4'-methoxyacetophenone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro Ames test

The test substance was determined to be not muatgenic.

In vitro HPRT

The read across substance p-methoxybenzyl acetate (CAS no. 104-21-2) was determined to be not muatgenic.

In vitro micronucleus test

The read across substance p-methoxybenzyl acetate (CAS no. 104-21-2) was determined to be not muatgenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-05-02 to 2017-06-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

May 30, 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium: histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate (S9) from Phenobarbital/ß-naphtoflavone pretreated rats

Test concentrations with justification for top dose:

In a pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. Since toxic effects were observed six or seven concentrations were tested in the main experiment. 5000 μg/plate, respectively 2500 μg/plate were chosen as maximal concentration. The following concentrations were tested in the final test:
Strain WP2 uvrA: 3; 10; 33; 100; 333; 1000; and 2500 μg/plate
The remaining strains: 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

With and without (vehicle control) DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-mainoanthracene (2-AA) and 4-nitro-o-phenylene-diamine, 4-NOPD

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- Pre-experiment for toxicity: In agar (plate incorporation)
- Experiment I: In agar (plate incorporation)
- Experiment II: Preincubation
- Selective Agar: Plates with selective agar (without histidine/tryptophan) were used

DURATION
- Preincubation period: Mix of 100 μL test solution (solvent or reference mutagen solution (positive control)), 500 μL S9 mix / S9 mix substitution buffer and 100 μL bacterial suspension incubated at 37 °C for 60 minutes
- Exposure duration: 48 hours at 37 °C in the dark after solidification of the plates

NUMBER OF REPLICATIONS:
- two independent experiments with three replications

DETERMINATION OF CYTOTOXICITY
- Method: Number of revertant colonies

Evaluation criteria:

A test item was considered as a mutagen if a biologically relevant increase in the number of revertants exceeded the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control was observed. A dose dependent increase was considered biologically relevant if the threshold was exceeded at more than one concentration. An increase exceeding the threshold at only one concentration was judged as biologically relevant if reproduced in an independent second experiment. A dose dependent increase in the number of revertant colonies below the threshold was regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remained within the historical range of negative and solvent controls such an increase was not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory. Therefore, no statistics were performed.

Key result

Species / strain:

other: Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA

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

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
No precipitation of the test item occurred up to the highest investigated dose.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Experiment I: Bacteriotoxic towards the strain TA 100 at 5000 µg/plate without S9 mix and towards WP2 uvrA at 2500 - 5000 µg/plate both with and without S9 mix.

- Experiment II: Bacteriotoxic towards the strains TA 1537 (without S9 mix), TA 100 (without S9 mix) and TA 98 (with S9 mix) at 5000 µg/plate.Bacteriotoxic towards the strain WP2 uvrA (with and without S9 mix) at 2500 µg/plate and towards the strain T100 at 2500-5000 with S9 mix.

