4-phenylbutenone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

1) AMES-Test (S. typhimurium TA 98, TA 100, TA 1535, TA 1537, E. coli WP2 uvrA ; Japan MHLW, 2009), positive with metabolic activation in TA 100.

2) Mouse lymphoma mutation assay, Seifried, 2006, positive without metabolic activation at a dose of 50 µg/mL

3) in vitro Chromosomal aberration test, Japan MHLW, 2009, no ability to induce chromosome abnormality, but has the ability to induce chromosomal structure abnormality

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well-documented publication, which meets basic scientific principles

Principles of method if other than guideline:

The toxicity of each chemical was determined both with and without liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats. S9 mix was prepared according to the procedure of Clive et al.. The mutagenicity assay was performed according to the procedure described by Clive and Spector.

GLP compliance:

not specified

Type of assay:

mammalian cell gene mutation assay

Target gene:

tymidine-kinase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

L5178Y TK+/- 3.7.C mouse lymphoma cells were originally obtained from Dr. Donald Clive, Burroughs Wellcome Co. (Research Triangle Park, NC). The cells were grown in Fischer’s medium for leukemic cells of mice (Gibco, Grand Island, NY, or Quality Biological, Gaithersburg, MD) supplemented with 10% horse serum (Gibco or Hyclone, Logan, UT) and 0.02% pluronic F-68 (BASF Wyandotte Corp., Wyandotte, MI). Cells were screened for the presence of mycoplasma after cryopreservation. New cultures were initiated at approximately 3 month intervals from cells stored in liquid N2.

Metabolic activation:

with and without

Metabolic activation system:

The toxicity of each chemical was determined both with and without liver S9 prepared from Aroclor 1254-induced male Sprague-Dawley rats.

Test concentrations with justification for top dose:

10, 20 ,30, 40 and 50 µg/ mL (tests without metabolic activation).
20, 30, 40, 50 and 60 µg (tests with metabolic acitvation).
The doses of chemical selected for testing were within the range yielding approximately 0-90% cytotoxicity).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: not given

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

7,12-dimethylbenzanthracene

9,10-dimethylbenzanthracene

ethylmethanesulphonate

methylmethanesulfonate

Remarks:

ethyl methylsulfonate at 4.7 * 10E-6 M (or methyl methanesulfonate at 10-20 µg/mL) (test without metabolic activation) & 3-methylcholanthrene at 1.86 * 10-5 M (or dimethylbenz[aanthracene at 0.5 - 4 µg/mL (tests with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 48 h
- Selection time (if incubation with a selection agent): trifluorothymidine (TFT, final concentration 3 µg/mL)
- Fixation time (start of exposure up to fixation or harvest of cells): Plates were incubated at 37 (1 °C in 5% CO2 in air for 10-12 days and then counted with an Artek automated colony counter (Artek 982, DynaTech) or ProtoCol colony counter (Synbiosis, Frederick, MD). Only colonies larger than 0.2 mm in diameter were counted. Mutant frequencies were expressed as mutants per 106 surviving cells.

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

OTHER:
S9 mix was prepared according to the procedure of Clive et al..

Evaluation criteria:

Results from this study were interpreted using a doubling of the mutant frequency over the concurrent solvent-treated control value as an indication of a positive effect, together with evidence of a dose-related increase.
Only doses yielding total growth values of 10% were used in the analysis of induced mutant frequency. Doses yielding less than 10% total growth were used in determining dose response. The size of mutant mouse lymphoma colonies was also determined using an Artek 982 colony counter/sizer or the ProtoCol colony counter. An internal discriminator was set to step sequentially to exclude increasingly larger colonies in approximate increments of 0.1 mm in colony diameter. The size range used was from 0.2 to 1.1 mm.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the highest and second highest concentration tested the average viable cell number was reduced ( 82 and 84 or 122 and 128, compared to 143 in the controls, respectively.

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the highest concentration tested the average viable cell number was reduced ( 96 and 100, compared to 118 in the controls, respectively)

Vehicle controls validity:

valid

Positive controls validity:

valid

Table 1 - Mouse Lymphoma Test Data
			Non-Activated Cultures					S9-Activated Cultures
Chemical Name	Solvent	Dose  (µg/mL)	Average TFT	Average VC	Mut Freq	RTG	Dose  (µg/mL)	Average TFT	Average VC	Mut Freq	RTG
		or µL/mL					or µL/mL
Benxylideneacetone (Methyl styryl ketone)	Ethanol	10 to 60
		10	20	148	0,27	92	20	23	119	0,39	73
			17	166	0,2	120		18	109	0,33	66
		20	31	130	0,48	66	30	35	129	0,54	35
			33	153	0,43	89		29	125	0,46	37
		30	35	137	0,51	51	40	26	141	0,37	21
			30	139	0,43	63		29	126	0,46	23
		40	46	122	0,75	38	50	32	123	0,52	14
			46	128	0,72	32		32	116	0,55	13
		50	63	82	1,54	11	60	28	100	0,56	8
			61	84	1,45	10		33	96	0,69	8
		Solvent	18	143	0,25		Solvent	21	118	0,34
		Positive	338	71	9,52	37	Positive	150	69	4,35	31

Conclusions:

Positive without metabolic activation: a clear positive result was obtained in the mouse lymphoma assay without metabolic activation at the dose level of 50 µg/mL.
Negative with metabolic activation: a negative results was obtained in the mouse lymphoma assay with metabolic acitvation at dose levels up to 60 µg/mL.

Benzylideneacetone was found to induce a clearly positive result in L5178 TK +/- 3.7C mouse lmyphoma cells at dose levels of 50 µg/mL when the test was conduced without metabolic activation. However, negative results were obtained in the tests with metabolic activation. As the data comes from a well-documented publication and the solvent controls and positive controls were valid, the source is considered to be of high quality and reliability (Klimisch 2).

Executive summary:

Seifried and collagues compiled in their publication the results of genetic mutations assays (AMES-tests) and Mouse-lymphoma Test data of more than 450 chemicals (Seifried et al., 2006). The test results were obtained from the data banc of the National Cancer Institute (NCI), which is responsible for the selection of the most significant chemicals for carinogenicity testing by the National Toxicology Program (NTP). As only some of the testing data available at the NCI has been made available in summary form in the Chemical Carcinogenisis Research Information System

(CCRIS), which is searchable on the NLM TOXNET system, they decided to compile the data of these chemicals in a summary table that presents the results for each compound. Benzylideneacetone (CAS 122 -57 -6) is given with a positive result in the Salmonella typhimurium strain TA 100 with metabolic activation (either rat S9 or hamster S9). Moreover, benzylideneacetone was found to be positive in the non activated mouse lymphoma test (doses 10 - 50 µg/mL, resulted in a lowest effective dose of 20 and 50). In case of test with metabolic activation (20 - 60 µg/mL), the results were either weakly positive or negative.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-01-15 - 2009-10-16

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

not specified

Remarks:

Not specified but it is assumed that GLP criteria have been applied.

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

Test concentrations with justification for top dose:

19.5 - 5000 µg/plate

6 times of 39.1~1250 μg/plate were carried out for S. typhimurium TA strain when not metabolically activated, for S. typhimurium TA 100, TA 1535 and TA 1537 when metabolically activated; 6 times of 156~5000 μg/plate were carried for S. typhimurium TA 98 when metabolically activated, for E. coli WP2 uvrA with or without metabolic activation.

