimal glucose agar medium which was examined for signs of contamination at the end of incubation.

The dose-determination was carried out on a single plate. All plates in the main tests were run in duplicate to confirm the reproducibility of test results.

Evaluation criteria:

The test substance is judged to be mutagenic (positive) when the test substance induces a dose-dependent and reproducible increase in the average number of revertant colonies on a plate containing the test substance to at least twice as many as that of the negative control in the present assay as to at least one strain out of the five tester strains, tested with and without the S9 mix.

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

Positive controls validity:

valid

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

Positive controls validity:

valid

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

Positive controls validity:

valid

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

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:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

CYTOTOXICITY

Without S9 mix:
>= 1000 µg/plate for TA98, TA100, TA1535, TA1537 (main test)
>= 1500 µg/plate for E. coli WP2 uvrA (dose range finding test)

With S9 mix:
>= 1500 µg/plate for all strains (dose range finding test)

DOSE RANGE FINDING TEST

The test was conducted in accordance with “OECD Test Guideline 471” in the dose range of 50.0 – 5000 μg/plate calculated by using a dilution factor of approx. 3.

As a result, bacterial growth inhibition was observed at 1500 μg/plate and above in all tester strains regardless of the presence or absence of the S9 mix dose. No precipitation from the test substance was observed at any dose.

Accordingly, the highest dose was determined to be 2000 μg/plate for tests both with and without the S9 mix.

MAIN TEST

Two mutagenicity tests were run with and without the S9 mix at six dose levels determined using a common ratio of 2 in the range from 62.5 to 2000μg/plate.

It was found as a result that the test substance did not increase the number of revertant colonies more than twice that of the negative control in any tester strain subject to the main test run in duplicate.

No contamination by foreign bacteria was observed in test solutions at the highest dose nor in the S9 mix in any test.

In positive control groups, the positive control substance was found to be mutagenic as to all tester strains. The number of revertant colonies in the positive controls were all within the fluctuations of the background data (mean value ± 3 x standard deviation). These results indicated that the assays were performed appropriately.

Based on the above results, 4-ethylphenol was assessed to be non-mutagenic (negative) under the conditions of the present test.

Mean revertant colonies per plate (I)

	TA 98	TA98	TA 100	TA 100	TA 1535	TA 1535	TA 1537	TA 1537	WP2 uvrA	WP2 uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	24	31	138	159	10	9	14	14	24	27
test item 62.5 µg	23	27	137	176	11	11	11	15	26	30
test item 125 µg	21	33	144	170	11	11	6	14	19	25
test item 250 µg	19	30	127	177	10	14	10	12	22	24
test item 500 µg	20	33	137	161	9	12	13	17	18	20
test item 1000 µg	9	22	50	129	3	6	2	9	20	18
test item 2000 µg	0	0	0	0	0	0	0	0	0	0
pos. control	640	503	573	1073	718	423	473	369	232	879

Mean revertant colonies per plate (II)

	TA 98	TA98	TA 100	TA 100	TA 1535	TA 1535	TA 1537	TA 1537	WP2 uvrA	WP2 uvrA
	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9	-S9	+S9
solvent control	23	33	142	141	12	11	9	15	26	22
test item 62.5 µg	25	35	141	161	10	12	12	17	20	29
test item 125 µg	22	32	154	188	14	16	12	10	13	26
test item 250 µg	20	26	149	164	10	13	9	19	19	22
test item 500 µg	23	39	150	158	12	12	10	15	13	26
test item 1000 µg	8	28	75	124	3	5	3	12	17	28
test item 2000 µg	0	0	0	0	0	0	0	0	0	0
pos. control	615	444	588	967	718	372	567	368	236	847

Conclusions:

4-Ethylphenol was concluded to be non-mutagenic under the conditions of the test.

Executive summary:

The bacteria reversed mutation assay was conducted on 4-ethylphenol, as a results of which 4-ethylphenol was assessed to be negative.

After a dose finding test, the main test was performed in two replicates for each dose using the pre-incubation method in five strains, viz. Salmonella typhimurium strains TA100, TA1535,

TA98, TA1537 and Escherichia coli strain WP2 uvrA in the presence and absence of an S9 mix. The dose finding test was conducted in the range of 50.0 - 5000 μg/plate with and without S9

metabolic activation, in which bacterial growth inhibition was observed at 1500 μg/plate and above in any tester strain. Therefore, six doses in the range from 62.5 to 2000 μg/plate were

selected for the main test to be conducted with and without the S9 mix.

Throughout the assay, 4-ethylphenol did not induce any increases in the number of revertant colonies which is at least twice as many as that of the negative control for any of the five tester

strains at any dose level. Therefore, 4-ethylphenol was concluded to be non-mutagenic under the conditions of the test.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

July - October 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Justification for read-across: see attachment

Reason / purpose for cross-reference:

read-across source

Remarks:

Vinylphenol

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT-Locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

EXPERIMENT I
- without metabolic activation:
0.01, 0.025, 0.05, 0.10, 0.25, 0.50, 0.75, 1.0, 1.2, 1.4 mM
- with metabolic activation:
0.05, 0.10, 0.25, 0.50, 0.75, 1.00, 1 .25, 1.50, 1.75, 2.0 mM
VERIFICATION EXPERIMENT
- without metabolic activation:
0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4 mM
- with metabolic activation:
1.0, 1.5, 1.7, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 mM
EXPERIMENT II
- without metabolic activation:
0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5,2.0 mM

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

V79 cells in vitro have been widely used to examine the ability of chemicals to induce cytogenetic changes and thus identify potential carcinogens or mutagens. These cells are characterized by their high proliferation rate (12 - 14 h doubling time of the Eurofins Munich stock cultures) and their high cloning efficiency of untreated cells, usually more than 50%. These facts are necessary for the appropriate performance of the study.
The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank of Eurofins Munich. This allows the repeated use of the same cell culture batch in experiments. Each cell batch was routinely checked for mycoplasma infections (PCR). Freshly thawed cells from stock cultures were maintained in plastic culture flasks in minimal essential medium (MEM).
For purifying the cell population of pre-existing HPRT mutants cells were exposed to HAT medium containing 100 µM hypoxanthine, 0.4 µM aminopterin, 16 µM thymidine and 10.0 µM glycine for several cell doublings (2-3 days).

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY:
A mutation assay is considered acceptable if it meets the following criteria:
- Negative and/or solvent controls fall within the performing laboratories historical control data range;
- The absolute cloning efficiency: ([number of positive cultures x 100] /total number of seeded cultures) of the negative and /or solvent controls is > 50%;
- The positive controls (EMS and DMBA) induce significant increases (at least 3-fold increase of mutant frequencies related to the comparable negative control values and higher than the historical range of negative controls) in the mutant frequencies.


EVALUATION:
A test is considered to be negative if there is no biological relevant increase in the number of mutants.
There are several criteria for determining a positive result:
- a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
- a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of the mutant frequency is not observed;
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

Statistics:

According to the 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.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

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

PRECIPITATION

No precipitation of the test item was noted in any of the main experiments.

TOXICITY

A biologically relevant growth inhibition (reduction of relative growth below 70%) was observed after the treatment with the test item in experiment land the verification experiment with and without metabolic activation as well as in experiment II without metabolic activation.

In experiment I without metabolic activation the relative growth was 12.5% for the highest concentration (1.4 mM) evaluated. The highest biologically relevant concentration evaluated with metabolic activation was 2.0 mM with a relative growth of 14.7%.

In the verification experiment without metabolic activation the relative growth was 7.9% for the highest concentration (1.4 mM) evaluated. The highest concentration evaluated with metabolic activation was 2.5 mM with a relative growth of 11 .2%.

