4-methoxybenzyl alcohol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material did not induce the frequency of revertant colonies in bacteria with and without metabolic activation. Furthermore, The test item did not induce gene mutations at the HPRT locus in V79 cells under the conditions chosen. The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 March 2002 and 07 November 2003

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

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 mix induced by phenobarbitone/p-naphthoflavone in livers of rats

Test concentrations with justification for top dose:

100, 333, 1000, 2500 and 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:

other: +S9: N-ethyl-N'-nitro-N-nitrosoguanidine (TA100 and TA1535), 9-Aminoacridine (TA1537), Mitomycin C (TA102), 4-Nitroquinoline-l-oxide (TA98); -S9: 2-Aminoanthracene (TA100, TA1535, TA1537), Benzo(a)pyrene (TA98), 1,8-Dihydroxyanthraquinone (TA102)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3 (two independent experiments)

DETERMINATION OF CYTOTOXICITY
- Method: counting numbers of revertants

Evaluation criteria:

The test material may be considered positive if the following criteria are met:
- The test material should have induced a reproducible, dose-related and statistically significant increase in the revertant count in at least one strain of bacteria.

Statistics:

Dunnett's method of linear regression.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES: A preliminary study was carried out using both the plate incorporation and pre-incubation methods with the following concentrations: 0,0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA100. The test material was non-toxic to the strain of Salmonella used (TA100). The test material formulation and the S9-mix used in this experiment were both shown to be effectively sterile. Based on the result, the following concentrations of the test susbstance in the main study was used: 100, 333, 1000, 2500 and 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA: done

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate.

Table 1: Spontaneous mutation rates (concurrent negative controls)

Dose µg/plate I/II	TA 100		TA 1535		TA102		TA98		TA1537
	I	II	I	II	I	II	I	II	I	II
Negative control	107	105	34	20	380	357	18	18	11	14

Table 2: Number of revertants (mean number of three plates) without metabolic activation

Dose µg/plate I/II	TA 100		TA 1535		TA102		TA98		TA1537
	I	II	I	II	I	II	I	II	I	II
Solvent Control	133	108	33	31	347	260	18	16	14	13
100	115	95	34	33	339	265	19	12	13	14
333	105	97	34	29	338	256	16	19	17	7
1000	115	101	36	35	327	278	18	12	21	9
2500	109	107	37	39	347	276	16	12	17	11
5000	107	99	39	30	312	240	13	12	18	8
Positive controls
ENNG (3.0)	500	474
ENNG (5.0)			900	205
MMC (0.5)					2414	1886
4NQO (0.2)							168	133
9AA (80)									991	1353

ENNG: N-ethyl-N'-nitro-N-nitrosoguanidine, 4NQO: 4-Nitroquinoline-1 -oxide, 9 -AA: 9-Aminoacridine, MMC: Mitomycin C

Table 3: Number of revertants (mean number of three plates) with metabolic activation

Dose µg/plate I/II	TA 100		TA 1535		TA102		TA98		TA1537
	I	II	I	II	I	II	I	II	I	II
Solvent Control	123	90	21	18	371	298	31	33	20	12
100	129	105	16	11	352	267	31	29	16	17
333	117	104	14	20	361	277	28	33	17	17
1000	123	106	15	16	375	281	31	35	19	15
2500	107	96	16	11	392	297	34	29	23	14
5000	113	106	20	13	369	295	35	29	19	16
Positive controls
2-AA (1.0)	1520	1463
2-AA (2.0)			341	267					316	483
DAN (10)					765	922
BP (5)							220	254

2 -AA: 2-Aminoanthracene, BP: Benzo(a)pyrene, DAN: 1,8-Dihydroxyanthraquinone

Conclusions:

The test substance was considered to be non-mutagenic with and without metabolic activation in bacteria.