Rationale: Based on dose-finding test results, as the highest dose level of the test substance in the main test, the 1250 μg / plate dose was selected for S. typhimurium TA without metabolic activation and S. typhimurium TA 100, TA 1535, TA 1537 with metabolism activation, 5000 μg/plate for S. typhimurium TA 98 with metabolism activation and for E. coli WP2 uvrA with/without metabolic activation. These highest doses were diluted 5 times (using a common ratio of 2) to provide a total of 6 dose levels. Main test was performed twice at the same doses.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

furylfuramide

other: ICR191,2AA

Details on test system and experimental conditions:

Test Duration:
- Preincubation period: 20 min (only in the second experiment)
- Exposure duration: 48 h (the first main test), 48.5 h (the second main test)

Number of Replications: 2 plates were used in the dose setting test and 3 plates were used in the 2 main tests

Evaluation criteria:

if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged positive. Statistical analysis was not done.
When the number of revertant colonies on the test plates shows an increase of 2 times or more in comparison with that of spontaneously revertant colonies (negative control value), when dose response and reproducibility are observed or even when it does not show clear dose response but it shows an increase of more than twice the number of spontaneously revertant colonies, it is judged positive when the reproducibility is confirmed in two main tests.
For the measurement results, average values ± standard deviation are also described.

Statistics:

Statistical analysis was not done.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535 pSK1002

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

 Observation result after completion of incubation: precipitation and discoloration on the test substance plates were not observed in any dose with or without metabolic activation. Through stereoscopic microscope observation, growth limitation was confirmed, by S. typhimurium TA strain without metabolically activation and S. typhimurium TA 100, TA 1535, TA 1537 with metabolism activation at above 1250 μg/plate, by S. typhimurium TA 98 with metabolic activation and E. coli WP2 uvrA with or without metabolic activation at above 2500 μg/plate.

Number of revertant colonies: in both of the two tests, an dose-dependent increase in the number of revertant colonies which is more than twice as much as the negative control value, was observed in S. typhimurium TA 100 with metabolically activated, indicating reproducibility.

Validity: the number of revertant colonies with positive control is more than twice that of the negative control of each strain, and the average number of revertant colonies in negative control group and in positive control group are within the control limit of background data (Average value ± 3SD), and no abnormality such as contamination of germs for the sterility and the test operation was observed, it was therefore judged this test was properly carried out.

Conclusions:

It was judged that methyl styryl keton has the ability to reverse mutagenize against S. typhimurium TA 100 when metabolically activated (is positive) under the present test conditions.

Executive summary:

Mutagenicity potential of methyl styryl keton was assessed with Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA, with or without metabolic activation, in the preincubation method.

For the test, test substance treatment dose range is 19.5~5000 µg/plate.

6 times of 39.1~1250μg/plate were carried out for S. typhimurium TA strain when not metabolically activated, for S. typhimurium TA 100, TA 1535 and TA 1537 when metabolically activated; 6 times of 156~5000 μg/plate were carried for S. typhimurium TA 98 when metabolically activated, for E. coli WP 2uvrA with or without metabolic activation.

1) Precipitation and coloring by the test substance

Precipitation and discoloration on the plate by the test substance were not observed in any dose with or without metabolic activation.

2) Growth Inhibition

Through stereoscopic microscope observation, growth limitation was confirmed, by S. typhimurium TA strain without metabolic activation and S. typhimurium TA 100, TA 1535, TA 1537 with metabolic activation at above 1250 μg/plate, by S. typhimurium TA 98 with metabolic activation and E. coli WP2 uvrA with or without metabolic activation at above 2500 μg/plate .

3) Number of revertant colonies

In both of the two tests, an dose-dependent increase in the number of revertant colonies was observed inS. typhimurium TA 100 when metabolically activated, which is twice more than the negative control value, indicated reproducibility.  When the specific mutation colony number was twice more than the negative control value and the reproducibility was observed, the specific activity value was determined to be 258 (Rev / mg) as the maximum, and the mutagenicity of this test substance was judged to be weak. 

Based on the above test results, it was judged that Methyl styryl keton has the ability to reverse mutagenize against bacteria (is positive) under the present test conditions (in S. typhimurium TA 100 when metabolically activated).

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-01-30 - 2009-02-26

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

not specified

GLP compliance:

not specified

Remarks:

it is not specified but it can be assumed that the study was conducted according to GLP criteria

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Remarks:

CHLIIU cells

Details on mammalian cell type (if applicable):

Cell line: Chinese hamster lung fibroblasts (CHLIIU cells), received on Nov. 2 of 2004. 19th generation was used for Cell proliferation inhibition test, 23rd generation for Chromosome aberration test (short-time treatment).
Reason for choice: low naturally occurred chromosomal aberrant rate, high sensitivity to various chemical substances, rich background data, widely used for chromosome aberration tests.
Culture condition:
CO2 incubator, CO2 concentration: 5%, 37°C under humid condition

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

Cell proliferation inhibition test: the maximum concentration was set at 1,500 μg/mL (equivalent to 10 mM), and concentrations of 750, 375, 188, 93.8, 46.9, 23.4 and 11.7 μg/mL (common ratio 2) were set. In addition, a negative control group was provided.
Chromosome aberration test with metabolic activation: for short-term treatment with metabolic activation was 150 μg/mL as highest concentration, then diluted by factor 1.5 to 5 groups; for short-term treatment without metabolic activation was 150 μg/mL, then diluted by factor 1.5 to 5 groups; for 24hr continuous treatment method was 40.0 μg/mL as highest concentration, then diluted by factor 1.5 to 5 groups; for 48 hr continuous treatment method was 26.7 μg/mL as highest concentration, then diluted by factor 1.5 to 6 groups. Since a clear positive result was obtained in the short-time treatment, the continuous treatment method and the confirmation / additional test were not carried out.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Reason for choice: it was confirmed that the test substance was hardly soluble in water and the test substance was dissolved at 150 mg/mL in DMSO.

Untreated negative controls:

no

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium on plate

Cell proliferation inhibition test:
1) short-time treatment method
(1) Negative control group and test substance treated group each with and without metabolic activation were provided. Two plates were used for each group.
(2) 2*10E4 cells per plate. After three days culturing, observation under microscope and confirmation of no abnormality in the cells were made. Then after addition of S9/test substance in each group, the cells were cultured for 6 hours.
(3) Six hours later, precipitation of the test substance was checked visually, and the state of the cells was visually checked under microscope. Subsequently, the cells were washed with a saline solution to which bovine serum had been added, 5.0 mL of a new culture media was added, then further cultured for 18 hours.
(4) Subsequently, precipitation of the test substance was checked visually, and the state of the cells was visually checked under microscope. The cell density was measured using a monolayer cell density measuring apparatus, and the value of the negative control group was taken as 100%, and for each group with/without metabolic activation, 50% Cell proliferation inhibitory concentration (approximate value) was determined.

Chromosome aberration test:
(1) Negative control group and test substance treated group for each of the 24-hour treatment and the 48-hour treatment. Two plates were used for each group.
(2) 2*10E4 cells per plate. After three days culturing, observation under microscope and confirmation of no abnormality in the cells were made. Then after addition of test substance in each group, the cells were cultured for 24 hours and 48hr respectively.
(3) 24 hr/48hr later, precipitation of the test substance was checked visually, and the state of the cells was visually checked under microscope. After washing, fixing, staining and cell density measuring, 24hr and 48hr 50% Cell proliferation inhibitory concentration (approximate value) was determined.