Table 1: Summary of Experiment I

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		8 ± 3	13 ± 2	28 ± 7	156 ± 7	44 ± 2
	Untreated		13 ± 2	12 ± 2	28 ± 8	170 ± 22	36 ± 8
	Test Item	3 µg	7 ± 1	9 ± 3	20 ± 6	159 ± 29	37 ± 2
		10 µg	6 ± 1	11 ± 2	24 ± 9	165 ± 13	40 ± 11
		33 µg	10 ± 1	13 ± 3	24 ± 5	171 ± 12	46 ± 10
		100 µg	17 ± 3	13 ± 3	28 ± 8	167 ± 15	44 ± 7
		333 µg	7 ± 3	11 ± 4	24 ± 8	166 ± 22	35 ± 9
		1000 µg	8 ± 1	15 ± 4	18 ± 3	171 ± 14	27 ± 3
		2500 µg	8 ± 2	15 ± 1	22 ± 4	129 ± 13	11 ± 1
		5000 µg	9 ± 3	8 ± 2	13 ± 4	57 ± 16	3 ± 1
	NaN3	10 µg	1120 ± 231			1993 ± 36
	4-NOPD	10 µg			300 ± 32
	4-NOPD	50 µg		65 ± 9
	MMS	2.0 µL					996 ± 11
With Activation	DMSO		10 ± 2	16 ± 6	28 ± 9	158 ± 24	49 ± 18
	Untreated		9 ± 2	16 ± 7	41 ± 5	148 ± 10	53 ± 10
	Test Item	3 µg	10 ± 4	15 ± 1	38 ± 3	149 ± 7	51 ± 12
		10 µg	13 ± 3	15 ± 1	29 ± 8	142 ± 11	62 ± 8
		33 µg	8 ± 2	16 ± 4	22 ± 2	137 ± 6	40 ± 2
		100 µg	9 ± 2	16 ± 4	29 ± 3	144 ± 19	54 ± 8
		333 µg	10 ± 3	15 ± 6	34 ± 10	134 ± 15	41 ± 8
		1000 µg	12 ± 0	27 ± 2	28 ± 8	147 ± 3	31 ± 9
		2500 µg	11 ± 1	20 ± 6	27 ± 9	130 ± 15	17 ± 4
		5000 µg	7 ± 4	12 ± 3	21 ± 1	75 ± 7	8 ± 3
	2-AA	2.5 µg	441 ± 19	212 ± 36	3255 ± 284	4212 ± 229
	2-AA	10 µg					296 ± 14

Key to Positive Controls:

NaN3: sodium azide

2-AA: 2 -aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

Table 2: Summary of Experiment II

Metabolic Activation	Test Group	Dose Level (per plate)	Revertant Colony Counts (Mean ±SD)
			TA 1535	TA1537	TA 98	TA 100	WP2 uvrA
Without Activation	DMSO		10 ± 3	10 ± 1	27 ± 10	120 ± 10	41 ± 7
	Untreated		11 ± 2	9 ± 09 ± 0	37 ± 8	213 ± 3	31 ± 10
	Test Item	3 µg					38 ± 8
		10 µg					38 ± 8
		33 µg	11 ± 5	8 ± 2	29 ± 8	106 ± 14	36 ± 9
		100 µg	13 ± 4	8 ± 2	23 ± 4	139 ± 3	32 ± 4
		333 µg	11 ± 4	9 ± 4	33 ± 12	119 ± 3	31 ± 5
		1000 µg	12 ± 3	10 ± 4	24 ± 3	96 ± 4	22 ± 3
		2500 µg	13 ± 3	6 ± 1	24 ± 4	67 ± 12	17 ± 6
		5000 µg	9 ± 3	4 ± 0	18 ± 5	0 ± 1
	NaN3	10 µg	1413 ± 31			2123 ± 55
	4-NOPD	10 µg			354 ± 7
	4-NOPD	50 µg		115 ± 21
	MMS	2.0 µL					636 ± 43
With Activation	DMSO		14 ± 1	11 ± 2	30 ± 5	125 ± 11	45 ± 9
	Untreated		12 ± 1	16 ± 7	48 ± 5	174 ± 11	50 ± 17
	Test Item	3 µg					55 ± 5
		10 µg					46 ± 2
		33 µg	12 ± 2	10 ± 2	36 ± 10	113 ± 12	54 ± 8
		100 µg	13 ± 2	10 ± 4	36 ± 12	131 ± 7	48 ± 13
		333 µg	13 ± 5	11 ± 3	32 ± 2	139 ± 15	37 ± 6
		1000 µg	11 ± 1	16 ± 1	38 ± 11	83 ± 8	35 ± 4
		2500 µg	10 ± 1	15 ± 3	31 ± 9	37 ± 5	19 ± 2
		5000 µg	7 ± 0	7 ± 1	6 ± 0	3 ± 1
	2-AA	2.5 µg	424 ± 18	110 ± 14	4237 ± 917	3262 ± 234
	2-AA	10 µg					453 ± 29

Key to Positive Controls:

NaN3: sodium azide

2-AA: 2 -aminoanthracene

4-NOPD: 4-nitro-o-phenylene-diamine

MMS: methyl methane sulfonate

Conclusions:

The test substance was not mutagenic to Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA in the presence and absence of a metabolizing system.

