1-Ethoxy-2,3-difluor-4-[4-(trans-4-propylcyclohexyl)-1-cyclohexen-1-yl]-benzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material was non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

The test material is considered to be non-clastogenic in the chromosome aberration test with and without S9 mix, when tested up to precipitating concentrations.

The test item is considered to be non-mutagenic in the HPRT assay.

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:

07.09.2007- 18.02.2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

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

Principles of method if other than guideline:

None

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

HIS operon (S. thyphimurium), TRY operon (E. coli)

Species / strain / cell type:

S. typhimurium TA 1535

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 1537

Details on mammalian cell type (if applicable):

his C 3076, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 98

Details on mammalian cell type (if applicable):

his D 3052, uvrB, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 100

Details on mammalian cell type (if applicable):

his G 46, uvrB, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

S. typhimurium TA 102

Details on mammalian cell type (if applicable):

his G 428, rfa + R-factor

Additional strain / cell type characteristics:

other: mutations in the histidine operon

Species / strain / cell type:

E. coli WP2

Details on mammalian cell type (if applicable):

his C 3076, uvrB, rfa

Additional strain / cell type characteristics:

other: mutations in the tryptophan operon

Metabolic activation:

with and without

Metabolic activation system:

rat liver homogenate (S9 mix) with standard co-factors with metabolic activation (Aroclor)

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3 - 5000 μg/plate. The pre-experiment is reported as experiment 1. Since no relevant toxic effects were observed 5000 μg/plate were chosen as maximal concentration. Due to the observed precipitation eight concentrations were tested in experiment II. The concentration range included two logarithmic decades. The following concentrations were tested: 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO (> 99 %)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene, 4-nitro-o-phenylene-diamine

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed. A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range.

Statistics:

No statistical evaluation of the data is required.

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:

not examined

True negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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:

not examined

True negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Please refer to confidential file under "Attachments".

Conclusions:

The test material was non-mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.

Executive summary:

The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD 471. This study was performed to investigate the potential of the test material to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate in pre-experiment/experiment I and II. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), occurred in the test groups with and without metabolic activation. Only in strain TA 1537 a minor reduction in the number of revertants (below the induction factor of 0.5) was observed at 5000 μg/plate in exp. I with S9 mix and in exp. II without S9 mix. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test material is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 04, 2007 - Feb 22, 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Principles of method if other than guideline:

none

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (Seromed, Berlin) including 10% (v/v) FCS (PAA Laboratories GmbH, Cölbe)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix (induction using PB/NBF)

Test concentrations with justification for top dose:

Experiment S9 Treatment+Recovery Vehicle Concentration
I - 4+14 hours DMSO 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, and 50 µg/mL
II - 18 hours DMSO 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, 50.0 and 100 µg/mL
II - 28 hours DMSO 3.1, 6.3, 12.5, 25.0, 50.0 and 100 µg/mL

I + 4+14 hours DMSO 0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, and 50 µg/mL

Vehicle / solvent:

Name: DMSO
Purity: 99.5 % (GC)
Final concentration in medium: 0.5 % (v/v)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

other: Griseofulvin

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Continous for 20 and 44 hours
Pulse for 3 + 14 and 3+41 hours

STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Solvent control: 4; others: 2

NUMBER OF CELLS EVALUATED: 100 metaphases (structural abberations)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; relative total growth

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A test article is considered as positive in this assay if:
1. the proportions of cells with structural aberrations at one or more concentration exceeds the normal range in both replicate cultures, and
2. a statistically significant increase in the proportion of cells with structural aberrations (excluding gaps) occurs at these doses. Increased incidence of cells with gaps or increased proportions of cells with structural aberrations not exceeding the normal range, or occurring only at very high or very toxic concentrations are likely to be concluded as "equivocal". Full assessment of the biological importance of such increases is likely only to be possible with reference to data from other test systems. Evidence of a dose-related effect is considered useful but not essential in the evaluation of a positive result. Cells with exchange aberrations or cells with greater than one structural aberration occur very infrequently in negative control cultures. Their appearance is therefore considered to be of particular biological significance.