Chromosome aberration test:
1) Negative control group, test substance treated group and positive control group were provided for each of the 24-hour treatment and the 48-hour treatment, with or without metabolic activation. 4 plates per group.
2) 2*104 cells per plate. After three days culturing, observation under microscope and confirmation of no abnormality in the cells were made. Then after addition of test substance etc. precipitation and the color were confirmed by naked eyes and cultured for 6 hours.
3) Six hours later, precipitation of the test substance was checked visually, and the state of the cells was visually checked under microscope. Subsequently, the cells were washed with a saline solution to which bovine serum had been added, 5.0 mL of a new culture media was added, then further cultured for 18 hours.
4) Two specimens were prepared per plate (2 plates per group) for chromosome observation.
5) For remaining 2 plates per group, specimen stained with Krysuknole violet according to the cell proliferation inhibition test was prepared and the cell density was measured using a monolayer cell density measuring apparatus.

Observation of specimen:
Two hundreds well-spread metaphases (100 metaphases per dish) obtained from each dose group were observed under the microscopes, the type of structural abnormality and the number of cells with abnormality were recorded. At the same time, the number of polyploid was recorded. These observations were performed under blind method for objectivity.
The type of chromosome aberration:
Structure aberrations
Gap (g)
Chromatid break (ctb)
Chromatid exchange (cte) (quadriradial exchange, etc.)
Chromosome break (csb)
Chromosome exchange (cse)
Others (fragmentation, frg)

Numerical aberration: Polyploidy (endoreduplication incl.).

Evaluation criteria:

Evaluation Criteria: the statistical method was not used. It was judged as follows according to rate of abnormal chromosome structure and number of abnormal cells.
The frequencies of abnormal cells <5%: negative (-)
The frequencies of abnormal cells 5~10%: equivocal (±)
The frequencies of abnormal cells >10%: positive (+)

The total appearance rate of structural anomalies was divided into cases containing gaps (TAG) and those without gaps (TA), and overall judgment was made latter.
For the occurrence rate of abnormal cells, when dose dependency or reproducibility was recognized, it was judged as positive.

Statistics:

No statistical method was used.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Remarks:

CHLIIU cells

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

From the results, it was concluded that the substance has no ability to induce chromosome abnormality under this test condition, but has the ability to induce chromosomal structure abnormality.

Executive summary:

The ability of methyl styryl keton to induce chromosomal aberrations was examined by using Chinese hamster lung fibroblasts (CHLIIU cells).

Initially, the cell proliferation inhibition test was carried out with the highest concentration set at 1500 μg/mL (corresponding to 10 mM) as specified in the test guideline. The result was, the cell proliferation suppressing effect of the short-time treatment method was judged to be exceeding 50% at 93.8 μg/mL with metabolic activation and at 46.9 μg/mL without metabolic activation. Moreover, for the 24-hour treatment by continuous treatment method, 50% cytostatic effect was judged at 23.4 μg/mL. The 50% cell proliferation inhibitory concentration (approximate value) was 82.6 μg/mL with metabolic activation and 34.8 μg/mL without metabolic activation in the short-time treatment method, 23.4 μg/mL for 24-hour treatment in the continuous treatment method. For the 48-hour treatment of the continuous treatment method, the cell proliferation inhibiting effect not exceeding 50% was not recognized even at the lowest concentration of 11.7 μg/mL, the cell proliferation rate was 44%, the 50% cytostatic concentration (Approximate value) was below 11.7 μg/mL. Based on these results, according to the provision [when the cytotoxicity of 50% or more is recognized, the concentration at which cell proliferation is obviously suppressed by more than 50% is taken as the highest concentration] of guideline, the maximum concentration of each treatment method of Chromosome aberration test for short-term treatment was 150 μg/mL with metabolic activation, 100 μg/mL without metabolic activation, for continuous treatment method was 40.0 μg/mL for 24-hour treatment and was 26.7 μg/mL for 48 hours treatment respectively.

The result of the chromosomal aberration test under metabolic activation of the short-time treatment, following one index of chromosome structural aberrations --- the frequency (TA value) of cells with structural aberrations excluding gaps, was positive at 100 μg/mL, equivocal at 66.7 μg/mL, and increase of TA value with concentration increase was recognized, therefore was judged positive. For the index value of chromosome aberrant induction intensity, the D20 value --- 20% of observed cells showing abnormality was 0.075 mg/mL, the TR value which is a comparative value of the rate of cells having chromatid exchange per unit dose (cte) was 560. In addition, it was also judged positive as it was positive at 44.4 and 29.6 μg/mL for the short-time treatment method without metabolic activation, and a rise in TA value with concentration increase was recognized; The D20 value was 0.032 mg/mL and the TR value was 1100. On the other hand, the frequency of occurrence of polyploidy cells in all treatment methods and all concentration were less than 5% the negative criterion.

And in all treatment methods, the frequency of occurrence of cells and polyploidy with chromosomal structural abnormality in the negative control group was less than 5% and was within the negative criteria. In contrast, induction of significant chromosomal structural abnormality was recognized in the positive control group, so it was considered that the test was performed appropriately.

From the above results, it was concluded that the substance has no ability to induce chromosome abnormality under this test condition, but has the ability to induce chromosomal structure abnormality.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

1) Comet assay in male Wistar rat, oral gavage, Vértesi (2022), 300, 600, 1200 mg/kg bw, 

2) micronuclues assay in bone marrow cells of the mouse, Roth (2012), 500, 1000 and 2000 mg/kg bw, negative

3) micronuclues assay, NTP (2012), negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: well known data banc (NCI), which collects data of potentially dangerous chemicals (NTP)

Principles of method if other than guideline:

This data comes from the well known data base from the National Cancer Institute, which contains data collected of potentially dangerous chemicals. The data is considered to be reliable, however, no details on the methods used is given. It is only indicated, that this results were obtained by conducting a micronucleus test in B6C3F1 mice.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Species:

mouse

Strain:

B6C3F1

Sex:

male

Details on test animals or test system and environmental conditions:

no details given

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: no details given

Details on exposure:

intraperitoneal injection

Duration of treatment / exposure:

72 hours

Frequency of treatment:

3 times

Post exposure period:

24 hours

Dose / conc.:

37.612 mg/kg bw/day (nominal)

Dose / conc.:

75.625 mg/kg bw/day (nominal)

Dose / conc.:

151.25 mg/kg bw/day (nominal)

Dose / conc.:

302.5 mg/kg bw/day (nominal)

Dose / conc.:

605 mg/kg bw/day (nominal)

No. of animals per sex per dose:

no data available

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control(s): none given
- Route of administration: intraperitoneal injection
- Doses / concentrations: 25 mg/kg

Tissues and cell types examined:

no details given

Details of tissue and slide preparation:

no details given

Evaluation criteria:

no details given

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Remarks:

The animals treated with 1210 and 2420 mg/kg died therefore no evaluation of results was possbile in these animals