Executive summary:

An in vitro reverse mutation assay study (Ames) was conducted under GLP-conditions according to OECD Guideline 471 and EU Method B13/B14 in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. Two separate Experiments were performed, one as a plate incorporation assay (Experiment I) and a second one as a preincubation assay (Experiment II). All tests were conducted in triplicate and concentrations between 3μg/plate and 5000μg/plate were tested. Strains were exposed to the test substance with and without metabolic activation with rat liver S9 mix. Cytotoxicity was observed in Experiment I towards the strain TA 100 at 5000 µg/plate without S9 mix and towards WP2 uvrA at 2500 - 5000 µg/plate both with and without S9 mix. In Experiment II: the test substance showed bacteriotoxic effects towards the strains TA 1537 (without S9 mix), TA 100 (without S9 mix) and TA 98 (with S9 mix) at 5000 µg/plate as well as towards the strain WP2 uvrA (with and without S9 mix) at 2500 µg/plate and towards the strain T100 at 2500-5000 with S9 mix. No substantial increase in revertant colony numbers of any of the tester strains was detected following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Therefore it was concluded that the test substance did not induce gene mutations and it was considered to be non-mutagenic.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See read-across justification attached to section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

An in vitro gene mutation study (HPRT) was conducted in V79 cells of the Chinese hamster. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

See read-across justification attached to section 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 26 September 2014

Key result

Species / strain:

other: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, it is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating concentrations.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-10 to 2015-12-03

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Guidelines: “Kanpoan No. 287 -- Environment Protection Agency“ “Eisei No. 127 -- Ministry of Health & Welfare“ “Heisei 09/10/31 Kikyoku No. 2 -- Ministry of International Trade & Industry“

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)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium) containing Hank’s salts supplemented with 10 % foetal bovine serum (FBS), neomycin (5 μg/mL) and amphotericin B (1 %)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Pre-test: 14.1, 28.2, 56.3, 112.6, 225.3, 450.5, 901.0, 1802.0 μg/mL
Main test: 28.1, 56.3, 112.5, 225.0, 450.0, 900.0 μg/mL

Vehicle / solvent:

- Vehicle used: DMSO; The final concentration of DMSO in the culture medium was 0.5% (v/v).
- Justification for choice of solvent: The solvent was chosen to its solubility properties and as it’s tolerated by the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h
- Expression time: 7 days
- Fixation time: The colonies used to determine the cloning efficiency were fixed and stained 6 to 8 days after treatment.

SELECTION AGENT: 6-TG (6-thioguanine)
STAIN: 10 % methylene blue in 0.01 % KOH solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: all colonies with more than 50 cells

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

A pre-test was conducted to establish the doses for the main test and evalute cytotxicity, precipitation and pH changes.

Evaluation criteria:

Acceptability of the Assay
The gene mutation assay is considered acceptable if it meets the following criteria:
a) the mean values of the numbers of mutant colonies per 10^6 cells found in the solvent controls of both parallel cultures remain within the 95 % confidence interval of the laboratory historical control data range.
b) the positive control substances should produce a significant increase in mutant colony frequencies and remain within the historical control range of positive controls.
c) the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.

The data of this study comply with the above mentioned criteria.

Evaluation of Results
A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range. A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system. A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95 % confidence interval limits). The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares). However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate within the laboratory´s historical control data range, a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls was also taken into consideration.

Statistics:

A linear regression (least squares, calculated using Sum_neu_v2.xltm, version 2.0) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance was considered together.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS (evaluated in pre-test)
- Effects of pH: no relevant shift
- Effects of osmolality: no relevant shift
- Precipitation: at 450.5 μg/mL and above after 4 hours treatment with and without metabolic activation


ADDITIONAL INFORMATION ON CYTOTOXICITY: In the pre-experiment a relevant cytotoxic effect, indicated by a relative cloning efficiency of 50 % or below was observed at 901.0 μg/mL and above without metabolic activation. In the presence of metabolic activation no relevant cytotoxic effect was determined up to the highest concentration.