Executive summary:

A bacterial reverse mutation assay (OECD 471) was performed to investigate the mutagenic potential of the test substance in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA100, TA1535, TA102, TA1537 and TA98 with and without metabolic activation. Based on the results of the preliminary study, the following concentrations were chosen for the main study: 100, 333, 1000, 2500 and 5000 µg/plate. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the strains of Salmonella, at any dose level either with or without metabolic activation. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains. In conclusion, the test material was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016-01-05 and 2016-04-07

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:

28 July 2015

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

August 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian gene mutation assay

Target gene:

HPRT (hypoxanthine-guanine phosphoribosyl transferase) gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Especially the high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50%) both necessary for the appropriate performance of the study, recommend the use of this cell line.
- Doubling time: 12 – 16 h
- Modal number of chromosomes: 22


MEDIA USED
- Type and identity of media including CO2 concentration: Minimum Essential Medium (MEM) containing Hank’s salts, neomycin (5 µg/mL), 10% FBS, and amphotericin B (1%)
- 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:

Concentration used: 43.2, 86.4, 172.8, 345.5, 691.0 and 1382.0 µg/mL
The maximum was 1382.0 µg/mL or 10 mM whcih is in accordance with the requirements of the guideline.

Vehicle / solvent:

- Vehicle/solvent used: water
- Justification for choice of solvent/vehicle: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: 0.7 – 1.2 x 10E+7 cells

DURATION
- Exposure duration: 4 hours
- Expression time (cells in growth medium): 7 days
- Selection time: 8 days

SELECTION AGENT: 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: two independent experiments with each replicates

STAINING TECHNIQUE USED: The colonies were stained with 10% methylene blue in 0.01% KOH solution

NUMBER OF CELLS EVALUATED: Stained colonies with more than 50 cells.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

- OTHER:
In a pre-experiment the medium was checked for precipitation as well as pH and osmolarity changes.

Evaluation criteria:

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).

Statistics:

A linear regression (least squares, calculated using a validated Excel spreadsheet) 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. A t-test (using a test script of “R”, a language and environment for statistical computing and graphics) was used to check the data generated at 345.5 μg/mL in both parallel cultures with metabolic activation (the mutation frequency exceeded the 95% confidence interval at this concentration). A t-test is considered 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:

cytotoxicity

Remarks:

not below 50%

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: none

RANGE-FINDING/SCREENING STUDIES: Test item concentrations between 10.8 µg/mL and 1382 µg/mL (equal to a molar concentration of approximately 10 mM) were used in the pre-experiment. In the pre-experiment no relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was noted up to the highest concentration with and without metabolic activation. Based on the results of the pre-experiment the dose range for the main experiment was 43.2 to 1382.0 µg/mL.

HISTORICAL CONTROL DATA:
- Positive historical control data (in mutant colonies per 10E+6 cells):
EMS: range = 53.9 – 889.0, mean = 153.0, SD = 88.5
DMBA: range = 59.6 – 2042.6, mean = 424.6, SD = 291.4

- Negative (solvent/vehicle) historical control data (in mutant colonies per 10E+6 cells):
+ S9: Solvent control (water, DMSO, medium): range = 1.6 – 42.8, mean = 15.0, SD = 7.4, 95% CL = 0.2 – 29.7
- S9: Solvent control (water, DMSO, medium): range = 2.4 – 44.2, mean = 14.6, SD = 7.0, 95% CL = 0.6 – 28.7

Table 1: Summary of results

	Conc. μg/mL	P / PS	S9 mix	Relative cloning efficiency I	Relative cell density	Relative cloning efficiency II	Mutant colonies / 10^6cells	95% confidence interval	Relative cloning efficiency I	Relative cell density	Relative cloning efficiency II	Mutant colonies / 10^6cells		95 % confidence interval
				%	%	%			%	%	%
Column	1	2	3	4	5	6	7	8	9	10	11	12		13
Experiment I / 4h treatment				Culture I					Culture II
Solvent control with water			-	100.0	100.0	100.0	16.7	0.2 – 29.7	100.0	100.0	100.0		14.7	0.2 – 29.7
Positive control (EMS)	450.00		-	99.4	123.3	122.5	280.9	0.2 – 29.7	102.4	98.2	100.6		258.9	0.2 – 29.7
Test item	43.20	-	-	98.5	85.0	83.7	#		100.9	114.8	115.9		#
Test item	86.40	-	-	102.4	111.7	114.3	17.6	0.2 – 29.7	104.0	101.4	105.4		11.8	0.2 – 29.7
Test item	172.80	-	-	100.4	118.1	118.6	12.5	0.2 – 29.7	105.7	97.5	103.1		12.9	0.2 – 29.7
Test item	345.50	-	-	101.3	106.6	108.0	24.0	0.2 – 29.7	101.2	115.4	116.8		18.1	0.2 – 29.7
Test item	691.00	-	-	99.3	78.3	77.7	6.7	0.2 – 29.7	103.4	110.0	113.7		13.5	0.2 – 29.7
Test item	1382.00	-	-	99.8	105.0	104.8	20.6	0.2 – 29.7	103.8	97.3	101.0		14.8	0.2 – 29.7
Solvent control with water			+	100.0	100.0	100.0	22.0	0.6 – 28-7	100.0	100.0	100.0		19.1	0.6 – 28-7
Positive control (DMBA)	2.20		+	98.5	86.9	85.6	254.3	0.6 – 28-7	94.6	72.4	68.5		308.6	0.6 – 28-7
Test item	43.20	-	+	99.6	90.5	90.1	#		104.2	85.3	88.9		#
Test item	86.40	-	+	97.0	79.0	76.6	7.3	0.6 – 28-7	100.5	74.9	75.3		28.0	0.6 – 28-7
Test item	172.80	-	+	96.7	81.8	79.1	12.7	0.6 – 28-7	98.1	83.4	81.8		27.0	0.6 – 28-7
Test item	345.50	-	+	98.8	82.4	81.4	30.0	0.6 – 28-7	99.8	73.2	73.1		39.2	0.6 – 28-7
Test item	691.00	-	+	98.4	61.7	60.7	14.9	0.6 – 28-7	98.4	76.0	74.7		22.6	0.6 – 28-7
Test item	1382.00	-	+	97.5	74.4	72.5	20.3	0.6 – 28-7	98.6	67.2	66.3		23.0	0.6 – 28-7