Vehicle controls validity:

valid

Positive controls validity:

valid

Bone Marrow
	Study ID		Result
			Sample Collection Time	Dose	Male		Female
	A43741				Negative		Not Tested
-A43741-1	Start Date				Sex	Methodology Used	Route	Dosing Regimen
	12/05/1994		24 Hours		Male	Slide Scoring	Intraperitoneal Injection	IP/I J x 3, 72 Hours
				(mg/kg)		Polychromatic Erythrocytes Trend P = 0.536
						No. Examined	Total MN Cells	Percent PCE	MN Cells per 1000
Vehicle Control CRO				0	389030	2000	2	43.8	1.0
				0	389049	2000	5	43.3	2.5
				0	389113	2000	1	43.3	0.5
				0	389119	2000	3	55.8	1.5
				0	389155	2000	1	53.7	0.5
Average ±5 EM								49.87 ± 0.03	1.20 ± 0.37
Test Chemical				37.612	389069	2000	2	64.2	1.0
				37.612	389078^d	0	0	0	0.0
				37.612	389148	2000	1	53.8	0.5
				37.612	389164	2000	2	55.1	1.0
				37.612	389178	2000	2	33.2	1.0
Average ±5 EM								39.20 ± 0.00	0.88 ± 0.13
Pairwise P									0.7477
Test Chemical				75.625	389089	2000	2	58.5	1.0
				75.625	389035	2000	5	48.1	2.5
				75.625	389096	2000	4	50.8	2.0
				75.625	389141	2000	3	53.3	1.5
				75.625	389171	2000	1	54.3	0.5
Average ±5 EM								48.10 ± 0.00	1.50 ± 0.35
									0.2817
Test Chemical				151.25	389047	2000	1	49.1	0.5
				151.25	389101	2000	2	65.8	1.0
				151.25	389160	2000	3	52.9	1.5
				151.25	389189	2000	1	61.0	0.5
				151.25	389190	2000	3	46.6	1.5
Average ±5 EM								47.85 ± 1.25	1.00 ± 0.22
Pairwise P									0.6652
Test Chemical				302.5	389031	2000	4	57.3	2.0
				302.5	389090	2000	3	50.6	1.5
				302.5	389109	2000	2	57.0	1.0
				302.5	389110	2000	2	60.3	1.0
				302.5	389153	2000	2	55.9	1.0
Average ±5 EM								56.22 ± 1.58	1.30 ± 0.20
Pairwise P									0.4207
Test Chemical				605	389025	2000	0	48.7	0.0
				605	389063	2000	1	50.3	0.5
				605	389067	2000	2	55.4	1.0
				605	389123	2000	5	54.1	2.5
				605	389175	2000	3	52.5	1.5
Average ±5 EM								48.70 ± 0.00	1.10 ± 0.43
Pairwise P									0.5826
Test Chemical				1210	389035^d	0	0	N/A	0.0
				1210	389065^d	0	0	N/A	0.0
				1210	389104^d	0	0	N/A	0.0
				1210	389108^d	0	0	N/A	0.0
				1210	389157^d	0	0	N/A	0.0
Average ±5 EM								N/A ± N/A	0.00 ± 0.00
Pairwise P
Test Chemical				2420	389085^d	0	0	N/A	0.0
				2420	389098^d	0	0	N/A	0.0
				2420	389115^d	0	0	N/A	0.0
				2420	389125^d	0	0	N/A	0.0
				2420	389174^d	0	0	N/A	0.0
Average ±5 EM								N/A ± N/A	0.00 ± 0.00
Pairwise P
Positive Control CPA				25	389043	2000	25	48.4	12.5
				25	389046	2000	31	33 8	15 5
				25	389082	2000	24	45.2	12.0
				25	389176	2000	17	47.9	8.5
				25	389180	2000	36	42.1	18.0
Average ±5 EM								44.68 ± 1.66	13.30 ±1.62
Pairwise P									< 0.0001
Abbreviations:
CRO = Corn Oil
d = dead
* = insufficient animals scored to calculate p-value
CPA = Cyclophosphamide

Conclusions:

The NTP is a reliable source to obtain data for several substances. This source is considered to be of high reliability (kimlisch 2). Moreover the positive and solvent controls were valid. Therefore the data is considered to be suitable to fulfill the requirements for the registration under REACH. The substance is considered to be negative for mutagenicity.

Executive summary:

This micronucleus test conducted with B6C3F1 mice was obtained from the National Toxicology Program, which collects data for possible dangerous substances. Its reliability is considered to be high (Klimisch 2). The test revealed no positive results for methyl styryl ketone (CAS No. 122-57-6).

Reference 2

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Remarks:

Comet assay / OECD 489

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 DEC 2021 - 19 AUG 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)

Version / remarks:

adopted 29th July, 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian comet assay

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Rat, HAN:WIST of Wistar origin

Rats are routinely used in this test and the chosen Wistar rat was selected due to a wide range of experience with this strain of rat in corresponding toxicity studies and historical control data at TOXI-COOP ZRT

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103 Budapest, Cserkesz u. 90.
- Age at study initiation: 6-10 weeks at commencement of the treatment
- Weight at study initiation: The weight variation in animals involved at the starting point of the test will not exceed ± 20 %.
- Assigned to test groups randomly: yes, under following basis: All animals will be sorted according to body weight by computer and grouped according to weight ranges. There will be an equal number of animals from each weight group in each of the experimental groups during the randomization. The grouping will be controlled by SPSS/PC computer program according to the actual body weight verifying the homogeneity and deviations among the groups and cages.
- Fasting period before study: no
- Housing: 2 - 3 animals/cage (Type III polypropylene/polycarbonate)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70 %
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: sunflower oil
- Concentration of test material in vehicle: 240, 120 and 60 mg/mL
- Amount of vehicle (if gavage or dermal): 5 mL/kg bw
- Lot/batch no. (if required): 8007391006

The positive control It will be dissolved and applied in ultrapure water (ASTM Type I), at a concentration of 20 mg/mL just before the treatment.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The planned doses are 1200, 600 and 300 mg/kg bw/day, consequently the test item will be formulated in the vehicle, sunflower oil in concentrations of 240, 120 and 60 mg/mL. The formulations will be prepared in the laboratory of the Test Facility just before each treatment.

Duration of treatment / exposure:

2 days (test item)
1 day (positive control)

Frequency of treatment:

once daily

Post exposure period:

the sampling will be performed 3-4 hours after the second treatment

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

600 mg/kg bw/day (nominal)

Dose / conc.:

1 200 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 animals in the dose groups and negative control group, respetively;
4 animals in the positive control group.