	concentration [µg/mL]	Phase separation	S9 mix	relative cloning efficiency I [%]	relative cell density [%]	relative adjusted cloning efficiency I [%]	mutant colonies/10exp6 cells	95 % confidence interval	relative cloning efficiency I [%]	relative cell density [%]	relative adjusted cloning efficiency I [%]	mutant colonies/10exp6 cells	95 % confidence interval
Main test with 4 h treatment				culture I					culture II
Solvent control (DMSO)			-	100	100	100	20.3	0.0-29.7	100	100	100	21.1	0.0-29.7
Positive control (EMS)	150		-	104.2	112.8	117.6	203.3	0.0-29.7	94.3	94.8	89.4	244	0.0-29.7
Test item	28.1		-	99.3	94.1	culture was not continued			99.2	89.5	culture was not continued
	56.3		-	103.1	112.8	116.3	23	0.0-29.7	91.9	64.9	59.6	11.2	0.0-29.7
	122.5		-	102.4	73.6	75.3	14.3	0.0-29.7	89.8	75.3	67.6	20.6	0.0-29.7
	225		-	98.5	129.1	127.1	14.8	0.0-29.7	96	72.7	69.8	16.7	0.0-29.7
	450	PS	-	94.4	100.4	94.8	21.3	0.0-29.7	95.7	88	84.1	12.8	0.0-29.7
	900	PS	-	99.5	68.5	68.2	8.4	0.0-29.7	92.9	83.3	77.4	25.3	0.0-29.7
Solvent control (DMSO)			+	100	100	100	19.5	0.0-27.7	100	100	100	8.6	0.0-27.7
Positive control (DMBA)	2.2		+	89.2	104.7	93.4	190	0.0-27.7	98.9	88	87.1	176.7	0.0-27.7
Test item	28.1		+	76	107.3	culture was not continued			101.9	96.7	culture was not continued
	56.3		+	78.5	107.5	84.4	29	0.0-27.7	97.3	85.9	83.6	18.6	0.0-27.7
	122.5		+	92.2	78.1	72	21.6	0.0-27.7	100.4	82.3	82.6	21.7	0.0-27.7
	225		+	59.2	91.5	54.2	11.4	0.0-27.7	103.5	76.1	78.7	14.6	0.0-27.7
	450	PS	+	80.8	93.9	75.9	15.3	0.0-27.7	96.7	80.3	77.6	14.3	0.0-27.7
	900	PS	+	43.2	117.6	50.7	20	0.0-27.7	98.7	83.4	82.4	22.3	0.0-27.7

Some cultures were not continued as a minimum of only four analysable concentrations was required.

Conclusions:

An in vitro gene mutation study (HPRT) was conducted in V79 cells of the Chinese hamster. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.

Executive summary:

An in vitro gene mutation study (HPRT) according to OECD Guideline 476 was conducted in V79 cells of the Chinese hamster. A pre-experiment was performed with and without metabolic activation for the selection of the test substance concentration. Concentrations between 14.1 μg/mL and 1802 μg/mL were tested. For the main experiments a concentration range between 28.1 and 900 μg/mL was used. The main assay was performed in duplicates. The cells were exposed to the test item for 4 hours with and without metabolic activation. In the main experiment with and without S9 mix the range of the solvent controls was from 8.6 up to 21.1 mutants per 10^6 cells and the range of the groups treated with the test item was from 8.4 up to 29.0 mutants per 10^6 cells. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic.

Reference 5

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-11-04 to 2015-12-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

adopted 26 September 2014

Deviations:

yes

Remarks:

For details on deviations please refer to "Principles of method".

Principles of method if other than guideline:

Deviations: A series of in-house non-GLP validation experiments was performed to get distinct responses of statistical significance when using the specified positive controls. To achieve such response the test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified comparing the current proposal given in the OECD Guideline 487.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

primary culture, other: Human lymphocytes, blood was collected from a male donor (30 years old) for Experiment I and from a male donor (23 years old) for Experiment II.