PS = Phase separation at the end of treatment

P = Precipitation at the end of treatment

# = culture was not continued as a minimum of only four analysable concentrations is required

Conclusions:

The test item did not induce gene mutations at the HPRT locus in V79 cells under the conditions chosen.

Executive summary:

A GLP conform study according to OECD TG 476 was performed to assess the potential of the test item to induce gene mutations at the HPRT locus in V79 cells. An appropriate cell number was treated with test item concentrations of 43.2, 86.4, 172.8, 345.5, 691.0 and 1382.0 µg/mL for 4 hours in the absence and presence of S9 mix. No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation. No relevant and reproducible increase in mutant colony numbers per 10E+6 cells was observed in the main experiment up to the maximum concentration. The 95% confidence interval was exceeded at 345.5 µg/mL in both cultures of the main experiment with metabolic activation. However, this increase was not dose dependent and not statistical significant. Furthermore, the absolute values remained within the historical solvent control data range. Thus this increase was judged as irrelevant. The postive controls showed a distinct increase in induced mutant colonies. In conclusion, the test item did not induce gene mutations at the HPRT locus in V79 cells and thus is not considered to be mutagenic in this HPRT assay.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 2016 - April 2016

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:

26 September 2014

Deviations:

yes

Remarks:

The test design, specifically for the treatment, the recovery phase and harvest time, was slightly modified to get the optimum in response.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Healthy non-smoking female donors not receiving medication.
- Suitability of cells: Human lymphocytes are the most common cells in the micronucleus test and have been used successfully for a long time in in vitro experiments. They show stable spontaneous micronucleus frequencies at a low level.
- Sex, age and number of blood donors: one female (27 years old) for experiment I and one female (33 years old) for experiment II
- Whether whole blood or separated lymphocytes were used: whole blood were used
- Methods for maintenance in cell culture: Human lymphocytes were stimulated for proliferation by the addition of the mitogen PHA to the culture medium for a period of 48 hours.
Blood cultures were established by preparing an 11 % mixture of whole blood in medium within 30 hrs after blood collection.

MEDIA USED
- Type and identity of media including CO2 concentration: 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). All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Concentrations used: 9.0, 15.7, 27.5, 48.1, 84.2, 147.4, 257.9, 451.3, 789.7 and 1382.0 µg/mL
With regard to the molecular weight of the test item, 1382.0 μg/mL (approx. 10 mM) were applied as top concentration which is in accordance with the Guideline.

Vehicle / solvent:

- Vehicle/solvent used: water
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: demecolcin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 hours (pulse exposure) and 20 hours (continuous exposure)

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Cells were fixed with an ice-cold mixture of methanol and glacial acetic acid. The cells were stained with Giemsa.

NUMBER OF CELLS EVALUATED: 1000 binucleate cell per culture

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: The micronuclei were counted in cells showing a clearly visible cytoplasm area.