Control animals:

yes, concurrent vehicle

Positive control(s):

ethylmethanesulphonate
- Justification for choice of positive control(s): In this study a separate positive control group with a known mutagen will be used for the tissues.
- Route of administration: oral gavage
- Doses / concentrations: 200 mg/kg bw

Tissues and cell types examined:

Glandular Stomach Cells, Duodenum Cells, Liver Cells, Gonadal cells (isolated, not examined)

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
For dose selection the corresponding OECD 489 guideline proposal, the information provided by Sponsor; furthermore, the results of non-GLP preliminary dose-range finding tests are taken into consideration. For the correct identification of the maximum tolerated dose (MTD) where no death, evidence of pain, suffering or distress occurs, non-GLP dose range finding tests were performed in the testing laboratory, using the same species, strain, sex, and treatment regimen (administration of doses by oral gavage twice: once on the day 0 and 24 hours thereafter) to be used in the main study. Each test was performed with two animals. The dose range-finding tests were started with 2000 mg/kg bw/day. Because of the observed toxic effects, the dose was lowered gradually to 1000 mg/kg bw/day. At the dose of 2000 mg/kg bw/day definite behavioral changes (reduced activity or hyperactivity, incoordination) appeared after the first treatment and these symptoms escalated to motionless, lying animals without reactions to the end of the observation period after the second treatment. These behavioral changes appeared at the investigated lower dose levels (at 1800 and 1500 mg/kg bw/day) at different timepoints within the observation periods (e.g.: after the first treatment the signs appeared equivocally at 1800 mg/kg bw/day, and no signs were noticed at 1500 mg/kg bw/day), but they reached the same intensity (motionless lying animals) to the end of the observation period after the second treatment. Incoordination, slight piloerection and slightly reduced activity were noticed at the end of the observation period (3-hour) after the second treatment at 1200 mg/kg bw/day, and incoordination and slight piloerection at 1000 mg/kg bw/day. Necropsy was performed on animals of the dose 1500 mg/kg bw/day. At the macroscopic examination of stomach in the forestomach tympanites, hyperaemia, bruising were described; furthermore faint structured glandular stomach was noticed; however, the latter signs were rather subjective observations. The non-GLP preliminary dose-range finding tests were not performed in compliance with the GLP-Regulations and will be excluded from the Statement of Compliance in the final report, but the raw data of these tests will be archived under the study code of the present study.
Based on the results of the preliminary dose-range finding tests the maximum dose will be 1200 mg/kg bw/day, and in addition to the maximum dose two additional doses, 600 and 300 mg/kg bw/day are selected.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The test item will be administered in doses by oral gavage twice: once on the day 0 and 24 hours thereafter (on day 1). The negative control animals will be treated concurrently with the vehicle only. The positive control animals will be treated by oral gavage once during the experiment on the day 1.
The sampling time is a critical variable because it is determined by the period needed for the test chemicals to reach maximum concentration in the target tissue and for DNA strand breaks to be induced but before those breaks are removed, repaired or lead to cell death. A suitable compromise for the measurement of genotoxicity is to sample at 2-6 hour after the last treatment. In this particular test and based on the experience of the laboratory over previous years the sampling will be performed 3-4 hours after the second treatment (the animals will be euthanized consistent with the effective animal welfare legislation and 3Rs
principles and the cells of the target tissues will be isolated) and care will be taken to necropsy all animals at the same time after the last dose.

DETAILS OF SLIDE PREPARATION:
Isolation of Glandular Stomach Cells
The stomach will be cut open and washed free shortly from food using cold phosphate buffered saline. The forestomach will be removed and discarded. The glandular stomach will then be placed into cold mincing buffer and incubated on ice in a short period of time. After the incubation, the surface epithelia will be gently scraped using a scalpel blade. This layer will be discarded and the gastric mucosa will be rinsed with cold mincing buffer. The stomach epithelia will than carefully scraped at least 4-5 times with scalpel blade. The cell suspension will be stored on ice for about 30 seconds to allow the large clumps to settle. The supernatant will be used to prepare the comet slides.

Isolation of Duodenum Cells
A representative duodenum sample will be removed, cut lengthwise open and washed thoroughly with cold phosphate buffered saline. The duodenum sample will then be placed into cold mincing buffer and incubated on ice in a short period of time (< 1 minute), thereafter the surface epithelia will be gently scraped using tweezers and/or scalpel blade. The obtained cell suspension will be stored on ice for about 30 seconds to allow the large clumps to settle. The supernatant will be used to prepare the comet slides.

Isolation of Liver Cells
A portion of the left lateral lobe of the liver will be removed and washed in the cold mincing buffer until as much blood as possible will be removed; thereafter placed in mincing buffer (ice cold Hank’s Balanced Salt Solution (HBSS) containing 20 mM EDTA and 10 % DMSO), minced with a pair of fine scissors to release the cells. The cell suspension will be stored on ice for about 30 seconds to allow the large clumps to settle. The supernatant will be used to prepare the comet slides.

Isolation of Gonadal Cells
The left testis will be removed, rinsed with cold phosphate buffered saline; the tunica layers will be opened; removed, thereafter the seminiferous tubules will be placed into ice cold phosphate buffered saline and the tightly coiled seminiferous tubules will be gently agitated, shaken with tweezers for getting a loose structure. The loosen structure of seminiferous tubules will be cut with a pair of fine scissors; thereafter the obtained parts (a volume of ~0.5 mL) transferred with tweezers, pipettes into an Eppendorf tube. The seminiferous tubules will be suspended shortly with pipette (using an adequate tip). The cell suspension will be stored on ice for about 30 seconds to allow the large clumps to settle. The supernatant will be used to prepare the comet slides. The cell suspensions will be stored frozen until potential analysis. In the case of additional analysis, its details will be provided in an amendment to the study plan. If additional analysis will not be required, the samples will be discarded after delivery of the final report.

METHOD OF ANALYSIS:
Coded slides will be stained and blind scored. The comets will be measured via a digital camera linked to an image analyzer system using a fluorescence microscope equipped with an appropriate excitation filter at a magnification of 200X. For image analysis, the Komet software (Andor Technology) will be used. For each tissue sample, fifty cells per slide will be randomly scored i.e. 150 cells per animal (750 analyzed cells per test item treatment, per vehicle control and 450 per positive controls).
DNA strand breaks in the Comet Assay will be measured by independent endpoints such as % tail DNA, tail moment and tail length. Olive Tail Moment (OTM): is expressed in arbitrary units, is calculated by multiplying the percentage of DNA (fluorescence) in the tail by the length of the tail in μm. The tail length is measured between the center of the comet head and the end of the comet tail. The tail % DNA (also known as tail intensity) is recommended for the evaluation and interpretation of the results and determined by the DNA fragment intensity in the tail expressed as a percentage of the cell’s total intensity.

Evaluation criteria:

Providing that all validity criteria are fulfilled, the test chemical is clearly negative if:
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- there is no concentration-related increase when evaluated with an appropriate trend test;
- all results are inside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administration;
- direct or indirect evidence supportive of exposure of, or toxicity to, the target tissue(s) is demonstrated.

Providing that all validity criteria are fulfilled, the test chemical is clearly positive if:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control;
- the increase is dose-related when evaluated with an appropriate trend test;
- any of the results are outside the distribution of the historical negative control data for given species, vehicle, route, tissue and number of administration;

There is no requirement for verification of the clearly negative or positive response. In the case of equivocal results, the further process will be discussed with the Sponsor’s monitor.

Statistics:

The animal is the experimental unit and therefore both, individual animal data and the summarized results will be presented in tabular form. The median % tail DNA for each slide will be determined and the mean and the median values will be calculated for each animal. The mean of the individual animal means is then determined to give a group mean. The treatment group values will be compared with the negative control data with appropriate statistical analysis and also discussed in the context of the lab’s historical control database. The data will be checked for normality and for homogeneity of variance with the adequate statistical test methods.

Sex:

male

Genotoxicity:

not determined

Remarks:

final result not yet available

Toxicity:

yes

Vehicle controls validity:

not examined

Remarks:

final result not yet available

Negative controls validity:

not examined

Positive controls validity:

not examined

Remarks:

final result not yet available

Remarks on result:

other: The final results of slide analysis are not yet available. Please refer to attached statement.

