3-phenylpropan-1-ol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was not mutagenic in the bacterial reverse mutation assay and in the in vitro mammalian cell gene mutation assay (HPRT). The test item did not induce micronuclei in the in vitro micronucleus test in human lymphocytes.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-09-20 to 2017-11-17

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:

July 2016

Deviations:

yes

Remarks:

please refer to 'Principles of method if other than guideline'

Principles of method if other than guideline:

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:

lymphocytes: human (primary culture)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood samples were drawn from healthy non-smoking donors not receiving medication. For this study, blood was collected from a male donor (27 years old) for Experiment I and from a male donor (24 years old) for Experiment II.
- Suitability of cells: The lymphocytes of the respective donors have been shown to respond well to stimulation of proliferation with PHA and to positive control substances. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes.
- Cell culture conditions: 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).
All incubations were done at 37 °C with 5.5 % CO2 in humidified air.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/beta-naphthoflavone induced rat liver S9-mix

Test concentrations with justification for top dose:

Experimtent I (4 h exposure): 8.8, 15.5, 27.1, 47.4, 83.0, 145, 254, 445, 778, 1362 µg/mL, with and without S9 mix
Experiment II (40 h exposure): 8.8, 15.5, 27.1, 47.4, 83.0, 145, 254, 445, 778, 1362 µg/mL, with S9 mix

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- 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:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Demecolcine (without metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4 h (pulse exposure), 20 h (continuous exposure, without S9 mix)
- Expression time (cells in growth medium): 16 h recovery period followed by another 20 h (4 h group), and 20 h (20 h exposure group)
- Fixation time (start of exposure up to fixation or harvest of cells): 40 h

SPINDLE INHIBITOR: Cytochalasin B

STAIN: Giemsa

NUMBER OF REPLICATIONS: 2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: The slides were prepared by dropping the cell suspension in fresh fixative onto a clean microscope slide.

NUMBER OF CELLS EVALUATED: 500 cells per culture

CRITERIA FOR MICRONUCLEUS IDENTIFICATION: Evaluation of the slides was performed using microscopes with 40 x objectives. The micronuclei were counted in cells showing a clearly visible cytoplasm area. The criteria for the evaluation of micronuclei are described in the publication of Countryman and Heddle (1976). 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.

DETERMINATION OF CYTOTOXICITY
- Method: see below
- Any supplementary information relevant to cytotoxicity:
To describe a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI (Cytokinesis-block proliferation index) of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis.
Cytostasis % = 100 – 100 [(CBPI(test item) – 1) / (CBPI(solvent control) – 1)]

CBPI = (Mononucleate cells x 1) + (Binucleate cells x 2) + (Multinucleate cells x 3)

Rationale for test conditions:

according to guideline

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in all of the experimental conditions examined:
- 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 (95% control limit realized as 95% confidence interval)
The test item is then considered unable to induce chromosome breaks and/or gain or loss in this test system.

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly positive if, in any of the experimental conditions examined:
- At least one of the test item concentrations exhibits a statistically significant increase compared with the concurrent solvent control
- The increase is concentration-related in at least one experimental condition
- The results are outside the range of the laboratory historical solvent control data (95% control limit realized as 95% confidence interval)
When all of the criteria are met, the test item is then considered able to induce chromosome breaks and/or gain or loss in this test system.

Statistics:

Statistical significance was confirmed by the Chi Square 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 was 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:

cytotoxicity

Remarks:

from 778 µg/mL onwards

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: none
- Effects of osmolality: none
- Precipitation: no precipitation of the test item in the culture medium was observed

RANGE-FINDING/SCREENING STUDIES:
Dose selection was based on a preliminary test for cytotoxicity (Experiment I).