Details on mammalian cell type (if applicable):

- Type and identity of media: Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection. The culture medium was Dulbecco's Modified Eagles Medium/Ham's F12 (DMEM/F12, mixture 1:1) already supplemented with 200 mM GlutaMAX. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL/100 μg/mL), the mitogen PHA (3 μg/mL), 10 % FBS (fetal bovine serum), 10 mM HEPES and the anticoagulant heparin (125 U.S.P.-U/mL).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable
- Periodically checked for karyotype stability: not applicable
- Periodically "cleansed" against high spontaneous background: not applicable
- Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours. The cell harvest time point was approximately 2 – 2.5 x AGT (average generation time). Any specific cell cycle time delay induced by the test item was not accounted for directly.

Additional strain / cell type characteristics:

other: established low incidence of micronuclei

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

4 h exposure: 11.7, 20.5, 35.8, 62.7, 109.8, 192.1, 336.2, 588.4, 1029.7, 1802.0 μg/mL
20 h exposure: 35.8, 62.7, 109.8, 192.1, 336.2, 588.4, 1029.7, 1802.0 μg/mL

Vehicle / solvent:

- Vehicle used: DMSO; The final concentration of DMSO in the culture medium was 0.5 %.
- Justification for choice of solvent: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

for pulse treatment, without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Demecolcin

Remarks:

for continous treatment, without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 (pulse exposure) and 20 h (continuous exposure)
- Expression time: 16 h (pulse exposure only)
- Recovery period: 20 h
- Fixation time: 40 h

SPINDLE INHIBITOR: Cytochalasin B
STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 1000 binucleate cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is characterized by the percentages of reduction in the CBPI (Cytokinesis-block proliferation index) in comparison with the controls (% cytostasis) by counting 500 cells per culture.

A pre-test was conducted to establish the doses for the main test.

Evaluation criteria:

The test item was considered to be clearly negative if, in all of the experimental conditions examined:
− None of the test item concentrations exhibited a statistically significant increase compared with the concurrent solvent control
− There was no concentration-related increase
− The results in all evaluated test item concentrations was within the range of the laboratory historical solvent control data
The test item was then considered unable to induce chromosome breaks and/or gain or loss in this test system.
The test item was considered to be clearly positive if, in any of the experimental conditions examined:
− At least one of the test item concentrations exhibited a statistically significant increase compared with the concurrent solvent control
− The increase was concentration-related in at least one experimental condition
− The results were outside the range of the laboratory historical solvent control data

When all of the criteria were met, the test item was then considered able to induce chromosome breaks and/or gain or loss in this test system.

Statistics:

Statistical significance was confirmed by using the Chi-squared test (α < 0.05) using the validated R Script CHI2.Rnw for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Key result

Species / strain:

other: Human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS (evaluated in pre-test)
- Effects of pH: no relevant shift
- Effects of osmolality: no relevant shift
- Precipitation: no
- Phase seperation: experiment I at 1029.7 μg/mL and above in the absence and presence of S9 mix; experiment II at 1802.0 μg/mL in the absence of S9 mix at the end of treatment

Experiment	Preparation interval [h]	Test item [µg/mL]	Proliferation index CBPI	Cytostasis [%]	Micronucleated cells [%]
Exposure period 4 h without S9
I	40	DMSO (0.5 %)	1.92		0.45
		MMC (1.0)	1.27	70.2	8.4
		336.2	1.85	6.9	0.3
		588.4	1.83	9.0	0.35
		1029.7	1.87	5.1	0.35
Exposure period 20 h without S9
II	40	DMSO (0.5 %)	1.95		0.35
		Demecolcin (125.0)	1.56	41.8	3.45
		588.4	1.91	4.3	0.05
		1029.7	1.92	3.7	0.35
		1802.0	1.82	13.7	0.00
Exposure period 4 h with S9
I	40	DMSO (0.5 %)	1.92		0.25
		CPA (17.5)	1.43	53.2	2.90
		336.2	1.96	n.c.	0.75
		588.4	1.90	2.3	0.40
		1029.7	1.96	n.c.	0.60

n.c. Not calculated as the CBPI is equal or higher than the solvent control value

Conclusions:

Under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, it is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating concentrations.