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000, 1800, 1500, 1200, 1000 mg/kg bw/d
- Clinical signs of toxicity in test animals: At the dose of 2000 mg/kg bw/day definite behavioral changes (reduced activity or hyperactivity, incoordination) appeared after the first treatment and these symptoms escalated to motionless, lying animals without reactions to the end of the observation period after the second treatment. These behavioral changes appeared at the investigated lower dose levels (at 1800 and 1500 mg/kg bw/day) at different timepoints within the observation periods (e.g.: after the first treatment the signs appeared equivocally at 1800 mg/kg bw/day, and no signs were noticed at 1500 mg/kg bw/day), but they reached the same intensity (motionless lying animals) to the end of the observation period after the second treatment. Incoordination, slight piloerection and slightly reduced activity were noticed at the end of the observation period (3-hour) after the second treatment at 1200 mg/kg bw/day, and incoordination and slight piloerection at 1000 mg/kg bw/day.
- Evidence of cytotoxicity in tissue analysed: Necropsy was performed on animals of the dose 1500 mg/kg bw/day. At the macroscopic examination of stomach in the forestomach tympanites, hyperaemia, bruising were described; furthermore faint structured glandular stomach was noticed; however, the latter signs were rather subjective observations.
- Rationale for exposure: For the correct identification of the maximum tolerated dose (MTD) where no death, evidence of pain, suffering or distress occurs non-GLP dose range finding tests were performed in the testing laboratory, using the same species, strain, sex, and treatment regimen (administration of doses by oral gavage twice: once on the day 0 and 24 hours thereafter) to be used in the main study. Each test was performed with two animals.

RESULTS OF DEFINITIVE STUDY
The observed symptoms were in correspondence to already observed signs in the dose-range finding tests. After the second treatment: incoordination, motionless lying animals at the highest examined dose level, and no or slight symptoms at the lower doses. At the necropsy no signs of test item effect were noticed at the controls and lower doses, but signs of local test item effects (tympanites, significant amount of bedding material, liquid in the stomach, but no signs of affected mucous membranes) were noticed in animals at 1200 mg/kg bw dose.
The viability values of liver, stomach and duodenum cell suspensions (Trypan blue technique, screening) remained in the same vehicle control range at all test item treatment doses and positive control. The screened average viability values varied between 94-98 % at the liver cell preparations, between 72-80 % at the stomach cell preparations, it was 73-76 % at the duodenum preparations (slight, dose dependent average viability decrease (the average viability value was 10% lower at the highest test item dose, than in the vehicle control).

The final results of slide analysis are not yet available.

Conclusions:

The final results of slide analysis are not yet available.

Executive summary:

A study according to OECD Guideline 489 and under GLP conditions was performed with the registered substance. Male Wistar rats were treated with the test item dissolved in sunflower oil at doses of 1200, 600 and 300 mg/kg bw twice (at 0 h and at 24 h). A concurrent vehicle control and positive control (Ethyl methanesulfonate) was included.

The observed symptoms were in correspondence to already observed signs in the dose-range finding tests. After the second treatment: incoordination, motionless lying animals at the highest examined dose level, and no or slight symptoms at the lower doses. At the necropsy no signs of test item effect were noticed at the controls and lower doses, but signs of local test item effects (tympanites, significant amount of bedding material, liquid in the stomach, but no signs of affected mucous membranes) were noticed in animals at 1200 mg/kg bw dose.
The viability values of liver, stomach and duodenum cell suspensions (Trypan blue technique, screening) remained in the same vehicle control range at all test item treatment doses and positive control. The screened average viability values varied between 94-98 % at the liver cell preparations, between 72-80 % at the stomach cell preparations, it was 73-76 % at the duodenum preparations (slight, dose dependent average viability decrease (the average viability value was 10% lower at the highest test item dose, than in the vehicle control).

The final results of slide analysis are not yet available.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

experimental starting date: 2012-03-28, experimental completion date: 2012-05-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

yes

Remarks:

The relative humidity in the animal rooms ranged between 35 – 65% instead of 45 – 65%. This deviation, however, does not affect the validity of the study.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

The relative humidity in the animal rooms ranged between 35 – 65% instead of 45 – 65%. This deviation, however, does not affect the validity of the study.

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, Germany

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Pre-experiment: Harlan Laboratories B.V., Horst / The Netherlands
Main experiment: Charles River Laboratories, Research Models and Services Germany GmbH, Sulzfeld, Germany
- Number of Animals used: 42
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: mean value 35.0 g (SD +/- 1.7 g)
- Assigned to test groups randomly: [yes]
- Housing: single
- Diet (e.g. ad libitum): ad libitum (pelleted standard diet)
- Water (e.g. ad libitum): ad libitum (tap water)
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2 °C
- Humidity (%): 35 - 65 %
- Photoperiod (hrs dark / hrs light): artificial light 6.00 a.m. - 6.00 p.m.

According to the suppliers assurance the animals were in healthy condition. The animals were under acclimation in the animal house of Harlan CCR for a minimum of five days after their arrival. During this period the animals did not show any signs of illness or altered behaviour.
The animals were distributed into the test groups at random and identified by cage number.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: DMSO; polyethylene glycol
- Justification for choice of solvent/vehicle: The vehicle was chosen to its relative non-toxicity for the animals.
- Concentration of test material in vehicle: On the day of the experiment, the test item was dissolved in 30% DMSO and 70% PEG 400.
- Amount of vehicle (if gavage or dermal): All animals received a single standard volume of 10 mL/kg body weight orally.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: On the day of the experiment, the test item was dissolved in 30% DMSO and 70% PEG 400. All animals received a single standard volume of 10 mL/kg body weight orally.

Duration of treatment / exposure:

once orally (Amount given: 10 mL/ kg bw)

Frequency of treatment:

once

Post exposure period:

24 or 48 hours, respectively

Remarks:

Doses / Concentrations:
0 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
500 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
1000 mg/kg bw
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.

No. of animals per sex per dose:

7 males/ dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: orally once
- Doses / concentrations: 40 mg/kg b.w. (Amount given: 10 mL/kg b.w.)
- Other: The stability of CPA at room temperature was sufficient. At 25 °C only 3.5 % of its potency is lost after 24 hours.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items.
The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.
The administered volume was 10 mL/kg b.w..
Three adequately spaced dose levels spaced by a factor of 2 were administered, and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): Sampling of the bone marrow was done 24 and 48 hours after treatment, respectively. The animals were sacrificed using CO2 followed by bleeding. The femora were removed, the epiphyses were cut off and the marrow was flushed out with foetal calf serum using a syringe.

DETAILS OF SLIDE PREPARATION:
The cell suspension was centrifuged at 1500 rpm (390 x g) for 10 minutes and the supernatant was discarded. A small drop of the re-suspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald /Giemsa. Cover slips were mounted with EUKITT. At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in polychromatic erythrocytes per 2000 erythrocytes. The analysis was performed with coded slides.

Evaluation criteria:

The study is considered valid if the following criteria are met:
- at least 5 animals per group can be evaluated.
- PCE to erythrocyte ratio should not be less than 20 % of the negative control.
- the positive control shows a statistically significant and biological relevant increase of micronucleated PCEs compared to the negative control.