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
Mitomycin C (78 experiments): micronucleated cells mean 12.48 (95 % Ctrl limit): 1.44 - 23.52
Demecolcine (81 experiments): micronucleated cells mean 3.72 (95 % Ctrl limit): 1.43 - 6.01
Cyclophosphamide (165 experiments): micronucleated cells mean 5.16 (95 % Ctrl limit): 0.84 - 9.49
- Negative (solvent/vehicle) historical control data:
with metabolic activation: 96 experiments: micronucleated cells mean 0.62 (95 % Ctrl limit): 0.15 - 1.3
without metabolic activation: 78 experiments: micronucleated cells mean 0.60 (95 % Ctrl limit): 0.08 - 1.12

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

Table 1: Cytotoxicity indicated as cytokinesis-block proliferation indexand cytostasis; exposure period 4 hrs without S9 mix, Experiment I

Treatmentgroup	Conc.per mL	S9mix	Exposure /preparation	Cell proliferation culture 1*			ProliferationIndex	Cell proliferation culture 2*			ProliferationIndex
				c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI	Cytostasis
												mean	[%]
Solv. control^#	0.5 %	-	4 / 40 hrs	79	374	47	1.94	90	365	45	1.91	1.92
Pos. control^##	0.8 µg	-	4 / 40 hrs	147	339	14	1.73	157	330	13	1.71	1.72	21.7
Test item	445 µg	-	4 / 40 hrs	97	356	47	1.90	112	346	42	1.86	1.88	4.7
²	778 µg	-	4 / 40 hrs	59	396	45	1.97	71	367	62	1.98	1.98	n.c.
²	1362 µg	-	4 / 40 hrs	48	426	26	1.96	107	373	20	1.83	1.89	3.5

*          c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#                DMSO

^##         MMC

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

 

Table 2: Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 4 hrs with S9 mix, Experiment I

Treatmentgroup	Conc.per mL	S9mix	Exposure /preparation	Cell proliferation culture 1*			ProliferationIndex	Cell proliferation culture 2*			ProliferationIndex
				c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI	Cytostasis
												mean	[%]
Solv. control^#	0.5 %	+	4 / 40 hrs	70	366	64	1.99	73	342	85	2.02	2.01
Pos. control^##	15.0 µg	+	4 / 40 hrs	279	208	13	1.47	250	238	12	1.52	1.50	50.7
Test item	445 µg	+	4 / 40 hrs	79	343	78	2.00	97	344	59	1.92	1.96	4.5
²	778 µg	+	4 / 40 hrs	130	334	36	1.81	61	402	37	1.95	1.88	12.3
²	1362 µg	+	4 / 40 hrs	147	332	21	1.75	114	373	13	1.80	1.77	23.2

*             c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#                     DMSO

^##            CPA

 

Table 3: Number of micronucleated cells; exposure period 4 hrs without S9 mix, Experiment I

Treatment	Conc.	S9	Exposure/	Micronucleated cells
group	per mL	mix	preparation	Binucleate cells withnmicronuclei culture 1			sum culture 1	Binucleate cells withnmicronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
				1	2	>2		1	2	>2
Solv. control^#	0.5 %	-	4 / 40 hrs	8	1	0	9	7	0	0	7	16	0.80
Pos. control^##	0.8 µg	-	4 / 40 hrs	136	15	1	152	144	17	2	163	315	15.75
Test item	445 µg	-	4 / 40 hrs	8	0	0	8	3	0	0	3	11	0.55
²	778 µg	-	4 / 40 hrs	3	1	0	4	4	0	0	4	8	0.40
²	1362 µg	-	4 / 40 hrs	3	1	0	4	12	3	0	15	19	0.95

^#                                            DMSO

^##                   MMC

 

Table 4:Number of micronucleated cells; exposure period 4 hrs with S9 mix, Experiment I

Treatment	Conc.	S9	Exposure/	Micronucleated cells
group	per mL	mix	preparation	Binucleate cells withnmicronuclei culture 1			sum culture 1	Binucleate cells withnmicronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
				1	2	>2		1	2	>2
Solv. control^#	0.5 %	+	4 / 40 hrs	4	0	0	4	5	1	0	6	10	0.50
Pos. control^##	15.0 µg	+	4 / 40 hrs	52	6	1	59	54	3	0	57	116	5.80
Test item	445 µg	+	4 / 40 hrs	4	1	0	5	4	0	0	4	9	0.45
²	778 µg	+	4 / 40 hrs	5	0	0	5	5	0	0	5	10	0.50
²	1362 µg	+	4 / 40 hrs	4	2	0	6	5	1	0	6	12	0.60

#             DMSO

##           CPA

 

 

Table 5:Cytotoxicity indicated as cytokinesis-block proliferation index and cytostasis; exposure period 20 hrs without S9 mix, Experiment II