Statistics:

Standard statistical methods have been applied for data processing.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: yes (>= 50 µg/mL)
- Cytotoxicity: no

Please refer to attached document under "Overall remarks, attachments".

See attchment

Conclusions:

The test material is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix, when tested up to precipitating concentrations.

Executive summary:

The test item, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. Each concentration was prepared individually because of poor test item solubility. The following study design was performed:

Experiment	S9	Treatment	Recovery	Vehicle	Concentrations
I	-	4	14	DMSO	0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, and 50 µg/mL
II	-	18	0	DMSO	0.8, 1.6, 3.1, 6.3, 12.5, 25.0, 50.0 and 100 µg/mL
II	-	28	0	DMSO	3.1, 6.3, 12.5, 25.0, 50.0 and 100 µg/mL
I	+	4	14	DMSO	0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, and 50 µg/mL

In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosome aberrations, except for the positive controls in Experiment II without metabolic activation at preparation intervals 18 and 28 hours, where only 50 metaphases were scored.
In the pre-test on toxicity 900 µg/mL (approx. 2.5 mM) was used as a top concentration due to results obtained in a solubility test. However, no homogeneous solution could be prepared. Therefore, we considered to weigh each implemented concentration individually in the main experiments in order to achieve the aimed concentrations.
This study was performed according to GLP and the methods applied are fully compliant with OECD 473.

In the absence and the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration where precipitation occurred.
No clastogenicity was observed at the concentrations evaluated, either with or without metabolic activation. In Experiment I in the presence of S9 mix two statistically significant increases in the number of aberrant cells, excluding gaps (3.0 % each) were observed after treatment with 12.5 and 50 µg/mL. However, there was no dose-dependency and both values were within the range of the laboratory's historical control data (0.0 - 4.0 % aberrant cells, excluding gaps). Therefore, this observation has to be regarded as biologically irrelevant. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.
Therefore, the test material is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix, when tested up to precipitating concentrations.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 January 2018 until 07 February 2018

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

HPRT

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM
- 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:

Due to the limited capacity for metabolic activation of potential mutagens in in vitro methods an exogenous metabolic activation system is necessary.

Phenobarbital/b-naphthoflavone induced rat liver S9 was used as metabolic activation system. The S9 was prepared and stored according to the currently valid version of the Envigo SOP for rat liver S9 preparation. Each batch of S9 was routinely tested for its capability to activate the known mutagens benzo[a]pyrene and 2-aminoanthracene in the Ames test.

An appropriate quantity of S9 supernatant was thawed and mixed with S9 cofactor solution to result in a final protein concentration of 0.75 mg/mL in the cultures. S9 mix contained MgCl2(8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4).

The protein concentration of the S9 preparation was 32.7 mg/mL (Lot. No.: 270717) in the pre-experiment and in the main experiment.

Test concentrations with justification for top dose:

4 hours treatment without S9 mix: 1.6, 3.1, 12.5, 25.0 (p), 50.0 (p), 100.0 (p), 200.0 (p) µg/mL
4 hours treatment with S9 mix: 1.6, 3.1, 12.5, 25.0 (p), 50.0 (p), 100.0 (p), 200.0 (p) µg/mL

p = precipitation visible to the unaided eye at the end of treatment

The concentrations used in the main experiment were selected based on precipitation observed in the pre-experiment.
The cultures at 50.0 µg/mL and above with and without metabolic activation were not evaluated for mutagenicity to avoid analysis of too many precipitating concentrations.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: tetrahydrofuran (THF)
- Justification for choice of solvent/vehicle: solubility properties of test item in THF and aqueous media

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

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation, Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): 6-Thioguanine

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: >1.5x10exp. 6

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

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. However, both, biological and statistical significance were considered together.