Evaluation of Results
A test item is classified as mutagenic if it induces either a dose-related increase or a clear increase in the number of micronucleated polychromatic erythrocytes in a single dose group in comparison to the laboratory’s historical data range. Statistical methods (nonparametric Mann-Whitney test (8)) will be used as an aid in evaluating the results. However, the primary point of consideration is the biological relevance of the results.
A test item that fails to produce a biological relevant increase in the number of micronucleated polychromatic erythrocytes is considered non-mutagenic in this system.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1000 mg/kg bw
- Solubility: test item was dissolved in 30 % DMSO / 70 % PEG 400
- Clinical signs of toxicity in test animals: none (no ruffled fur, no reduction of spontaneous activity

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): no
- Ratio of PCE/NCE (for Micronucleus assay): test item PCE with micronuclei: 0.042 versus 0.05 % in the vehicle control

Pre-Experiment on Toxicity

A preliminary study on acute toxicity was performed with two animals per sex under identical conditions as in the mutagenicity study concerning: animal strain, vehicle, route, frequency, and volume of administration.

The animals were treated once orally with the test item and examined for acute toxic symptoms at intervals of approximately 1 h, 2-4 h, 6 h, 24 h, 30 h, and 48 h after administration of the test item.

Since no gender specific differences on acute toxic symptoms were observed in agreement with the sponsor the main study was performed using males only.

Dose Selection

It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly or 2000 mg/kg as the upper limit for non-toxic test items.

The maximum tolerated dose level is determined to be the dose that causes toxic reactions without having major effects on survival within 48 hours.

The administered volume was 10 mL/kg b.w..

Three adequately spaced dose levels spaced by a factor of 2 were administered, and samples were collected at the central sampling interval 24 h after treatment. For the highest dose level an additional sample was taken at 48 h after treatment.

Pre-Experiments for Toxicity

The animals treated in the pre-experiments received the test item 4-phenylbutenone dissolved in 30% DMSO / 70% PEG 400 once orally. The volume administered was 10 mL/kg b.w.. The following dose levels were tested and expressed toxic reactions were shown in the table:

Table 1: results of the pre-experiments for toxicity

	hours post-treatment
	1	2-4	6	24	30	48
1^stPre-experiment: 1000 mg/kg b.w. ; males / females
reduction of spontaneous activity	0/0	0/2	0/2	0/2	0/2	0/0
ruffled fur	0/0	0/2	0/2	0/2	0/2	0/0

On the basis of these data 2000 mg/kg b.w. were estimated to be suitable as highest dose.

No substantial sex specific differences were observed with regard to clinical signs. In agreement with the sponsor the main study was performed using males only.

Toxic Symptoms in the Main Experiment

In the main experiment for each test item dose groups 7 males received the test item 4-phenylbutenone dissolved in 30% DMSO / 70% PEG 400 once orally. The volume administered was 10 mL/kg b.w.. The symptoms of toxicity observed following treatment are shown in the following table for each dose group, which indicates the number of males with findings. The animals treated with the negative control (30% DMSO / 70% PEG 400) did not express any toxic reaction.

Table 2: Toxic symptoms in the main experiment

	hours post-treatment (males)
	1	2-4	6	24	48*  h
High dose: 2000 mg/kg b.w. (24 h and 48 h)
reduction of spontaneous activity	3	4	4	2	0
abdominal position	3	1	1	0	0
eyelid closure	3	2	2	2	0
ruffled fur	3	3	3	4	0
death	0	1**	0	0	0
Medium dose: 1000 mg/kg b.w. (24 h)
reduction of spontaneous activity	1	1	1	0	-
abdominal position	1	0	0	0	-
eyelid closure	1	1	1	0	-
ruffled fur	1	1	1	1	-
Low dose: 500 mg/kg b.w. (24 h)
abdominal position	3	0	0	0	-
eyelid closure	4	0	0	0	-
excitement	2	0	0	0
ruffled fur	4	0	0	0	-

*   data only from 7 male animals

**  animal no. 40

-   no observation made

Table 3: Summary of Micronucleus Test Results

test group	dose mg/kg b.w.	sampling time (h)	PCEs with micronuclei (%)	range	PCE per 2000 erythrocytes
vehicle	0	24	0.050	0 -2	1288
test item	500	24	0.114	0 -3	1333
test item	1000	24	0.100	0 -5	1245
test item	2000	24	0.093	1 -3	1234
positive control	40	24	1.964	32 -49	1240
test item	2000	48	0.042	0 -2	1099

Conclusions:

The in vivo micronucleus test was conducted in NMRI mice, according to OECD 474 and EU Method B12 (with only non-significant deviations) and therefore considered to be of the highest quality. No increases in the incidence of micronucleated PCEs were observed in males exposed to 4-phenylbutenone. It is therefore considered that 4-phenylbutenone is not clastogenic (negative) in the mouse micronucleus test.

Executive summary:

The test item 4-phenylbutenone was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse (Roth, 2012). The test item was dissolved in 30% DMSO / 70% PEG 400, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w. and 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis. Seven males per test group were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei. To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes.

As estimated by pre-experiments 2000 mg 4-phenylbutenone per kg bw was suitable as top dose. The following dose levels of the test item were investigated: 24 h preparation interval: 500, 1000, and 2000 mg/kg bw and 48 h preparation interval: 2000 mg/kg bw.

As a result , the mean number of polychromatic erythrocytes was not substantially decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that 4-phenylbutenone did not have any cytotoxic properties in the bone marrow. However, one male (animal no. 40) of the high dose group (48 h treatment interval) died approximately 3 hours after treatment with the test item.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with 4-phenylbutenone were near to the value of the vehicle control group. However, the statistically significant increase above the vehicle control group observed in the low dose group was considered to have no biological relevance, as the value was well within the laboratory’s historical vehicle control data. Additionally, no dose dependent increase in the frequency of detected micronuclei was observed with increasing dosages.

Cyclophosphamide administered once orally (40 mg/kg b.w.) was used as positive control which showed a substantial and biologically relevant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Therefore, 4-phenylbutenone is considered to be non-mutagenic (negative) in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

The mutagenicity potential of methyl styryl keton was assessed with Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA, with or without metabolic activation, in the preincubation method by the Japanese Ministry of Health,Labour anf Welfare.

For the test, test substance treatment dose range is 19.5~5000 μg/plate.

6 times of 39.1~1250μg/plate were carried out for S. typhimurium TA strain when not metabolically activated, for S. typhimurium TA 100, TA 1535 and TA 1537 when metabolically activated; 6 times of 156~5000 μg/plate were carried for S. typhimurium TA 98 when metabolically activated, for E. coli WP2 uvrA with or without metabolic activation.

1) Precipitation and coloring by the test substance

Precipitation and discoloration on the plate by the test substance were not observed in any dose with or without metabolic activation.

2) Growth Inhibition

Through stereoscopic microscope observation, growth limitation was confirmed, by S. typhimurium TA strain without metabolic activation and S. typhimurium TA 100, TA 1535, TA 1537 with metabolic activation at above 1250 μg/plate, by S. typhimurium TA 98 with metabolic activation and E. coli WP2 uvrA with or without metabolic activation at above 2500 μg/plate .