In experiment II without metabolic activation the relative growth was 14.9% for the highest concentration (2 .0 mM) evaluated.

MUTAGENICITY

In experiment I without metabolic activation the mutant values of the negative controls, the solvent controls and most mutant values of the test item concentrations found were within the historical control data of the test facility Eurofins Munich (about 2-43 mutants per 10⁶ cells). No dose-response relationship could be observed.

Mutation frequencies with the negative control were found to be 21.71 and 26.80, of the solvent control 23.97 and 18.31 mutants/10⁶ cells and in the range of 16.67 to 76.82 mutants/10⁶ cells with the test item, respectively.

At a concentration of 1.2 mM an increased number of mutant colonies was observed (76.82 mutants per 10⁶ cells), exceeding the historical data range and resulting in a mutation rate of 3.63 (compared to the solvent control values) with a relative growth of 23.3%. For confirmation of this elevated mutant value, which is enclosed from concentrations with normal or low mutant values, a verification experiment was performed.

With metabolic activation the mutant values of the negative controls, the solvent controls and various mutant values of the test item concentrations found were within the historical control data of the test facility Eurofins Munich (about 5-44 mutants per 10⁶ cells). The mutant values of some of the

test item concentrations exceed the historical data range, but no dose-response relations hip could be observed.

Mutation frequencies with the negative control were found to be 29.72 and 17.60, of the solvent control 31.08 and 27.85 mutants/10⁶ cells and in the range of 13.24 to 111.26 mutants/10⁶ cells with the test item, respectively.

At a concentration of 2.0 mM an increased number of mutant colonies was observed (111.26 mutants per 10⁶ cells), exceeding the historical data range and resulting in a mutation rate of 3.78 (compared to the solvent control values) with a relative growth of 14.7%. For confirmation of this elevated mutant value, which is next to concentrations with normal or low mutant values, a verification experiment was performed.

In the verification experiment without metabolic activation the mutant values of the negative controls, the solvent controls and most mutant values of the test item concentrations found were within the historical control data of the test facility Eurofins Munich (about 2-43 mutants per 10⁶ cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls.

Mutation frequencies with the negative control were found to be 16.17 and 38.44, of the solvent control 33.81 and 25.30 mutants/10⁶ cells and in the range of 4.08 to 46.30 mutants/10⁶ cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of 1.57 was found at a concentration of 0.7 mM with a relative growth of 47.2%.

In the verification experiment with metabolic activation the mutant values of the negative controls, the solvent controls and most mutant values of the test item concentrations found were within the historical control data of the test facility Eurofins Munich (about 5-44 mutants per 10⁶ cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls.

Mutation frequencies with the negative control were found to be 37.08 and 41.49, of the solvent control 22.59 and 33.90 mutants/10⁶ cells and in the range of 25.10 to 81.41 mutants/10⁶ cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of

2.88 was found at a concentration of 2.4 mM with a relative growth of 16.7%.

In experiment II without metabolic activation (Iong term exposure experiment) the mutant values of the negative controls, the solvent controls and many mutant values of the test item concentrations found were within the historical control data of the test facility Eurofins Munich (about 2-43 mutants per 10⁶ cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls.

Mutation frequencies with the negative control were found to be 40.74 and 40.79, of the solvent control 30.42 and 35.54 mutants/10⁶ cells and in the range of 26.79 to 66.43 mutants/10⁶ cells with the test item, respectively. The highest mutation rate (compared to the solvent control values) of

2.01 was found at a concentration of 0.6 mM with a relative growth of 31 .5%.

The observed elevated mutation rates of 3.63 and 3.78 in experiment I (without and with metabolic activation) at concentrations of 1.2 mM and 2.0 mM could not be reproduced in the verification experiment, which was performed to cover the specific concentration range resulting in a relative

growth of approx. 10-50%. For none of the concentrations tested an increased number of mutants were observed resulting in a mutation factor above 3.0. There was only one concentration (2.4 mM with metabolic activation) resulting in an elevated mutant rate of 2.88, but this value is enclosed from concentrations with lower mutant rates.

Therefore, the elevated mutation rates observed in the first experiment are considered to be not biologically relevant.

DMBA (1 .0 and 1.5 µg/mL) and EMS (300 µg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

EXPERIMENT I without metabolic activation

dose group [mM]	cell density [cells/mL]	rel. growth [%]	CE [%]	mean mutant colonies	mutant colonies / 10E6 cells	MF
0 (NC 1)	869000	105.7	76	6.6	21.71
0 (NC 2)	856000	104.1	77	8.2	26.80
0 (S 1)	871000	100.0	67	6.4	23.97
0 (S 2)	774000	100.0	74	5.4	18.31
0.01	846000	102.9	82	9.6	29.45	1.39
0.025	887000	107.8	81	5.4	16.67	0.79
0.05	873000	106.1	87	8.0	22.99	1.09
0.10	833000	101.3	85	13.4	39.64	1.88
0.25	496000	60.3	84	9.8	29.34	1.39
0.50	419000	50.9	75	6.0	20.00	0.95
0.75	341000	41.5	80	8.6	26.96	1.28
1.0	315000	38.3	77	10.4	33.66	1.59
1.2	192000	23.3	72	22.2	76.82	3.63
1.4	103000	12.5	77	13.6	44.01	2.08
300 (EMS)	658000	80.0	76	93.0	306.93	14.52

EXPERIMENT I with metabolic activation

dose group [mM]	cell density [cells/mL]	rel. growth [%]	CE [%]	mean mutant colonies	mutant colonies / 10E6 cells	MF
0 (NC 1)	1370000	105.8	81	9.6	29.72
0 (NC 2)	1290000	99.6	85	6.0	17.60
0 (S 1)	1230000	100.0	74	9.2	31.08
0 (S 2)	1360000	100.0	79	8.8	27.85
0.05	1330000	102.7	72	3.8	13.24	0.45
0.10	1410000	108.9	74	8.8	29.83	1.01
0.25	1290000	99.6	70	17.6	62.86	2.13
0.50	1250000	96.5	69	16.8	61.09	2.07
0.75	1010000	78.0	80	12.8	40.00	1.36
1.00	756000	58.4	78	12.6	40.51	1.38
1.25	503000	38.8	73	21.2	73.10	2.48
1.50	478000	36.9	76	18.2	59.87	2.03
1.75	320000	24.7	75	12.2	40.94	1.39
2.00	190000	14.7	76	33.6	111.26	3.78
1.0 (DMBA)	1190000	91.9	68	95.0	349.26	11.85
1.5 (DMBA)	845000	65.3	73	188.6	648.11	22.00

VERIFICATION EXPERIMENT without metabolic activation

dose group [mM]	cell density [cells/mL]	rel. growth [%]	CE [%]	mean mutant colonies	mutant colonies / 10E6 cells	MF
0 (NC 1)	409000	55.8	84	5.4	16.17
0 (NC 2)	784000	106.9	77	11.8	38.44
0 (S 1)	642000	100	87	11.8	33.81
0 (S 2)	825000	100	83	8.4	25.30
0.5	409000	55.8	85	13.0	28.24	1.29
0.6	386000	52.6	87	13.4	38.51	1.30
0.7	346000	47.2	78	14.4	46.30	1.57
0.8	322000	43.9	76	7.6	24.92	0.84
0.9	368000	50.2	90	12.4	34.35	1.16
1.0	324000	44.2	84	7.8	23.35	0.79
1.1	245000	33.4	69	5.0	18.05	0.61
1.2	201000	27.4	81	6.4	19.81	0.67
1.3	120000	16.4	80	5.0	15.67	0.53
1.4	57700	7.9	74	1.2	4.07	0.14
300 (EMS)	1120000	152.7	71	102.0	357.89	12.11