DETERMINATION OF CYTOTOXICITY
- Method: Cytokinesis-block proliferation index (CBPI)
- Any supplementary information relevant to cytotoxicity: CBPI was determined in 500 cells per culture.

Evaluation criteria:

The micronuclei have to be stained in the same way as the main nucleus. The area of the micronucleus should not extend the third part of the area of the main nucleus. At least 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides.

Negative result:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data

Positive result:
− None of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
− There is no concentration-related increase
− The results in all evaluated test item concentrations should be within the range of the laboratory historical solvent control data

Statistics:

Statistical significance was confirmed by using the Chi-squared test (α < 0.05) using a validated test script of “R”, a language and environment for statistical computing and graphics. Within this test script a statistical analysis will be conducted for those values that indicated an increase in the number of cells with micronuclei compared to the concurrent solvent control.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: none
- Definition of acceptable cells for analysis: The micronuclei were counted in cells showing a clearly visible cytoplasm area.
- Other confounding effects: none

RANGE-FINDING/SCREENING STUDIES: A pre-test was performed with test item concentrations ranging from 9.0 to 1382.0 µg/mL (with and without S9 mix). Using a reduced Cytokinesis-block proliferation index (CBPI) as an indicator for toxicity, no cytotoxic effects were observed in Experiment I after 4 hours treatment in the absence and presence of S9 mix. Therefore, 1382.0 μg/mL was chosen as top treatment concentration for Experiment II.

CYTOKINESIS BLOCK
- Distribution of mono-, bi- and multi-nucleated cells: Comparable to the the negative control.

NUMBER OF CELLS WITH MICRONUCLEI
- Number of cells for each treated and control culture: 1000 binucleate cells per culture (in total 2000 binucleated cells per concentration and controls)

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
-S9 mix (MMC; pulse treatment): mean = 11.66, range = 4.15 - 24.00, 95% CL = 1.48 – 21.85, SD = 5.09
-S9 mix (Demecolcin, continuous treatment): mean = 3.55, range = 2.10 – 6.40, 95% CL = 1.69 – 5.41, SD = 0.93
+S9 mix (CPA; pulse treatment): mean = 4.80, range = 2.25 - 11.30, 95% CL = 0.88 – 8.73, SD = 1.96

- Negative (solvent/vehicle) historical control data:
-S9 mix (pulse treatment): mean = 0.61, range = 0.15 - 1.25, 95% CL = 0.02 – 1.15, SD = 0.27
-S9 mix (continuous treatment): mean = 0.55, range = 0.05 - 1.43, 95% CL = 0.05 – 1.05, SD = 0.25
+S9 mix (pulse treatment): mean = 0.64, range = 0.15 - 1.35, 95% CL = 0.08 – 1.20, SD = 0.28

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: CBPI

Table 1: Summary of results

Exp.	Preparation interval	Test item concentration in μg/mL	Proliferation index CBPI	Cytostasis in %*	Micronucleated cells in %**
Exposure period 4 hrs without S9 mix
I	40 hrs	Solvent control^1	1.90		0.55
		Positive control^2	1.81	10.0	9.65^S
		451.3	1.90	n.c.	0.05
		789.7	1.98	n.c.	0.20
		1382.0	1.91	n.c.	0.15
Exposure period 20 hrs without S9 mix
II	40 hrs	Solvent control^1	1.94		0.85
		Positive control^3	1.79	15.4	4.95^S
		451.3	1.90	3.9	0.90
		789.7	1.86	8.2	0.80
		1382.0	1.72	23.7	1.25
Exposure period 4 hrs with S9 mix
I	40 hrs	Solvent control^1	1.93		0.40
		Positive control^4	1.56	39.9	4.30^S
		451.3	1.91	1.7	0.75
		789.7	1.93	n.c.	0.85
		1382.0	1.98	n.c.	0.90^S

*     For the positive control groups and the test item treatment groups the values are related to the solvent controls

**    The number of micronucleated cells was determined in a sample of 2000 binucleated cells

s    The number of micronucleated cells is statistically significantly higher than corresponding control values

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

1    Deion. water 10.0 % (v/v)

2    MMC 1.0 μg/mL

3    Demecolcin 50.0 ng/ml

4    CPA 15.0 μg/mL

Conclusions:

The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, the test item is considered to be non-mutagenic (non-clastogenic and non-aneugenic) in this test, when tested up to the highest required concentration.