Treatmentgroup	Conc.per mL	S9mix	Exposure /preparation	Cell proliferation culture 1*			ProliferationIndex	Cell proliferation culture 2*			ProliferationIndex
				c1	c2	c4-c8	CBPI	c1	c2	c4-c8	CBPI	CBPI	Cytostasis
												mean	[%]
Solv. control^#	0.5 %	-	20 / 40 hrs	40	397	63	2.05	68	380	52	1.97	2.01
Pos. control^##	75 ng	-	20 / 40 hrs	251	226	23	1.54	279	206	15	1.47	1.51	49.6
Test item	254 µg	-	20 / 40 hrs	109	359	32	1.85	98	367	35	1.87	1.86	14.6
²	445 µg	-	20 / 40 hrs	126	340	34	1.82	132	342	26	1.79	1.80	20.4
²	778 µg	-	20 / 40 hrs	200	292	8	1.62	250	245	5	1.51	1.56	44.1

*             c1: mononucleate cells; c2: binucleate cells; c4-c8: multinucleate cells

^#                     DMSO

^##            Demecolcine

 

Table 6: Number of micronucleated cells; exposure period 20 hrs without S9 mix, Experiment II

Treatment	Conc.	S9	Exposure/	Micronucleated cells
group	per mL	mix	preparation	Binucleate cells withnmicronuclei culture 1			sum culture 1	Binucleate cells withnmicronuclei culture 2			sum culture 2	sum in 2000 binucleate cells	[%]
				1	2	>2		1	2	>2
Solv. control^#	0.5 %	-	20 / 40 hrs	8	0	0	8	2	1	0	3	11	0.55
Pos. control^##	75 ng	-	20 / 40 hrs	36	7	3	46	19	9	3	31	77	3.85
Test item	254 µg	-	20 / 40 hrs	7	0	0	7	3	0	0	3	10	0.50
²	445 µg	-	20 / 40 hrs	3	0	0	3	2	0	0	2	5	0.25
²	778 µg	-	20 / 40 hrs	3	0	0	3	2	0	0	2	5	0.25

^#                     DMSO

^##                   Demecolcine

Conclusions:

The test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Executive summary:

The test item (dissolved in DMSO) was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. 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 analyzed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis. The highest treatment concentration in this study, 1362 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test. In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II in the absence of S9 mix after continuous treatment, moderate cytotoxicity of 44.1 % cytostasis was observed at the highest evaluated concentration (778 µg/mL). Due to a steep cytotoxic gradient caused by the test item the exact requested among of cytotoxicity was not met. The next higher tested and highest applied concentration (1362 µg/mL), however, which was separated by a smaller factor than requested by the guideline, showed clear cytotoxic effects and were not evaluable for cytogenetic damage (no cells available). In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. Demecolcine (75 ng/mL), MMC (0.8 µg/mL) and CPA (15.0 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2002-07-21 to 2002-08-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP and Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: besides being histidine auxotrophic, all strains carry an additional mutation (loss of outer lipopolysaccharide barrier) which increases cell permeability

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix, Aroclor 1254 induced

Test concentrations with justification for top dose:

0, 15, 50, 150, 500, 1500 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:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: with S9 mix: 2-aminoanthracene (all strains)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Expression time (cells in growth medium): 48 - 72 h

NUMBER OF REPLICATIONS: 3 each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

The number of spontaneous revertants observed using each of the five strains and the results with the positive controls were compared to historical control data. Differences between the number of revertants in the negative controls and the test plates were tested for significance.

Statistics:

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 was performed using a X2-test.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed in strains TA 98, 100, 102 and 1537 at the highest test concentrations without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed in strains TA 98, 100, 102 and 1537 at the highest test concentrations without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed in strains TA 98, 100, 102 and 1537 at the highest test concentrations without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed in strains TA 98, 100, 102 and 1537 at the highest test concentrations without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity was observed in strains TA 98, 100, 102 and 1537 at the highest test concentrations without metabolic activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Conclusions:

The test substance was not mutagenic in the bacterial reverse mutation assay.