VERIFICATION EXPERIMENT with metabolic activation

dose group [mM]	cell density [cells/mL]	rel. growth [%]	CE [%]	mean mutant colonies	mutant colonies / 10E6 cells	MF
0 (NC 1)	991000	102.9	82	12.2	37.08
0 (NC 2)	1030000	107.0	81	13.4	41.49
0 (S 1)	995000	100	91	8.2	22.59
0 (S 2)	931000	100	74	10.0	33.90
1.0	526000	54.6	78	13.2	42.31	1.50
1.5	430000	44.7	80	11.8	36.99	1.31
1.7	289000	30.0	74	8.0	27.21	0.96
1.9	224000	23.3	67	10.4	38.66	1.37
2.0	234000	24.3	64	6.4	25.10	0.89
2.1	209000	21.7	78	13.6	43.59	1.54
2.2	188000	19.5	83	10.2	30.91	1.09
2.3	182000	18.9	75	12.2	40.53	1.44
2.4	161000	16.7	78	25.4	81.41	2.88
2.5	108000	11.2	71	16.0	56.14	1.99
1.0 (DMBA)	861000	89.4	79	122.4	387.34	13.71
1.5 (DMBA)	539000	56.0	78	163.8	523.32	18.53

EXPERIMENT II without metabolic activation

dose group [mM]	cell density [cells/mL]	rel. growth [%]	CE [%]	mean mutant colonies	mutant colonies / 10E6 cells	MF
0 (NC 1)	1530000	107.7	81	13.2	40.74
0 (NC 2)	1550000	109.2	76	12.4	40.79
0 (S 1)	1440000	100	77	9.4	30.42
0 (S 2)	1400000	100	83	11.8	35.54
0.1	1320000	93.0	84	17.0	50.90	1.54
0.2	1060000	74.6	80	8.6	26.79	0.81
0.4	727000	51.2	81	16.6	51.55	1.56
0.6	447000	31.5	72	19.0	66.43	2.01
0.8	340000	23.9	71	8.4	29.79	0.90
1.0	319000	22.5	70	17.4	62.14	1.88
1.5	265000	18.7	74	9.6	32.54	0.99
2.0	211000	14.9	77	8.4	27.45	0.83
300 (EMS)	1390000	97.9	70	203.6	732.37	22.21

Conclusions:

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item 4-Vinylphenol is considered to be non-mutagenic in the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

The test item 4-Vinylphenol was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster.

The selection of the concentrations was based on data from the pre-experiments.

Experiment l and the verification experiment with and without metabolic activation were performed as 4 h short-term exposure assay. Experiment II without metabolic activation was performed as a 20 h long-term exposure assay.

The test item was investigated at the following concentrations:

Experiment I

- without metabolic activation: 0.01 , 0.025, 0.05, 0.10, 0.25, 0.50, 0.75, 1.0, 1.2, 1.4 mM

- with metabolic activation: 0.05, 0.10, 0.25, 0.50, 0.75, 1.00, 1.25, 1.50, 1.75, 2.0 mM

Verification Experiment

- without metabolic activation: 0.5, 0.6, 0.7,0.8,0.9,1.0, 1.1, 1.2,1 .3,1.4 mM

- with metabolic activation: 1.0, 1.5,1 .7, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5 mM

Experiment II

- without metabolic activation: 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0 mM

No precipitation of the test item was noted in the main experiments.

Biologically relevant growth inhibition was observed in experiment l and the verification experiment with and without metabolic activation . In experiment I without metabolic activation the relative growth was 12.5% for the highest concentration (1.4 mM) evaluated. The highest biologically

relevant concentration evaluated with metabolic activation was 2.0 mM with a relative growth of 14.7%. In the verification experiment without metabolic activation the relative growth was 7.9% for the highest concentration (1.4 mM) evaluated. The highest concentration evaluated with metabolic activation was 2.5 mM with a relative growth of 11 .2%. In experiment II without metabolic activation the relative growth was 14.9% for the highest concentration (2 .0 mM) evaluated.

In the experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.

DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

mammalian cell line, other: Chinese hamster lung cells CHL/IU

Details on mammalian cell type (if applicable):

CHL/IU cells (obtained from the JCRB cell bank) were incubated in Eagle's Medium, MEM
(Nissui Pharmaceutical) containing 10% of bovine calf serum (Cansera International, Lot No. :
2608311) in a CO2 incubator (5% CO2, 37 °C). Cells sub-cultured for less than 10 passages after thawing were used for the test.

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

MAIN EXPERIMENT
- without metabolic activation, 6 h treatment: 0.050, 0.10, 0.20, 0.40, 0.80 mg/mL
- with metabolic activation, 6 h treatment: 0.019, 0.038, 0.075, 0.15, 0.30 mg/mL
- without metabolic activation, 24 h treatment: 0.019, 0.038, 0.075, 0.15, 0.30 mg/mL

Vehicle / solvent:

DMSO (Wako Pure Chemical Industries, Lot No.: ACL5008)

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

propylene glycol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

see "Any other information on materials and methods"

Evaluation criteria:

Chromosomal evaluation was carried out in accordance with the classification set by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagen Society (Ed): “The Atlas of Chromosomal Aberrations Induced by Chemicals”, Asakura Publishing, Tokyo (1988)

Statistics:

Fisher's exact test, Cochran-Armitage trend test

Key result

Species / strain:

mammalian cell line, other: CHL/IU

Remarks:

6h treatment

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mammalian cell line, other: CHL/IU

Remarks:

6h treatment

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mammalian cell line, other: CHL/IU

Remarks:

24h treatment

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

4EP induced structural chromosomal aberrations at the maximum observation dose (0.075 mg/mL) in short-term tests in the presence of an S9 mix (the frequency of aberration including gaps: 11.0%).

It also induced structural chromosomal aberrations in 24-h continuous tests whereby the frequency of aberration was determined to be 21.0% when tested at a concentration of 0.075 mg/mL.

In contrast thereto, the frequency of structural chromosomal aberration in a short-term test in the absence of an S9 mix was at the same level as that found with the vehicle control group.

A significant increase (p < 0.01) was observed in cells subject to a short-term treatment by the test substance at mid-range and high doses (0.038 mg/mL, 0.075 mg/mL resp.) in the evaluation of polyploid cells, the frequency of which scored low at 1.50 % and 1.63%, resp. Therefore, the substance is judged negative in this respect. Other test series showed no evidence of inducing polyploid cells.

D20 value at which structural aberrations are found in 20% of metaphases was determined by type of treatment. D20 in groups subject to a short-term test in the presence of an S9 mix was determined to be 0.14 mg/mL, that in groups subject to a 24h continuous test was 0.077 mL.