3) Number of revertant colonies

In both of the two tests, an dose-dependent increase in the number of revertant colonies was observed in S. typhimurium TA 100 when metabolically activated, which is twice more than the negative control value, indicated reproducibility. When the specific mutation colony number was twice more than the negative control value and the reproducibility was observed, the specific activity value was determined to be 258 (Rev / mg) as the maximum, and the mutagenicity of this test substance was judged to be weak. 

Based on the above test results, it was judged that Methyl styryl keton has the ability to reverse mutagenize against bacteria (is positive) under the present test conditions (in S. typhimurium TA 100 when metabolically activated).

Seifried et al. compiled in their publication the results of genetic mutations assays (AMES-tests) and mouse-lymphoma test data of more than 450 chemicals (Seifried et al., 2006). The test results were obtained from the data banc of the National Cancer Institute (NCI), which is responsible for the selection of the most significant chemicals for carcinogenicity testing by the National Toxicology Program (NTP). As only some of the testing data available at the NCI has been made available in summary form in the Chemical Carcinogenesis Research Information System (CCRIS), which is searchable on the NLM TOXNET system, they decided to compile the data of these chemicals in a summary table that presents the results for each compound. 4 -phenylbutenone (CAS 122 -57 -6) is given with a positive result in an AMES test in the Salmonella typhimurium strain TA 100 with metabolic activation (either rat S9 or hamster S9). Moreover, 4 -phenylbutenone was found to be positive in the non activated mouse lymphoma test (doses 10 - 50 µg/mL, resulted in a lowest effective dose of 20 and 50). In case of test with metabolic activation (20 - 60 µg/mL), the results were either weakly positive or negative.

The ability of methyl styryl keton to induce chromosomal aberrations was examined by using Chinese hamster lung fibroblasts (CHLIIU cells) (Japan MHLW, 2009).

Initially, the cell proliferation inhibition test was carried out with the highest concentration set at 1500 μg/mL (corresponding to 10 mM) as specified in the test guideline. The result was, the cell proliferation suppressing effect of the short-time treatment method was judged to be exceeding 50% at 93.8 μg/mL with metabolic activation and at 46.9 μg/mL without metabolic activation. Moreover, for the 24-hour treatment by continuous treatment method, 50% cytostatic effect was judged at 23.4 μg/mL. The 50% cell proliferation inhibitory concentration (approximate value) was 82.6 μg/mL with metabolic activation and 34.8 μg/mL without metabolic activation in the short-time treatment method, 23.4 μg/mL for 24-hour treatment in the continuous treatment method. For the 48-hour treatment of the continuous treatment method, the cell proliferation inhibiting effect not exceeding 50% was not recognized even at the lowest concentration of 11.7 μg/mL, the cell proliferation rate was 44%, the 50% cytostatic concentration (Approximate value) was below 11.7 μg/mL. Based on these results, according to the provision [when the cytotoxicity of 50% or more is recognized, the concentration at which cell proliferation is obviously suppressed by more than 50% is taken as the highest concentration] of guideline, the maximum concentration of each treatment method of Chromosome aberration test for short-term treatment was 150 μg/mL with metabolic activation, 100 μg/mL without metabolic activation, for continuous treatment method was 40.0 μg/mL for 24-hour treatment and was 26.7 μg/mL for 48 hours treatment respectively.

The result of the chromosomal aberration test under metabolic activation of the short-time treatment, following one index of chromosome structural aberrations --- the frequency (TA value) of cells with structural aberrations excluding gaps, was positive at 100 μg/mL, equivocal at 66.7 μg/mL, and increase of TA value with concentration increase was recognized, therefore was judged positive. For the index value of chromosome aberrant induction intensity, the D20 value --- 20% of observed cells showing abnormality was 0.075 mg/mL, the TR value which is a comparative value of the rate of cells having chromatid exchange per unit dose (cte) was 560. In addition, it was also judged positive as it was positive at 44.4 and 29.6 μg/mL for the short-time treatment method without metabolic activation, and a rise in TA value with concentration increase was recognized; The D20 value was 0.032 mg/mL and the TR value was 1100. On the other hand, the frequency of occurrence of polyploidy cells in all treatment methods and all concentration were less than 5% the negative criterion.

And in all treatment methods, the frequency of occurrence of cells and polyploidy with chromosomal structural abnormality in the negative control group was less than 5% and was within the negative criteria. In contrast, induction of significant chromosomal structural abnormality was recognized in the positive control group, so it was considered that the test was performed appropriately.

From the above results, it was concluded that the substance has no ability to induce chromosome abnormality under this test condition, but has the ability to induce chromosomal structure abnormality.

The test item 4-phenylbutenone was assessed in the micronucleus assay for its potential to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse (Roth, 2012).The test item was dissolved in 30% DMSO / 70% PEG 400, which was also used as vehicle control. The volume administered orally was 10 mL/kg b.w. and 24 h and 48 h after a single administration of the test item the bone marrow cells were collected for micronuclei analysis.Seven males per test group were evaluated for the occurrence of micronuclei. Per animal 2000 polychromatic erythrocytes (PCEs) were scored for micronuclei.To describe a cytotoxic effect due to the treatment with the test item the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and reported as the number of PCEs per 2000 erythrocytes. As estimated by pre-experiments 2000 mg 4-phenylbutenone per kg b.w. was suitable as top dose.The following dose levels of the test item were investigated:24 h preparation interval: 500, 1000, and 2000 mg/kg b.w. and 48 h preparation interval: 2000 mg/kg b.w..

As a result , the mean number of polychromatic erythrocytes was not substantially decreased after treatment with the test item as compared to the mean value of PCEs of the vehicle control indicating that 4-phenylbutenone did not have any cytotoxic properties in the bone marrow. However, one male (animal no. 40) of the high dose group (48 h treatment interval) died approximately 3 hours after treatment with the test item.

In comparison to the corresponding vehicle controls there was no statistically significant or biologically relevant enhancement in the frequency of the detected micronuclei at any preparation interval and dose level after administration of the test item. The mean values of micronuclei observed after treatment with 4-phenylbutenone were near to the value of the vehicle control group. However, the statistically significant increase above the vehicle control group observed in the low dose group was considered to have no biological relevance, as the value was well within the laboratory’s historical vehicle control data. Additionally, no dose dependent increase in the frequency of detected micronuclei was observed with increasing dosages.

Cyclophosphamide administered once orally (40 mg/kg b.w.) was used as positive control which showed a substantial and biologically relevant increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test item did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse. Therefore, 4-phenylbutenone is considered to be non-mutagenic in this micronucleus assay.

A micronucleus test conducted with B6C3F1 mice was obtained from the National Toxicology Program, which collects data for possible dangerous substances. The test is considerd to be reliable with restrictions (Klimisch2). The test revealed no positive results for methyl styryl ketone (CAS No. 122 -57 -6).

Justification for classification or non-classification

Benzalacetone caused mutations in Salmonella typhimurium TA 100 with and without S9. Benzalacetone was shown to cause a mutagenic response in mouse lymphoma cells treated with doses of 50 µg/mL in the absence of S9 and has the ability to induce chromosomal structure abnormality. Based on this information there is indication for genotoxicity of benzalacetone, although the test material did not induce chromosome aberrations in an in vivo micronucleus assay. To draw a final conclusion on classification according to Regulation (EC) No 1272/2008 the result of the currently running in vivo comet assay is awaited.