Executive summary:

To assess the potential of the test item to induce micronuclei in human lymphocytes, an in vitro micronucleus assay according to OECD 487 under GLP conditions was performed. 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 analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. Based on the results of the pre-experiment, cells were treated with test item concentrations of 451.3, 789.7 and 1382.0 µg/mL.To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity was described as % cytostasis. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. No precipitation occurred and no osmolality and pH changes were observed. 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.

One statistically significant increase (0.9%) in micronucleated cells was observed at the highest concentration in the absence of S9 mix. However, this finding can be considered as biologically relevant as the value is clearly within the 95% Cl. In the absence of S9 mix (continuous treatment) the value of 1.25% micronucleated cells at the highest evaluated concentration exceeded the 95 % control limit (0.05 – 1.05 %). Since the value is not statistically significant, the finding can be considered as biologically irrelevant.

The positive controls induced statistically significant increases in cells with micronuclei.

In conclusion, the test item did not induce micronuclei in human lymphocytes under the test conditions chosen. Thus, the test item is considered to be non-mutagenic (non-clastogenic and non-aneugenic), when test up to the highest required concentration.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Key study - Ames test

A bacterial reverse mutation assay (OECD 471) was performed to investigate the mutagenic potential of the test substance in the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA100, TA1535, TA102, TA1537 and TA98 with and without metabolic activation. Based on the results of the preliminary study, the following concentrations were chosen for the main study: 100, 333, 1000, 2500 and 5000 µg/plate. The test material caused no visible reduction in the growth of the bacterial background lawn at any dose level. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the strains of Salmonella, at any dose level either with or without metabolic activation. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies thus confirming the activity of the S9-mix and the sensitivity of the bacterial strains. In conclusion, the test material was considered to be non-mutagenic under the conditions of this test.

Supporting study - Ames test

The mutagenic potential of the test substance was investigated in an Ames plate incorporation assay in Salmonella typhimurium TA100 without metabolic activation. The test substance, dissolved in DMSO at concentrations of 50, 100, 200 and 500 µg/plate did not produce any mutagenic effects.

Key study - HPRT test

A GLP conform study according to OECD TG 476 was performed to assess the potential of the test item to induce gene mutations at the HPRT locus in V79 cells. An appropriate cell number was treated with test item concentrations of 43.2, 86.4, 172.8, 345.5, 691.0 and 1382.0 µg/mL for 4 hours in the absence and presence of S9 mix. No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50% in both cultures occurred up to the maximum concentration with and without metabolic activation. No relevant and reproducible increase in mutant colony numbers per 10E+6 cells was observed in the main experiment up to the maximum concentration. The postive controls showed a distinct increase in induced mutant colonies. In conclusion, the test item did not induce gene mutations at the HPRT locus in V79 cells and thus is not considered to be mutagenic in this HPRT assay.

Key study - MNT in vitro

To assess the potential of the test item to induce micronuclei in human lymphocytes, an in vitro micronucleus assay according to OECD 487 under GLP conditions was performed. 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 analyzed. Per culture 1000 binucleated cells were evaluated for cytogenetic damage. Based on the results of the pre-experiment, cells were treated with test item concentrations of 451.3, 789.7 and 1382.0 µg/mL.To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity was described as % cytostasis. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. No precipitation occurred and no osmolality and pH changes were observed. 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. The positive controls induced statistically significant increases in cells with micronuclei.

In conclusion, the test item did not induce micronuclei in human lymphocytes under the test conditions chosen. Thus, the test item is considered to be non-mutagenic (non-clastogenic and non-aneugenic), when test up to the highest required concentration.

Overall Conclusion

Based on the results of the Ames key study which was performed in accordance with OECD TG 471, the test item did not increased the frequency of revertant colonies in Salmonella typhimurium strains TA100, TA1535, TA102, TA1537 and TA98 with and without S9 mix. The result for S. typhimurium TA100 without S9 mix was confirmed in a supporting study. Additionally, the test item did not induce gene mutations at the HPRT locus in V79 cells and did not induce micronuclei in human lymphocytes. Therefore, the test item ist considered to be non-mutagenic in bacteria and mammalian cells. Furthermore, the test item is non-clastogenic and non-aneugenic in human lymphocytes.

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 results indicate that the substance is non-mutagenic, non-clastogenic and non-aneugenic. 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 eighth time in Regulation (EC) No 2016/918.