Short term experiment without metabolic activation 6h treatment

Dose group	Conc. [mg/mL]	Cell no.	No. of aberrations	Aberrant cells incl. gaps [%]	Aberrant cells excl. gaps [%]	Polyploid [%]	Cytotoxicity [%]
Neg. control	0	200	2	1	1	0.00	-
Solvent control	0	200	3	1	1	0.00	100
4EP	0.05	200	0	0	0	0.00	85
4EP	0.10	200	5	1	0.5	0.38	88.5
4EP	0.20	200	1	0.5	0.5	0.38	76
4EP	0.40	-	-	-	-	-	2
4EP	0.80	-	-	-	-	-	19
Pos. control (MC)	0.1 µg/mL	200	193	50.5	50.0	0.00	-

Short term experiment with metabolic activation 6h treatment

Dose group	Conc. [mg/mL]	Cell no.	No. of aberrations	Aberrant cells incl. gaps [%]	Aberrant cells excl. gaps [%]	Polyploid [%]	Cytotoxicity [%]
Solvent control	0	200	3	1.0	1.0	0.13	100
4EP	0.019	200	6	2.5	1.5	0.00	95
4EP	0.038	200	16	5.0	4.0	1.50	83.5
4EP	0.075	200	61	11.0	11.0	1.63	56
4EP	0.15	-	-	-	-	-	38
4EP	0.30	-	-	-	-	-	21
Pos. control (CPA)	5 µg/mL	200	253	62.0	60.5	0.00	-

Continuous experiment without metabolic activation 24h treatment

Dose group	Conc. [mg/mL]	Cell no.	No. of aberrations	Aberrant cells incl. gaps [%]	Aberrant cells excl. gaps [%]	Polyploid [%]	Cytotoxicity [%]
Solvent control	0	200	2	1.0	1.0	0.00	100
4EP	0.019	200	14	5.5	4.0	0.00	102
4EP	0.038	200	17	8.0	6.5	0.13	89
4EP	0.075	200	50	22.5	21.0	0.00	68.5
4EP	0.15	-	-	-	-	-	33
4EP	0.30	-	-	-	-	-	6.5
Pos. control (MC)	0.05 µg/mL	200	185	52.5	52.0	0.00	-

Conclusions:

As a result of chromosome analysis, it was observed that 4EP induces structural chromosomal aberration in cells in a short-term treatment in the presence of an S9 mix as well as with cells treated continuously for 24 hours in the absence of an S9 mix.

Therefore, 4-Ethylphenol is considered to be positive in this chromosome aberration test.

Executive summary:

4-Ethylphenol (4EP) induced chromosomal aberrations in CHL/IU cells (of Chinese hamsters, isolated from the lung).

50% growth-inhibitory concentrations (IC50) of 4EP were measured to be 0.15 mg/mL and 0.41 mg/mL resp. in cells in a short-term treatment in the presence of an S9 mix (6-h treatment in an S9 reaction solution with an 18-h recovery culture) and cells treated in the absence of an S9 mix (in EMM without an S9 reaction solution), and 0.15 mg/mL in cells treated continuously (for 24 hours in a fresh medium).

Hence, a value of approximately twice IC50 was selected as the maximum concentration in all test series in the chromosomal aberrations assay. By diluting it by a common factor of two, five

concentrations in total were selected for the test. The highest concentrations applicable to chromosome evaluation are 0.075 mg/mL and 0.20 mg/mL resp. in cells in a short-term treatment in the presence and absence of an S9 mix, and 0.075 mg/mL in cells treated continuously for 24 hours. Therefore, the said three concentrations were used in the test.

As a result of chromosome analysis, it was observed that 4EP induces structural chromosomal aberration in cells in a short-term treatment in the presence of an S9 mix as well as with cells treated continuously for 24 hours in the absence of an S9 mix.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

July - November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Justification for read-across: see attachment

Reason / purpose for cross-reference:

read-across source

Remarks:

Vinylphenol

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

lymphocytes: human

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

MAIN EXPERIMENT
- without metabolic activation, 4 h treatment, 24 h preparation interval: 0.1, 0.25, 0.50, 1.0, 2.0, 3,0, 3,5, 4.0, 4.5 and 5 mM
- with metabolic activation, 4 h treatment, 24 h preparation interval: 0.5, 1.0, 2.0, 3.0, 4.0, 4.5, 5.0, 5.5 and 6 mM

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

propylene glycol

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

TEST SYSTEM

Human peripheral blood Iymphocytes from healthy and non-smoking donors with no known recent exposure to genotoxic chemicals and radiation were used to examine the ability of chemicals to induce cytogenetic damage and thus to identify potential carcinogens or mutagens in vitro. For this study for the main experiment blood was collected only from two donors due to organisationel reasons.
Blood samples were drawn by venous puncture and collected in heparinized tubes. Before use the blood was stored under sterile conditions at 4 °C for a maximum of 4 h. Whole blood samples treated with an anti-coagulant (e. g. heparin) were pre-cultured in the presence of mitogen (phytohaematogglutinin, PHA).

Complete Culture Medium
RPMI 1640 medium supplemented with:
- 15% fetal bovine serum (FBS)
- 100 U/100 µg/mL penicillin/streptomycin solution
- 0.24 g/mL PHA-L
Also used lor the long-term treatment and the post incubation.

Treatment Medium (short-term exposure)
Complete culture medium without FBS.
All incubations were done at 37°C in humidified atmosphere with 5% CO2.

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY:
The chromosomal aberration assay is considered acceptable if it meets the following criteria:
- the number of aberration found in the negative and/or solvent controls falls within the range of historical laboratory control data / is considered acceptable for addition to the laboratory historical negative control database.
- concurrent positive controls should induce responses that are compatible with those generated in the historical positive control data base and produce a statistically significant increase compared with the concurrent negative control
- the proliferation rate of the solvent control should be similar to the corresponding negative control value
- All three experimental conditions are tested unless one results in positive results
- Adequate number of cells and concentrations are analyzable.

EVALUATION:
A test chemical is considered to be clearly positive if:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data

A test chemical is considered clearly negative if:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data.

Statistics:

Fisher's exact test

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: human

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Precipitation

No precipitation of the test item was noted without and with metabolic activation at the concentrations evaluated.

Toxicity

No toxic effects of the test item were noted in the main experiment without metabolic activation considering the mitotic index in the concentrations reported. However, toxic effects were noted starting from concentrations of 0.5 mM and higher, as the cultures showed a brownish colouring, pointing towards a toxic / denaturating effect of the test material. With metabolic activation slightly toxic effects considering the mitotic index (decrease below 70% rel. mitotic index) were noted at a concentration of 0.5 mM and 2 mM (both 69%). Brownish colouring of the cultures was noted at concentrations of 2 mM and higher, indicating a toxic / denaturating effect of the test item.

Proliferation Index

The BrdU-technique was used for determining the proliferation index to detect a possible effect on the proliferation rate after treatment with the test item and thus indicating cell cycle delay. In the main experiment, the values of the proliferation index of the negative controls were:

- 1.32 (without metabolic activation)

- 1.04 (with metabolic activation)

- 1.44 (solvent control without metabolic activation)

- 1.03 (solvent control with metabolic activation).

The proliferation index of the highest dose groups evaluated were 1.14 (0.5 mM; without metabolic activation) and 1.02 (2 mM; with metabolic activation). No biologically relevant decrease of the proliferation index was indicated.

Clastogenicity

There are several criteria for determining a positive result, such as a concentration-related increase or a reproducible increase in the number of cells with chromosome aberrations for at least one of the dose groups, which is higher than the laboratory negative control range (0.0% - 3.0% aberrant cells (without and with metabolic activation)).

Without metabolic activation the aberration rates of the negative control (1.7%), the solvent control (2.3%) and the dose groups treated with the test item (2.0% (0.10,0.25 and 0.5 mM)) were within the historical control data of the testing facility (0.0% - 3.0%).

The number of aberrant cells found in the dose groups treated with the test item did not show a biologically relevant increase compared to the corresponding solvent control. In addition, no dose-response relationship was observed.

With metabolic activation the aberration rates of the negative control (1.7%), the solvent control (1.7%) and the dose group 0.5 mM (2.3%) were within the historical control data of the

testing facility (0.0% - 3.0%).

The number of aberrant cells lound at concentrations of 1 mM and 2 mM showed a biologically relevant increase compared to the corresponding solvent control (3.7% and 6.0%, respectively). In addition, a dose-response relationship was observed.

EMS (600 µg/mL) and CPA (10 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the ability of the test system to indicate potential clastogenic effects.

The Fisher's exact test was performed to verify the results in the experiment. A statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in the highest dose group (2 mM) of the test item evaluated in the main experiment with metabolic activation.

Polyploid Cells

No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item.