Executive summary:

The test substance was tested in the bacterial reverse mutation assay using the five Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. The test concentrations were 0, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test was carried out in two independent tests using the standard plate incorporation assay with and without metabolic activation (S9 mix from rat livers, Aroclor 1254 induced). Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene were used as positive controls. At the highest test concentration, bacteriotoxicity was observed in the absence of metabolic activation. Mutagenicity was not observed.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-17 to 2017-09-14

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:

2016

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation assay (HPRT)

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: stocks of the V79 cell line from Laboratory for Mutagenicity Testing; Technical University, 64287 Darmstadt, Germany
- Suitability of cells: The V79 cell line has been used successfully in in vitro experiments for many years. Recommend cell line for this assay.
- Cell cycle length, doubling time or proliferation index: doubling time 12 - 16 h in stock cultures, good cloning efficiency of untreated cells (as a rule more than 50%)
- Modal number of chromosomes: 22


MEDIA USED
- 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%). The cells were sub-cultured once or twice weekly. All incubations were done at 37°C with 1.5% carbon dioxide (CO2) in humidified air.
During treatment no FBS was added to the medium. For the selection of mutant cells the complete medium was supplemented with 11 μg/mL 6-thioguanine. All cultures were incubated at 37 °C in a humidified atmosphere with 1.5% CO2 (98.5% air).
- Properly maintained: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/β-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

In a pre-experiment relevant cytotoxic effect, indicated by a relative cloning efficiency of 50% or below was observed at 1362.0 μg/mL with and without metabolic activation. The individual concentrations in the main experiment were generally spaced by a factor of 2.0. A narrower spacing was used at the higher concentrations to cover the cytotoxic range more closely. The following concentrations were tested:
in µg/mL: 85.1, 170.3, 340.5, 681.0, 851.25, 1021.5, 1191.75, 1362.0 (both with and without metabolic activation)

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- 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:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding: Approximately 0.7 to 1.2×10^7 were seeded in plastic flasks. The cells were grown for 24 hours prior to treatment.

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 days
- Selection time: 8 days
- Fixation time: The colonies used to determine the cloning efficiency I were fixed and stained 6 to 8 days after treatment.

SELECTION AGENT: 11 μg/mL 6-thioguanine

STAIN (for cytogenetic assays): 10% methylene blue in 0.01% KOH solution

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: The stained colonies with more than 50 cells were counted.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

A test item is classified as clearly mutagenic if, in any of the experimental conditions examined, all of the following criteria are met:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
A test item is classified as clearly non-mutagenic if, in all experimental conditions examined, all of the following criteria are met:
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data (based 95% control limits).

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 was performed using a validated test script of “R”, a language and environment for statistical computing and graphics, to evaluate an isolated increase of the mutation frequency at a test point exceeding the 95% confidence interval. Again a t-test is judged as significant if the p-value (probability value) is below 0.05.
However, both, biological and statistical significance were 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:

at 1021.5 μg/mL with and without metabolic activation, at 851.3 µg/mL with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects observed
- Effects of osmolality: no effects observed
- Precipitation/Phase separation: Phase separation occurred at 1362.0 μg/mL after 4 hours treatment in the absence of metabolic activation.

Table 1: Main experiment, 4 hour exposure period, culture I and II

Test group [µg/mL]	S9 mix	Relative cloning efficiency 1 (survival) [%]		Relative cloning efficiency 2 (viability) [%]		Mutant Frequency (per 10^6 cells)
		Culture 1	Culture 2	Culture 1	Culture 2	Culture 1	Culture 2
Solvent control DMSO	-	100	100	100	100	39.8	8.1
Positive control (EMS, 300)	-	78.6	47.8	94.8	118.3	395.6	335.2
85.1	-	92.8	68.1	#	#	#	#
170.3	-	90.1	53.4	97.5	102.9	34.4	27.6
340.5	-	80.1	41.6	89.2	110.6	25.4	18.6
681.0	-	61.3	53	72	113.1	28.5	25.5
851.25	-	51.1	54.8	91.2	103.3	14.8	18.1
1021.5	-	7.2	7.3	93.3	88.6	30.1	13.8
1191.75	-	##	##	##	##	##	##
1362	-	##	##	##	##	##	##
Solvent control DMSO	+	100	100	100	100	18.9	18
Positive control (DMBA, 2.3)	+	89.6	111.7	105.4	67.9	115	133.2
85.1	+	#	124.3	#	#	#	#
170.3	+	83	101.4	90.2	82.7	21	23.9
340.5	+	86.6	89.2	100.4	91.6	22.4	17.7
681.0	+	54.4	98.1	99.7	104.4	17.3	15.3
851.25	+	66.5	17.9	87.7	93.1	27.5	5.4
1021.5	+	16.3	6.6	107.4	84.2	4.1	19.6
1191.75	+	8	##	##	##	##	##
1362	+	4.1	##	##	##	##	##