Executive summary:

An in vitro micronucleus test according to OECD Guideline 487 was conducted in primary human lymphocytes. Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity was described as % cytostasis. No precipitation of the test item in the culture medium was observed. Phase separation was observed in Experiment I at 1029.7 μg/mL and above in the absence and presence of S9 mix and in Experiment II at 1802.0 μg/mL in the absence of S9 mix at the end of treatment. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. However, in Experiment I in the presence of S9 mix one single statistically significant increase in micronucleated cells (0.75 %) was observed after treatment with 336.2 μg/mL. Since the value is in the range of the laboratory historical control data (0.15 – 1.45 % micronucleated cells), the finding can be regarded as biologically irrelevant. The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, it is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating concentrations.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Genetic toxicity in vitro

Ames Test

An in vitro reverse mutation assay study (2017) was conducted under GLP-conditions according to OECD Guideline 471 and EU Method B13/B14 in Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. Two separate Experiments were performed, one as a plate incorporation assay (Experiment I) and a second one as a preincubation assay (Experiment II). All tests were conducted in triplicate and concentrations between 3 μg/plate and 5000μg/plate were tested. Strains were exposed to the test substance with and without metabolic activation with rat liver S9 mix. Cytotoxicity was observed in Experiment I towards the strain TA 100 at 5000 µg/plate without S9 mix and towards WP2 uvrA at 2500 - 5000 µg/plate both with and without S9 mix. In Experiment II: the test substance showed bacteriotoxic effects towards the strains TA 1537 (without S9 mix), TA 100 (without S9 mix) and TA 98 (with S9 mix) at 5000 µg/plate as well as towards the strain WP2 uvrA (with and without S9 mix) at 2500 µg/plate and towards the strain T100 at 2500-5000 with S9 mix. No substantial increase in revertant colony numbers of any of the tester strains was detected following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Therefore it was concluded that the test substance did not induce gene mutations and it was considered to be non-mutagenic. 

HPRT

An in vitro gene mutation study (HPRT) according to OECD Guideline 476 was conducted (2015) in V79 cells of the Chinese hamster with the surrogate test substance p-methoxybenzyl acetate (CAS 104-21-2). A pre-experiment was performed with and without metabolic activation for the selection of the test substance concentration. Concentrations between 14.1 μg/mL and 1802 μg/mL were tested. For the main experiments a concentration range between 28.1 and 900 μg/mL was used. The main assay was performed in duplicates. The cells were exposed to the test item for 4 hours with and without metabolic activation. In the main experiment with and without S9 mix the range of the solvent controls was from 8.6 up to 21.1 mutants per 10⁶ cells and the range of the groups treated with the test item was from 8.4 up to 29.0 mutants per 10⁶ cells. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. It was concluded that the test substance did not induce gene mutations at the HPRT locus and therefore was considered to be non-mutagenic. 

Micronucleus assay

An in vitro micronucleus test (2015) according to OECD Guideline 487 was conducted in primary human lymphocytes with the surrogate test substance p-methoxybenzyl acetate (CAS 104-21-2). Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure period was 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item. In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity was described as % cytostasis. No precipitation of the test item in the culture medium was observed. Phase separation was observed in Experiment I at 1029.7 μg/mL and above in the absence and presence of S9 mix and in Experiment II at 1802.0 μg/mL in the absence of S9 mix at the end of treatment. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration. In the absence and presence of S9 mix, no relevant increase in the number of micronucleated cells was observed after treatment with the test item. However, in Experiment I in the presence of S9 mix one single statistically significant increase in micronucleated cells (0.75 %) was observed after treatment with 336.2 μg/mL. Since the value is in the range of the laboratory historical control data (0.15 – 1.45 % micronucleated cells), the finding can be regarded as biologically irrelevant. The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, it is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to phase separating concentrations.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The data did not indicate genetic mutation properties of the test substance and was concluded to be non clastogenic. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the fifteenth time in Regulation (EU) 2020/1182.