Table1: Main Experiment without metabolic activation (Experiment I, 4h treatment)

Dose group	Conc. [mM]	rel. mitotic index [%]	Proliferation index	% Aberrant cells incl. gaps	% Aberrant cells excl. gaps	Precipitation	Stat. significance
Neg. control	0	95	1.32	2.7	1.7	no	no
Solvent control	0	100	1.44	6.3	2.3	no	no
test item	0.10	125	/	4.0	2.0	no	no
test item	0.25	83	/	2.3	2.0	no	no
test item	0.5	102	1.14	3.7	2.0	no	no
Pos. control (EMS)	600 µg/mL	42	/	21.9	19.4	no	yes

Table 2: Main Experiment with metabolic activation (Experiment I, 4h treatment)

Dose group	Conc. [mM]	rel. mitotic index [%]	Proliferation index	% Aberrant cells incl. gaps	% Aberrant cells excl. gaps	Precipitation	Stat. significance
Neg. control	0	102	1.04	4.3	1.7	no	no
Solvent control	0	100	1.03	3.7	1.7	no	no
test item	0.5	69	/	3.7	2.3	no	no
test item	1	86	/	6.3	3.7	no	no
test item	2	69	1.02	8.0	6.0	no	yes
Pos. control (CPA)	10 µg/mL	98	/	23.1	23.1	no	yes

Conclusions:

In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item 4-Vinylphenol induced structural chromosomal aberrations in human Iymphocyte cells.

Therefore, 4-Vinylphenol is considered to be clastogenic in this chromosome aberration test.

Executive summary:

A chromosome aberration assay was carried out to investigate a possible potential of 4-Vinylphenol to induce structural chromosome aberrations in human Iymphocytes.

The metaphases were prepared 24 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation. Duplicate cultures were set up. Per culture 150 metaphases were scored for structural chromosomal aberrations.

The following concentrations were evaluated:

- without metabolic activation, 4 h treatment, 24 h preparation interval: 0.1, 0.25 and 0.5 mM

- with metabolic activation, 4 h treatment, 24 h preparation interval: 0.5, 1 and 2 mM

No precipitation of the test item was noted without and with metabolic activation in all dose groups evaluated in the experiment.

No toxic effects of the test item were noted in the main experiment without metabolic activation considering the mitotic index in the concentrations reported. However, toxic effects were noted

starting from concentrations of 0.5 mM and higher, as the cultures showed a brownish colouring, pointing towards a denaturating effect of the test material. With metabolic activation slightly toxic effects considering the mitotic index (decrease below 70% rel. mitotic index) were noted at a concentration of 0.5 mM and 2 mM (both 69%). Brownish colouring of the cultures was noted at concentrations of 2 mM and higher.

No biologically relevant decrease of the proliferation index was observed.

No biologically relevant increase of the aberration rates was noted after treatment with the test item in the main experiment without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

In the main experiment with metabolic activation a biologically relevant increase of the aberration rates was noted after treatment with the test item at concentrations of 1 mM and higher.

In the experiments I and II without and with metabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the solvent controls.

EMS (600 µg/mL) and CPA (10 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the efficiency of the test system to indicate potential clastogenic effects.

In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item 4-Vinylphenol induced structural

chromosomal aberrations in human Iymphocyte cells.

Therefore, 4-Vinylphenol is considered to be clastogenic in this chromosome aberration test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

4-Ethylphenol was tested in an in vivo micronucleus study according to OECD TG 474 in order to investigate it’s in vivo potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of rats. A single dose of 125, 250 and 500 mg/kg bw 4-ethylphenol in corn oil was applied with 500 mg/kg bw determined to be the MTD in a pre-experiment. Peripheral blood samples were collected for micronuclei analysis 48 h and 72 h after exposure. At the MTD, signs of systemic toxicity such as reduction of spontaneous activity, prone position, piloerection, ataxia, hunched posture and half eyelid closure demonstrated that the test substances became systemically available and could thus also reach the bone marrow as target organ. Under the experimental conditions reported, the test item 4-Ethylphenol did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the rat.

The observations described above are supported by results obtained with the structurally similar 4-vinylphenol. In an in vivo micronucleus test according to OECD TG 474, 4-vinylphenol did not show structural and/or numerical chromosomal damage in the immature erythrocytes of treated mice. 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2022-March-22 to 2022-May-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Gemany

Type of assay:

mammalian erythrocyte micronucleus test

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
Source: Charles River, 97633 Sulzfeld, Germany
Number of animals: 5 males per dose group (7 animals for 1 MTD)
Initial age at start of acclimatisation: 6 - 8 weeks
Age at start of treatment: Minimum 7 weeks


ENVIRONMENTAL CONDITIONS
Housing: 5 animals of identical sex per cage
Cage type: IVC cage (Polysulphone), Type III H
Bedding: Altromin saw fiber bedding (Batch: 0131)
Feed: Free access to Altromin 1324 (Batch: 0939) maintenance diet for rats and mice
Air change: At least 10 x per hour
Water: Free access to tap water, sulphur acidified to pH value of approx. 2.8 (drinking water, municipal residue control, micro-biologically controlled at frequent intervals)
Environment: Temperature 22 ± 3 °C
Relative humidity 55 ± 10%
Artificial light 6:00 - 18:00

Route of administration:

oral: gavage

Vehicle:

Name: Corn oil
Supplier: Sigma
Batch No.: MKCN9742

Details on exposure:

The test item was prepared in corn oil within 2 h before treatment. All animals received a single volume orally of 10 mL/kg bw.

Frequency of treatment:

All animals received a single volume orally of 10 mL/kg bw.

Post exposure period:

The sampling times were 48 h for all dose groups evaluated and additionally 72 h for the negative control and highest dose group.

Dose / conc.:

500 mg/kg bw/day

Remarks:

1 MTD

Dose / conc.:

250 mg/kg bw/day

Remarks:

0.5 MTD

Dose / conc.:

125 mg/kg bw/day

Remarks:

0.25 MTD

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Name: CPA; Cyclophosphamide
CAS No.: 50-18-0
Supplier: Sigma
Catalogue No.: C0768
Batch No.: MKCL2547
Dissolved in: physiological saline
Dosing: 10 mg/kg bw
Route and frequency of
administration: ip, single
Volume administered: 10 mL/kg bw

Tissues and cell types examined:

peripheral blood erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION
A pre-study on acute toxicity was performed with both gender of the same strain and under identical conditions as in the mutagenicity study. The maximum dose administered was 2000 mg/kg bw according to the OECD 474 guideline. The maximum volume which was administered at one time was 10 mL/kg bw. The animals received the test item once. The sampling times were 48 h for all dose groups evaluated and additionally 72 h for the negative control and highest dose group. Since no relevant differences in systemic toxicity were observed for male and female animals in the pre-experiment, the main experiment was only performed with male animals.

METHOD OF ANALYSIS
Evaluation of all samples, including those of positive (CPA) and negative controls, were performed using a flow cytometer (FACSLyric, BD Biosciences). Anti-CD71 antibodies were labeled with fluorescein isothiocyanate (FITC), anti-CD61 antibodies were labelled with phycoerythrin (PE). Particles were differentiated using forward scatter (FSC) and side scatter (SSC) parameters of the flow cytometer. Fluorescence intensities were recorded on PMT position E, D and B for FITC, PE and PI, respectively. 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes. To detect a possibly occurring cytotoxic effect of the test item, the ratio between immature and mature erythrocytes was determined. The result was expressed as relative PCE (rel. PCE = proportion of polychromatic (immature) erythrocytes among total erythrocytes).