Linear Regression Analysis:
experimental group / p-value *
without S9 mix: 0.495
with S9 mix: 0.507

*calculated for the mean mutant frequencies of culture I and II

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.40 in the solvent control versus pH 7.39 at 500.0 µg/mL)
- Effects of osmolality: No relevant increase (345 mOsm in the solvent control versus 371 mOsm at 500 µg/mL)
- Evaporation from medium: Not examined
- Precipitation: determined at 25.0 - 200.0 µg/mL
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
According to the current OECD Guideline for Cell Gene Mutation Tests at least four analysable concentrations should be used in two parallel cultures. For freely-soluble and non-cytotoxic test items the maximum concentration should be 2 mg/mL, 2 µL/mL or 10 mM, whichever is the lowest. For cytotoxic test items the maximum concentration should result in approximately 10 to 20 % relative survival or cell density at subcultivation and the analysed concentrations should cover a range from the maximum to little or no cytotoxicity. Relatively insoluble test items should be tested up to the highest concentration that can be formulated in an appropriate solvent as solution or homogenous suspension. These test items should be tested up to or beyond their limit of solubility. Precipitation or phase separation should be evaluated at the beginning and at the end of treatment by the unaided eye.
The pre-experiment was performed in the presence and absence of metabolic activation. Test item concentrations between 3.9 µg/mL and 500 µg/mL were used. The highest concentration was chosen based on the solubility properties of the test item.
No relevant cytotoxic effect, indicated by a relative cloning efficiency of approx. 50 % or below was observed up to the highest concentration with and without metabolic activation.
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 hours) before the test item was removed. Precipitation occurred at 125 µg/mL and above in the presence and absence of metabolic activation.
There was no relevant shift of pH and osmolarity of the medium even at the maximum concentration of the test item.
The concentrations used in the main experiment were selected based on precipitation observed in the pre-experiment. The individual concentrations were spaced by a factor of 2.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.

COMPARISON WITH HISTORICAL CONTROL DATA: Complies

ADDITIONAL INFORMATION ON CYTOTOXICITY:
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.

Summary Table

				relative	relative	rel. adjusted	mutant	95%
	conc.	P	S9	cloning	cell	cloning	colonies/	confidence
	µg/mL		mix	efficiency I	density	efficiency I	10^6cells	interval
				%	%	%
Solvent control with THF		-	-	100.0	100.0	100.0	13.7	1.7 - 30.2
Positive control (EMS)	300.0	-	-	93.0	86.1	80.2	191.8	1.7 - 30.2
Test item	1.6	-	-	98.6	86.2	84.9	19.2	1.7 - 30.2
Test item	3.1	-	-	97.8	86.6	84.5	22.4	1.7 - 30.2
Test item	6.3	-	-	98.2	81.1	79.4	11.9	1.7 - 30.2
Test item	12.5	-	-	88.3	83.8	73.0	21.1	1.7 - 30.2
Test item	25.0	P	-	87.0	57.8	50.9	11.5	1.7 - 30.2
Test item	50.0	P	-	#	#	#	#	#
Test item	100.0	P	-	#	#	#	#	#
Test item	200.0	P	-	#	#	#	#	#
Solvent control with THF		-	+	100.0	100.0	100.0	20.1	2.0 - 29.4
Positive control (DMBA)	2.3	-	+	69.0	99.2	68.0	135.4	2.0 - 29.4
Test item	1.6	-	+	90.4	92.7	84.4	13.7	2.0 - 29.4
Test item	3.1	-	+	83.3	95.6	80.3	20.6	2.0 - 29.4
Test item	6.3	-	+	86.6	94.4	82.2	23.7	2.0 - 29.4
Test item	12.5	-	+	84.8	92.6	79.0	18.0	2.0 - 29.4
Test item	25.0	P	+	75.3	87.2	66.9	22.1	2.0 - 29.4
Test item	50.0	P	+	75.9	104.0	78.9	#	#
Test item	100.0	P	+	#	#	#	#	#
Test item	200.0	P	+	#	#	#	#	#

P = precipitation visible to the unaided eye at the end of treatment

# culture was not continued to avoid analysis of too many precipitating concentrations

Conclusions:

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, the test item is considered to be non-mutagenic in this HPRT assay.

Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD 476.

The treatment period was 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment (500 µg/mL) was based on the solubility properties of the test item. The highest concentration in the main experiment (200.0 µg/mL) was limited by precipitation observed in the pre-experiment.

No relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed in the main experiment up to the maximum concentration.

No cytotoxic effects indicated by an adjusted cloning efficiency below 50 % was observed neither in absence nor presence of metabolic activation.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

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, the test item 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

OECD 471

The information for this endpoint study record was obtained from an experimental study. The OECD GLP criteria were met and the methods applied are fully compliant with OECD 471. This study was performed to investigate the potential of the test material to induce gene mutations according to the plate incorporation test (experiment I) and the preincubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102. The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate in pre-experiment/experiment I and II. The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used. No toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), occurred in the test groups with and without metabolic activation. Only in strain TA 1537 a minor reduction in the number of revertants (below the induction factor of 0.5) was observed at 5000 μg/plate in exp. I with S9 mix and in exp. II without S9 mix. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, the test material is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

 

OECD 473

The test item, suspended in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in two independent experiments. Each concentration was prepared individually because of poor test item solubility. The following study design was performed:

Experiment	S9	Treatment	Recovery	Vehicle	Concentrations
I	-	4	14	DMSO	0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, and 50 µg/mL
II	-	18	0	DMSO	0.8, 1.6, 3.1, 6.3, 12.5, 25.0, 50.0 and 100 µg/mL
II	-	28	0	DMSO	3.1, 6.3, 12.5, 25.0, 50.0 and 100 µg/mL
I	+	4	14	DMSO	0.4, 0.8, 1.6, 3.1, 6.3, 12.5, 25.0, and 50 µg/mL

In each experimental group two parallel cultures were set up. Per culture 100 metaphases were scored for structural chromosome aberrations, except for the positive controls in Experiment II without metabolic activation at preparation intervals 18 and 28 hours, where only 50 metaphases were scored.
In the pre-test on toxicity 900 µg/mL (approx. 2.5 mM) was used as a top concentration due to results obtained in a solubility test. However, no homogeneous solution could be prepared. Therefore, we considered to weigh each implemented concentration individually in the main experiments in order to achieve the aimed concentrations.
This study was performed according to GLP and the methods applied are fully compliant with OECD 473.

In the absence and the presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration where precipitation occurred.
No clastogenicity was observed at the concentrations evaluated, either with or without metabolic activation. In Experiment I in the presence of S9 mix two statistically significant increases in the number of aberrant cells, excluding gaps (3.0 % each) were observed after treatment with 12.5 and 50 µg/mL. However, there was no dose-dependency and both values were within the range of the laboratory's historical control data (0.0 - 4.0 % aberrant cells, excluding gaps). Therefore, this observation has to be regarded as biologically irrelevant. No relevant increase in the frequencies of polyploid metaphases was found after treatment with the test item as compared to the frequencies of the controls. Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations as determined by the chromosome aberration test in V79 cells (Chinese hamster cell line) in vitro.
Therefore, the test material is considered to be non-clastogenic in this chromosome aberration test with and without S9 mix, when tested up to precipitating concentrations.

 

OECD 476

The study was performed to investigate the potential of the test item to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster according to OECD 476.

The treatment period was 4 hours with and without metabolic activation.

The maximum test item concentration of the pre-experiment (500 µg/mL) was based on the solubility properties of the test item. The highest concentration in the main experiment (200.0 µg/mL) was limited by precipitation observed in the pre-experiment.

No relevant and reproducible increase in mutant colony numbers/10⁶ cells was observed in the main experiment up to the maximum concentration.

No cytotoxic effects indicated by an adjusted cloning efficiency below 50 % was observed neither in absence nor presence of metabolic activation.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system.

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, the test item is considered to be non-mutagenic in this HPRT assay.

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. Based on available data, the test item does not require classification for genetic toxicity according to Regulation (EC) No 1272/2008 (CLP).