p-methoxybenzyl acetate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro Ames test:
The test substance was not muatgenic.
In vitro HPRT:
The test substance was not muatgenic.
In vitro micronucleus:
The test substance was 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:

2000-07-16 and 2000-07-17

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:

1997

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 TA 102

Metabolic activation:

with and without

Metabolic activation system:

Liver homogenate (S9) from Aroclor 1254 pretreated male rats

Test concentrations with justification for top dose:

0, 15, 50, 150, 500, 1500, 5000 µg/plate

Vehicle / solvent:

- Vehicle/solvent used: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (TA 98, TA 100, TA 102, TA 1535, TA 1537)

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 to 72 h

NUMBER OF REPLICATIONS: two independent experiments with three replications

DETERMINATION OF CYTOTOXICITY
- Method: Number of revertant colonies

Evaluation criteria:

The test substance is evaluated as mutagenic if it significantly or dose dependently increases the mutation frequency of the tester strains.

Statistics:

For estimation of the statistical significance of the difference between the mean number of revertants in the negative controls and the plates at each dosage level a X2-test was used.

Key result

Species / strain:

S. typhimurium, other: TA1537, TA1535, TA98, TA100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Bacteriotoxic towards the strains TA 1537 and TA 102 at 5000 µg/plate without S9 mix.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Bacteriotoxic towards the strains TA1537 and TA102 at 5000μg/plate in the absence of S9-mix.

 Experiment 1:

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium
	TA98	TA100	TA102	TA1535	TA1537
	Results without S9
Untreated	30± 5	122 ± 9	279 ± 25	15 ± 1	8 ± 2
DMSO (50µL)	25 ± 5	134 ± 5	246 ± 12	15 ± 3	9 ± 6
15			252 ± 13
50	24 ± 6	123 ± 6	243 ± 16	15 ± 4	7 ± 3
150	19 ± 2	123 ± 6	241 ± 16	15 ± 3	5 ± 3
500	30 ± 6	139 ± 1	215 ± 11	17 ± 3	9 ± 4
1500	15 ± 6	134 ± 12	197 ± 19	12 ± 4	8 ± 3
5000	21 ± 6	147 ± 5	108 ± 5	13 ± 4	4 ± 3
NaN3 (0.7)		483 ± 15		428 ± 20
2-NF (2.5)	368 ± 24
9-AA (50)					187 ± 13
Mitomycin (0.15)			727 ± 7
	Results with S9
Untreated	26 ± 7	149 ± 6	267 ± 10	9 ± 1	17 ± 6
DMSO (50µL)	21 ± 4	131 ± 8	147 ± 4	10 ± 2	16 ± 2
15			243 ± 8		10 ± 5
50	21 ± 6	133 ± 3	247 ± 12	12 ± 3	17 ± 4
150	35 ± 5	132 ± 9	251 ± 10	14 ± 2	11 ± 3
500	19 ± 3	131 ± 2	235 ± 10	13 ± 4	9 ± 5
1500	29 ± 4	133 ± 6	249 ± 14	10 ± 3	14 ± 4
5000	19 ± 2	140 ± 8	230 ± 2	9 ± 3
2-AA (0.7)	1081 ± 29
2-AA (0.8)		2394 ± 162
2-AA (1.0)				523 ± 29
2-AA (1.5)			1253 ± 45		210 ± 17

2-NF: 2-nitrofluorene; 9-AA: 9-aminoacridine; 2-AA: 2-aminoanthracene

 

 

Experiment 2:

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium
	TA98	TA100	TA102	TA1535	TA1537
	Results without S9
Untreated	16 ± 2	115 ± 11	249 ± 6	16 ± 3	8 ± 4
DMSO (50µL)	14 ± 5	123 ± 4	237 ± 11	14 ± 3	9 ± 4
15			223 ± 9
50	14 ± 5	106 ± 9	240 ± 16	12 ± 4	9 ± 4
150	17 ± 2	116 ± 7	234 ± 9	12 ± 2	9 ± 4
500	13 ± 2	112 ± 11	198 ± 11	18 ± 4	6 ± 3
1500	14 ± 1	120 ± 10	195 ± 2	19 ± 4	9 ± 2
5000	10 ± 4	134 ± 4	111 ± 12	14 ± 3	5 ± 6
NaN3 (0.7)		334 ± 24		180 ± 15
2-NF (2.5)	339 ± 41
9-AA (50)					331 ± 38
Mitomycin (0.15)			1106 ± 112
	Results with S9
Untreated	25 ± 6	129 ± 11	255 ± 6	12 ± 3	19 ± 5
DMSO (50µL)	21 ± 2	120 ± 5	245 ± 8	10 ± 3	14 ± 6
50	20 ± 0	109 ± 1	223 ± 13	8 ± 2	13 ± 7
150	20 ± 3	109 ± 8	245 ± 7	13 ± 2	11 ± 2
500	24 ± 5	114 ± 3	230 ± 15	11 ± 5	11 ± 4
1500	21 ± 7	125 ± 9	204 ± 3	11 ± 3	13 ± 4
5000	21 ± 3	113 ± 13	193 ± 9	6 ± 2	14 ± 4
2-AA (0.7)	1207 ± 57	1665 ± 51
2-AA (1.0)				313 ± 8
2-AA (1.5)			1265 ± 25		282 ± 23

2-NF: 2-nitrofluorene; 9-AA: 9-aminoacridine; 2-AA: 2-aminoanthracene

Conclusions:

The test substance was not mutagenic to Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102 in the presence and absence of a metabolizing system.

Executive summary:

An in vitro reverse mutation assay study (Ames) was conducted according to OECD Guideline 471 in Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537). Experiments were performed as a plate incorporation assay. All tests were conducted in triplicate and concentrations between 15 μg/plate and 5000 μg/plate were tested. Strains were exposed to the test substance with and without metabolic activation with rat liver S9 mix. Cytotoxicity was observed without S9 in the strains TA 1537 and TA 102 at 5000 μg/plate. 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:

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 3

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 (Ames) was conducted according to OECD Guideline 471 in Salmonella typhimurium. All tests were conducted in triplicate and concentrations between 15 μg/plate and 5000 μg/plate were tested with and without metabolic activation. Cytotoxicity was observed without S9 in the strains TA 1537 and TA 102 at 5000 μg/plate. 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. 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 in V79 cells. The concentration range between 28.1 and 900 μg/mL was used. The assay was performed in duplicates. 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.

 

Micronucleus in vitro:

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. In each experimental group two parallel cultures were analysed. 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 is 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 ninth time in Regulation (EU) No 2016/1179.