Evaluation criteria:

Providing all acceptability criteria are fulfilled, a test item is considered clearly positive if:
- at least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
- this increase is dose-related at least at one sampling time when evaluated with an appropriate trend test, and
- any of these results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
If only the highest dose is examined at a particular sampling time, a test item is considered clearly positive if there is a statistically significant increase compared with the concurrent negative control and the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
Providing that all acceptability criteria are fulfilled, a test item is considered clearly negative if, in all experimental conditions examined:
- none of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated immature erythrocytes compared with the concurrent negative control,
- there is no dose-related increase at any sampling time when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits), and
- bone marrow exposure to the test item occurred.

Statistics:

Pairwise comparison of the proportion of PCE among total erythrocytes as well as the proportion of micronucleated polychromatic (immature) erythrocytes (PCE) among total PCE between the control groups and each of the treatment groups was performed by means of the non-parametric Mann-Whitney test at a statistical significance level of 5% (p < 0.05, two-tailed).
The X² test for trend at a statistical significance level of 5% (p < 0.05, two-tailed) was used to test whether there is a dose-related increase in the micronucleated cells frequency of the dose groups of the 44 h sampling time.
Statistical methods were performed using the software GraphPad Prism version 6.0.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Animals treated with the highest dose of 500 mg/kg bw showed toxic effects.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY

Clinical signs of toxicity in test animals:
In the pre-experiment a concentration of 200 mg/mL of the test item was evaluated. One male and one female rat received a single dose of 2000 mg/kg bw orally and showed strong toxicity such as reduction of spontaneous activity, hunched posture, piloerection, ataxia and half eyelid closure. Both animals were euthanised 1.5 h after application for animal welfare reasons. The dose was reduced to 500 mg/kg bw. Three male and three female rats received a single dose of 500 mg/kg bw orally and showed toxicity such as reduction of spontaneous activity, prone position, hunched posture, ataxia, piloerection and eyelid /half eyelid closure.

RESULTS OF DEFINITIVE STUDY

- Toxicity in the main experiment
500 mg/kg bw was tested as the maximum tolerated dose (1 MTD) in the main experiment. The volume administered orally was 10 mL/kg bw. Animals treated with the highest dose (1 MTD) showed toxic effects with reduction of spontaneous activity, prone position, piloerection, ataxia, hunched posture and half eyelid closure up to 4 h after application. Male rats treated with 250 mg/kg bw (0.5 MTD) and 125 mg/kg bw (0.25 MTD) showed no clinical signs of systemic toxicity after treatment with the test item


- Induction of micronuclei (for Micronucleus Assay):
For all dose groups, including positive and negative controls, 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes.
The negative controls (48 h and 72 h) evaluated were within the historical control limits of the negative control (0.03 – 0.14% for males). The mean values of micronuclei observed for the negative control were 0.09% after 48 h and 0.10% after 72 h .
The mean value of micronuclei observed after treatment with 0.25 MTD was 0.07%. The mean value observed was within the range of the concurrent negative control as well as within the historical control limits of the negative control.
The mean value noted for the 0.5 MTD dose group was 0.06%. The mean value observed was within the range of the concurrent negative control as well as within the historical control limits of the negative control.
The dose group treated with 1 MTD (48 h sampling) showed a mean value of 0.07%. The mean value observed was within the range of the concurrent negative control as well as within the historical control limits of the negative control. The mean value observed for the 1 MTD (72 h sampling) was 0.06%. The mean value was statistically significantly decreased compared to the concurrent negative control and within the historical control limits of the negative control. No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.


- Ratio of PCE
The relative PCE (rel. PCE = proportion of polychromatic (immature) erythrocytes among total erythrocytes) was determined for each animal. The relative PCE is the supportive endpoint to assess cytotoxicity, which helps to demonstrate a target cell exposure with the test item.
The negative control (48 h) was within the historical control limits of the negative control for the 48 h sampling time (0.64% - 2.49% for males). The mean value noted for the 48 h negative control was 2.20%. For the 72 h negative control a mean value of 2.56% was observed (Table 4 and Table 9). The value lay slightly above the upper control limit which can be attributed to blood loss 24 h earlier for the first sampling.
The animal group treated with 0.25 MTD showed a mean value of the relative PCE of 2.39% (Table 6). The mean value observed was within the range of the concurrent negative control and within the historical control limits of the negative control.
The dose group, which was treated with 0.5 MTD, showed a mean value of the relative PCE of 1.95% (Table 7). The mean value observed was within the range of the concurrent negative control and within the historical control limits of the negative control.
The animals who received 1 MTD (48 h sampling) showed a mean value of the relative PCE of 1.67% (Table 8). The mean value observed was within the range of the concurrent negative control and within the historical control limits of the negative control.


- Statistical evaluation
The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p< 0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated. Additionally, the X² Test for trend was performed to test whether there is a dose-related increase in the micronucleated cells frequency of the dose groups of the 48 h sampling time. No statistically significant increase in the frequency of micronucleated cells was observed.

Conclusions:

In conclusion, it can be stated that during the study and under the experimental conditions reported, the test item 4-Ethylphenol did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the rat.
Therefore, the test item 4-Ethylphenol is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the Mammalian Erythrocyte Micronucleus Test.

Executive summary:

In a Wistar rats peripheral blood micronucleus assay, five male  animals per dose group were treated orally with the test item at doses of 500, 250 and 125 mg/kg bw.  Peripheral blood cells were harvested at 48 h (all dose and control groups) and 72 h (negative control and 1 MTD group) post-treatment.  The vehicle was Corn Oil. The animals received the test item once orally. There were signs of toxicity during the study.  The animals treated with doses of 0.2 and 0.5 MTD showed no signs of systemic toxicity. The animals treated with a dose of 1 MTD showed signs of systemic toxicity such as reduction of spontaneous activity, prone position, piloerection, ataxia, hunched posture and half eyelid closure up to 4 h after application.

There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in peripheral blood cells after any treatment time.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5395; OECD 474 for in vivo cytogenetic mutagenicity data.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

December 2015 - January 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, number 420 "Acute Oral Toxicity - Fixed Dose Procedure"

Version / remarks:

(adopted: December 17, 2001)

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, number 423 "Acute Oral Toxicity - Acute Toxic Class Method"

Version / remarks:

(adopted: December 17, 2001)

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS
Species: Mouse (mus musculus)
Strain: NMRI, young healthy adult
Source: Charles River, 97633 Sulzfeld, Germany
Number of animals: 5 of each sex per dose group
Initial age at start of acclimatisation: 6 -12 weeks
Age at start of treatment: Minimum 7 weeks
The animals were derived from a controlled full barrier maintained breeding system (spf). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes and underwent an adequate acclimatisation period after arrival. The animals were randomly distributed to test groups.

HUSBANDRY
The animals were barrier maintained (semi-barrier) in an air conditioned room. The experiment was conducted under standard laboratory conditions.
Housing: 5 animals of identical sex per cage
Cage type: IVC cage (Polysulphone), Type 11 L
Bedding: Altromin sawfiber bedding (Bateh: 02102150820)
Feed: Free access to Altromin 1324 (Bateh: 0631) maintenance diet for rats and mice
Air change: At least 10 x per hour
Water: Free access to tap water, sulphur acidified to pH value of approx. 2.8 (drinking water, municipal residue control, micro-biologically controlled at frequent intervals)
Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich.
The animals were randomly distributed into the test groups. The animals of each test group and sex were housed in separate cages that were individually marked (study number, sex, control / test group). The animals of each cage were individually marked for identification by tai! and ear drawing.