# culture not continued as a minimum of only four analyzable concentrations are required

## culture not continued due to exceedingly severe cytotoxic effects

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:

This in vitro experiment was performed to assess the potential of the test item to induce gene mutations using the Chinese hamster cell line V79. Two parallel cultures were used throughout the assay.

The main experiment was performed with a treatment time of 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment was 1362μg/mL regarding the current OECD guideline 476 (10 mM). The analyzed concentration range of the main experiment was limited by cytotoxicity of the test item. The individual concentrations in the main experiment were generally spaced by a factor of 2.0. A narrower spacing was used at the higher concentrations to cover the cytotoxic range more closely. The following concentrations were tested [µg/mL]: 85.1, 170.3, 340.5, 681.0, 851.25, 1021.5, 1191.75, 1362.0 (both with and without metabolic activation, Phenobarbital/β-naphthoflavone induced rat liver S9 mix).

Appropriate reference mutagens, used as positive controls (ethylmethane sulfonate without metabolic activation and 7,12-dimethylbenz(a)anthracene with metabolic activation), induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore it is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test 

The test substance was tested in the bacterial reverse mutation assay using the five Salmonella typhimurium strains TA1535, TA1537, TA98, TA100, and TA102. The test concentrations were 0, 15, 50, 150, 500, 1500 and 5000 µg/plate. The test was carried out in two independent tests using the standard plate incorporation assay with and without metabolic activation (S9 mix from rat livers, Aroclor 1254 induced). Sodium azide, 2-nitrofluorene, 9-aminoacridine, mitomycin C, and 2-aminoanthracene were used as positive controls. At the highest test concentration, bacteriotoxicity was observed in the absence of metabolic activation. Mutagenicity was not observed.

HPRT 

An in vitro experiment was performed to assess the potential of the test item to induce gene mutations in mammalian cells using the Chinese hamster cell line V79. Two parallel cultures were used throughout the assay. The main experiment was performed with a treatment time of 4 hours with and without metabolic activation. The maximum test item concentration of the pre-experiment was 1362μg/mL regarding the current OECD guideline 476 (10 mM). The analyzed concentration range of the main experiment was limited by cytotoxicity of the test item. The individual concentrations in the main experiment were generally spaced by a factor of 2.0. A narrower spacing was used at the higher concentrations to cover the cytotoxic range more closely. The following concentrations were tested [µg/mL]: 85.1, 170.3, 340.5, 681.0, 851.25, 1021.5, 1191.75, 1362.0 (both with and without metabolic activation, Phenobarbital/β-naphthoflavone induced rat liver S9 mix).

Appropriate reference mutagens, used as positive controls (ethylmethane sulfonate without metabolic activation and 7,12-dimethylbenz(a)anthracene with metabolic activation), induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. No substantial and reproducible dose dependent increase of the mutation frequency was observed in the main experiment. In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. Therefore it is considered to be non-mutagenic in this HPRT assay.

MNT 

The test item, dissolved in DMSO, was further assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. 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 analyzed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in 500 cells per culture and cytotoxicity is described as % cytostasis. The highest treatment concentration in this study, 1362 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test. In Experiment I in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In Experiment II in the absence of S9 mix after continuous treatment, moderate cytotoxicity of 44.1 % cytostasis was observed at the highest evaluated concentration (778 µg/mL). Due to a steep cytotoxic gradient caused by the test item the exact requested among of cytotoxicity was not met. The next higher tested and highest applied concentration (1362 µg/mL), however, which was separated by a smaller factor than requested by the guideline, showed clear cytotoxic effects and were not evaluable for cytogenetic damage (no cells available). In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed. Demecolcine (75 ng/mL), MMC (0.8 µg/mL) and CPA (15.0 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei. In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes.

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. As a result the substance is not considered to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.