ENVIRONMENT
Temperature 22 ± 3 °C
Relative humidity 55 ± 10%
Artificial light 6:00 - 18:00

Route of administration:

intraperitoneal

Vehicle:

Propylene gycol and aqua ad iniectabilia

Details on exposure:

PREPARATION OF THE TEST ITEM
The test item was available as solution in propylene glycol with a content of 4-Vinylphenol of 9.7%. To obtain a non-toxic dose of propylene glycol the test item was further diluted in Aqua ad iniectabilia.
For all dose groups the test item was administered as split dose with a time period of 2 h between both applications. The volume administered ip for each application was 10 mL/kg bw.

Duration of treatment / exposure:

Single administration of a split dose with a time period of 2 h between applications.

Frequency of treatment:

The animals received the test item twice as split dose by the intraperitoneal route with a time period of 2 h between applications.

Post exposure period:

44 h and 68 h

Dose / conc.:

19.4 mg/kg bw (total dose)

Remarks:

0.2 MTD

Dose / conc.:

48.5 mg/kg bw (total dose)

Remarks:

0.5 MTD

Dose / conc.:

97 mg/kg bw (total dose)

Remarks:

1 MTD

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

- Name: CPA (cyclophosphamide)
- CAS No.: 50-18-0
- Supplier: Sigma
- Catalogue No.: C0768
- Batch No.: SLBG4216V
- Dissolved in: physiological saline
- Dosing: 40 mg/kg bw
- Route and frequency of administration: ip, single
- Volume administered: 10 mL/kg bw

The solution was aliquoted and stored at <=-15°C. On day of administration the solution was freshly thawed. The stability of CPA at room temperature is quite good (3.5% is hydrolysed per day in aqueous solution). It is acceptable that the positive control can be administered by a route different from the test agent and sampled at only a single time. The sampling time for the positive control is 44 h after treatment.

Tissues and cell types examined:

Blood cells / erythrocytes

Details of tissue and slide preparation:

see "Any other information on materials and methods"

Evaluation criteria:

There are several criteria for determining a positive result:
- dose-related increase in the number of micronucleated cells and/or
- biologically relevant increase in the number of micronucleated cells for at least one of the dose groups.
According to the OECO guideline, the biological relevance as well as the statistical significance of the results are the criteria for the interpretation.
A test item is considered to be negative if there is no biologically relevant and/or statistically significant increase in the number of micronucleated cells at any dose level.

Statistics:

For the statistics the nonparametric Mann-Whitney test was used.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

other: vehicle served as negative control

Positive controls validity:

valid

PRE-EXPERIMENT

The test item was available as solution in propylene glycol with a content of 4-Vinylphenol of 9.7%.

To rule out that propylene glycol per se causes systemic toxicity, the highest non-toxic dose level of propylene glycol was determined first. One male mouse received a single dose of 10 mL/kg bw pure propylene glycol ip and showed strong signs of systemic toxicity such as reduction of spontaneous activity, prone position, ataxia, bradykinesia, constricted abdomen and abnormal breathing. Based on animal welfare aspects the mouse was euthanized one hour after application. The dose was reduced to 2 mL/kg bw and applied ip to one male animal. The mouse showed strong systemic toxicity such as reduction of spontaneous activity, prone position, ataxia, bradykinesia and hunched posture and was euthanized two hours after application based on animal welfare aspects. The dose was reduced to 1 mL/kg bw and applied ip to one male animal. The mouse showed clear signs of systemic toxicity such as reduction of spontaneous activity, prone position, ataxia, bradykinesia and constricted abdomen and was euthanized 30 minutes after application based on animal welfare aspects. The dose was further reduced to 0.5 mL/kg bw and applied ip to one male animal. This animal did not show any clinical signs of systemic toxicity during a time period of two hours after application. Thus the animal received a second dose of 0.5 mL/kg bw propylene glycol ip two hours after the first application and did not show any symptoms during a time period of 72 h.

Based on these findings a cumulative intraperitoneal dose of propylene glycol of 1 mL/kg bw applied as split dose of two times 0.5 mL/kg bw with a time period of 2 h between applications was

determined as the highest non-toxic dose of propylene glycol.

To obtain a non-toxic dose of propylene glycol the test item was further diluted in Aqua ad iniectabilia at a ratio of 1:20 (w/v) leading to a concentration of the 4-Vinylphenol/propylene glycol

solution of 50 mg/mL corresponding to 4.85 mg/mL 4-Vinylphenol. The test item was administered two times at a dose of 500 mg/kg bw 4-Vinylphenol/propylene glycol solution corresponding to 48.5 mg/kg bw 4-Vinylphenol with a time period of 2 h between applications leading to a cumulative dose of 97 mg/kg bw 4-Vinylphenol.

The selection of the highest dose was conducted in accordance with the following current international guidelines for assessment of acute toxicity (OECD 420, OECD 423), particularly with respect to selection of dose spacing and animal welfare aspects.

In the pre-experiment a cumulative dose of 97 mg/kg bw of 4-Vinylphenol administered as split dose of two applications with 48.5 mg/kg bw each was evaluated. Three male and three female mice received a cumulative dose of 97 mg/kg bw ip and showed toxicity such as reduction of spontaneous activity, prone position, bradykinesia, constricted abdomen, ataxia, hunched posture, piloerection and half eyelid / eye closure.

Due to the results obtained in the pre-experiment 97 mg/kg bw 4-Vinylphenol was chosen as maximum tolerated dose (1 MTD) in the main experiment.

Signs of toxicity in the pre-experiment (97 mg/kg bw)

Signs	Time post-application / sex (3 male and 3 female mice)
	1. Application  30 min		1 h		2 h		2. Application 30 min		1 h		2 h		3 h		4 h		24 h		48 h		72 h
	m	f	m	f	m	f	m	f	m	f	m	f	m	f	m	f	m	f	m	f	m	f
Red. spont. activity	2	3	3	3	2	3	3	3	3	3	3	3	3	3	3	3	0	0	0	0	0	0
Prone position	2	0	1	0	0	0	0	0	3	0	3	0	2	0	2	0	0	0	0	0	0	0
Constricted abdomen	2	3	2	3	2	3	3	3	2	3	2	3	0	0	0	0	0	0	0	0	0	0
Bradykinesia	0	0	1	0	0	0	0	0	3	0	2	3	2	0	2	0	0	0	0	0	0	0
Ataxia	0	0	0	0	0	0	2	3	2	3	2	3	0	0	0	0	0	0	0	0	0	0
Hunched posture	2	3	2	3	2	3	2	3	2	3	0	3	0	0	0	3	0	0	0	0	0	0
Piloerection	0	0	1	0	1	0	0	0	1	3	0	3	1	3	1	0	1	0	0	0	0	0
Half eyelid closure	2	3	3	3	3	3	3	3	3	3	3	3	3	3	1	3	0	0	0	0	0	0
Eye closure	0	0	0	0	0	0	0	0	0	0	0	0	0	0	2	0	0	0	0	0	0	0

MAIN EXPERIMENT

Toxicity

All animals treated with the highest dose (1 MTD) showed strong toxic effects. Male animals showed symptoms until 44 h after the second application. Female animals showed toxic symptoms until 24 h after the second application. Mice treated with 48.5 mg/kg bw (0.5 MTD) showed the same signs of toxicity as displayed for the 1 MTD dose group animals execpt for ataxia,

hunched posture and eye closure. These signs of toxicity were in total less intensely developed (mild/moderate) than in the 1 MTD group animals. 24 h after the second application no toxic

symptoms were observed anymore in the male and female 0.5 MTD animals. The animals treated with 19.4 mg/kg bw (0.2 MTD) showed mild toxic effects after the treatment with the test item. 1 h after the second applieation no toxic symptoms were observed anymore in this group.

The weight variation of the animals did not exceed ± 20% of the mean weight of each sex, as recommended in OECD guideline 474.

Signs of toxicity in the main experiment (97 mg/kg bw)

Signs	Time post-application / sex (5 male and 5 female mice)
	1. Application  30 min		1 h		2 h		2. Application 30 min		1 h		2 h		3 h		4 h		24 h		48 h		72 h
	m	f	m	f	m	f	m	f	m	f	m	f	m	f	m	f	m	f	m	f	m	f
Red. spont. activity	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	5	0	0	0
Prone position	5	5	5	5	0	0	5	0	0	0	5	0	5	0	0	0	0	0	0	0	0	0
Constricted abdomen	0	5	5	5	5	0	5	5	5	5	0	5	0	5	0	5	0	0	0	0	0	0
Bradykinesia	5	5	5	5	0	0	5	5	5	5	5	5	5	5	0	5	0	0	0	0	0	0
Ataxia	0	0	5	5	0	0	5	5	5	5	5	0	5	0	5	0	0	0	0	0	0	0
Hunched posture	0	0	0	0	0	0	0	0	0	0	0	0	0	5	5	5	0	0	0	0	0	0
Half eyelid closure	0	5	5	5	5	5	0	5	5	5	0	0	5	5	5	5	5	5	0	0	0	0
Eye closure	5	0	0	0	0	0	5	0	0	0	5	5	0	0	2	0	0	0	0	0	0	0

Relative PCE

The relative PCE (rel. PCE = proportion of polychromatic (immature) erythrocytes among total erythrocytes) was determined for each animal. The relative PCE is the supportive end point to assess

cytotoxicity, which helps to demonstrate a target cell exposure with the test item.

The negative controls (44 h, 68 h) were within the historical control limits of the negative control (1.12% - 4.46% for males, 0.88% - 3.16% for females). The mean values noted for the 44 h negative control were 2.27% (male mice) and 1.50% (female mice). The mean values detected for the 68 h negative control were 3.05% (male mice) and 2.02% (female mice).

The animal group treated with 0.2 MTD showed mean values of the relative PCE of 1.91 % (male mice) and 2.38% (female mice). The mean value observed in the male group was decreased and the value noted in the female group was statistically significantly increased as compared to the concurrent negative control. However, the mean values were within the historical control limits of the negative control.

The dose groups which were treated with 0.5 MTD showed mean values of the relative PCE of 2.30% (male mice) and 1.68% (female mice). The values observed in both groups were slightly increased compared to the concurrent negative control, but these differences were not statistically significant. Moreover, the me an values were within the historical control limits of the negative control.

The animals who received 1 MTD (44 h sampling) showed mean values of 1.81% (male mice) and 0.98% (female mice). The values observed in both groups were decreased compared to the concurrent negative control, but these differences were not statistically significant. Moreover, the mean values were within the historical control limits of the negative control.

The animal group which was treated with 1 MTD (68 h sampling) showed mean values of the relative PCE of 1.12% (male mice) and 0.93% (female mice). The values observed in both groups

were statistically significantly decreased compared to the concurrent negative control.

The decrease and/or increase of PCE va lues in treated animals compared to control animals is a hint of a target cell exposure of the test item.

Micronucleated polychromatic erythrocytes

For all dose groups, including positive and negative controls, 10000 immature erythrocytes per animal were scored for the incidence of micronucleated immature erythrocytes.

The negative controls (44 hand 68 h) evaluated were within the historical control limits of the negative control (0.14 - 0.32% for males, 0.13% - 0.31 % for females). The mean values of micronuclei observed for the negative control (44 h) were 0.21 % (male mice) and 0.23% (female mice). The mean values of the 68 h negative control were 0.18% (male mice) and 0.21 % (female mice).

The mean values of micronuclei observed after treatment with 0.2 MTD were 0.31% (male mice) and 0.24% (female mice). The value observed in the male group was increased compared to the concurrent negative control, but this increase was not statistically significant. Moreover, the value was within the historical control limits of the negative control. The value observed in the female group was within the range of the concurrent negative control and within the historical control limits of the negative control.

The mean values noted for the 0.5 MTD dose group were 0.24% (male mice) and 0.24% (female mice). The values observed in the male and female group were within the range of the concurrent negative control and within the historical control limits of the negative control.

The dose group treated with 1 MTD (44 h sampling) showed mean values of 0.31 % (male mice) and 0.18% (female mice). The value observed in the male group was increased compared to the concurrent negative control, but this increase was not statistically significant. Moreover, the value was within the historical control limits of the negative control. The value observed in the female group was within the range of the concurrent negative control and within the historical control limits of the negative control.

The mean values observed for the 1 MTD (68 h sampling) were 0.23% (male mice) and 0.15% (female mice). The values observed in the male and female group were within the range of the concurrent negative control and within the historical control limits of the negative control.

No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.

Conclusions:

In conclusion, it can be stated that during the study and under the experimental conditions reported, the test item 4-Vinylphenol did not induce structural and/or numerical chromosomal damage in the immature erythrocytes of the mouse.
Therefore, the test item 4-Vinylphenol is considered to be non-mutagenic with respect to clastogenicity and/or aneugenicity in the Mammalian Erythrocyte Micronucleus Test.

Executive summary:

This study was performed to investigate the potential of 4-Vinylphenol to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse, which is the endpoint of this test

to assess genotoxicity.

The test item was available as solution in propylene glycol with a content of 4-Vinylphenol of 9.7%. To obtain a non-toxic dose of propylene glycol the test item was further diluted in Aqua ad iniectabilia. For all dose groups the test item was administered as split dose with a time period of 2 h between both applications. The volume administered ip for each application was 10 mL/kg bw. Peripheral blood samples were collected for micronuclei analysis 44 h and 68 h after the second application of the test item.

A pre-experiment was performed as dose range finding study based on the OECD guideline 474 and other relevant documents (OECD 420, OECD 423). Based on the outcome of the dose range finding study, a dose of 97 mg/kg bw was selected as maximum tolerated dose (MTD).

In the main experiment three dose levels were used covering a range from the maximum tolerated dose to little or no toxicity. The following dose groups were selected based on the toxicity observed

in the pre-experiment:

Doses	Concentration [mg/kg bw]
1 MTD	97
0.5 MTD	48.5
0.2 MTD	19.4

The animals treated with doses of 0.2 MTD and 0.5 MTD showed mild and moderate signs of systemic toxicity, respectively. The animals treated with a dose of 1 MTD showed strong signs of systemic toxicity such as reduction of spontaneous activity, prone position, constricted abdomen, bradykinesia, ataxia, hunched posture and half eyelid / eye closure.

For all dose groups, including positive and negative controls, 10000 polychromatic erythrocytes per animal were scored for incidence of micronucleated immature erythrocytes. The negative controls

(44 h, 68 h) were within the historical control limits of the negative control. The mean values noted for the dose groups which were treated with the test item (44 h, 68 h) were within the range of the

concurrent negative control except for the values of the 0.2 MTD and 1 MTD (44 h) male dose groups that were increased compared to the concurrent negative control. However, these increases

were not statistically significant and the values were within the historical control limits of the negative control. Thus the observed increases were considered as not biologically relevant.

No biologically relevant increase of micronuclei was found after treatment with the test item in any of the dose groups evaluated.

The nonparametric Mann-Whitney Test was performed to verify the results. No statistically significant increases (p< 0.05) of cells with micronuclei were noted in the dose groups of the test item evaluated. Additionally, the X2 Test for trend was performed to test whether there is a dose related increase in the micronucleated cells frequency of the dose groups of the 44 h sampling time.

No statistically significant increase in the frequency of micronucleated cells was observed.

Cyclophosphamide (40 mg/kg bw) administered ip was used as positive control, which induced a statistically significant increase in the micronucleus frequency. This demonstrates the validity of the

assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on available information, 4-ethylphenol is not to be classified according to Regulation (EC) No 1272/2008 in terms of genotoxicity.