4'-Ethyl-3-fluorbiphenyl-4-boronic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

For this compound, genotoxicity was studied in vitro in bacteria and in mammalian cells. The information was obtained from experimental studies. The OECD GLP criteria were met and the methods applied are fully compliant with OECD TG 471, 473, and 476.

The following results have been obtained:

OECD 471: negative

OECD 473: positive

OECD 476: negative

The cytogenetic potential of the test substance has to be further investigated. An in vivo Mammalian Erythrocyte Micronucleus Assay (OECD 474) was proposed by the registrant for the structural analaogue substance 4'-Ethyl-2,3 -difluorbiphenyl-4 -boronic acid. Due to animal welfare reasons and exposure considerations (use as intermediate, extended personal protective measures) the in vivo Mammalian Erythrocyte Micronucleus Assay proposed with the structural analogue substance will be used to conclude on the cytogenetic potential.

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:

2006-11-01 to 2006-11-10

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:

07-1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Official Journal of the European Communities L136, 8. June 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

HIS operon (S. thyphimurium)
TRP operon (E. coli)

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from Aroclor 1254-pretreated rats with standard co-factors

Test concentrations with justification for top dose:

The test material concentrations used were selected according to the EC and OECD guidelines for this test system and the requirements of the Labor Ministry of Japan:
1st series: 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate
2nd series: 50, 88.9, 158, 281 and 500 μg/plate
In the two series with S9 mix, 10 or 30 % S9 in the S9 mix were used in the 1st and 2nd series, respectively.

Vehicle / solvent:

DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

sodium azide

cumene hydroperoxide

other: daunomycin

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

The assessment of test material-induced effects is dependent on the number of spontaneous revertants of each bacterial strain (solvent controls) and the increase in the number of revertants at the test material concentration which shows the highest number of colonies. The following criteria, based upon the historical controls of the laboratory and statistical considerations, are established:

Mean Number of Colonies Maximal Mean Number of Colonies over
(Solvent Control) the Actual Solvent Control (Test Material)
----------------------------------------------------------------------------------------------
<=10 <=9 >=30
<=30 <=19 >=40
<=80 <=29 >=80
<=200 <=49 >=120
<=500 <=79 >=200
Assessment: No increase Clear increase
----------------------------------------------------------------------------------------------

All further results, ranging between "no" and "clear", are assessed as "weak in-creases".

Interpretations:

A test material is defined as non-mutagenic in this assay if:
- "no" or "weak increases" occur in the first and second series of the main experiment. ("Weak increases" randomly occur due to experimental variation.)

A test material is defined as mutagenic in this assay if:
- a dose-related (over at least two test material concentrations) increase in the number of revertants is induced, the maximal effect is a "clear increase", and the effects are reproduced at similar concentration levels in the same test system;
- "clear increases" occur at least at one test material concentration, higher concentrations show strong precipitation or cytotoxicity, and the effects are reproduced at the same concentration level in the same test system.

In all further cases, a third test series with the bacterial strain in question should be performed. If the criteria for a positive test result are not fulfilled in at least two out of the three series, the test material is defined as being non-mutagenic in this test system.

Evaluation criteria:

For details see results.

Statistics:

n.a.

Key result

Species / strain:

S. typhimurium TA 98

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 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:

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:

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 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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:

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, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Executive summary:

Study Design

The investigations for the mutagenic potential of the test material were performed using Salmonella typhimurium tester strains TA 98, TA 100, TA 102, TA 1535, TA 1537 and Escherichia coli WP2 uvrA. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. As solvent DMSO was used in two independent experimental series with concentrations ranging from 5 - 5000 µg/plate.

Results

Precipitation of the test material on the agar plates occurred at concentrations of >= 158 μg/plate. In the absence of S9 mix, toxicity to the bacteria was observed in the concentration range between 281 and 5000 µg/plate in tester strains TA 100, TA 102, TA 1535 and TA 1537. Each treatment with the test materials used as positive controls led to a clear increase in revertant colonies, thus, showing the expected reversion properties of all strains and good metabolic activity of the S9 mix used. In both series of experiments, each performed with and without the addition of rat liver S9 mix as the external metabolizing system, the test material showed no increase in the number of revertants of any bacterial strain. According to the criteria for negative and positive results, the test material was not mutagenic under the described experimental conditions.

Conclusion

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic under the experimental conditions described.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-01-16 until 2019-02-15

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Type and identity of media: Dulbeccos's modified Eagle's medium/Ham's F12 medium
- Properly maintained: yes

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

With metabolic activation:
Experiment I: 1.8, 3.2, 5.7, 9.9, 17.4, 30.5, 53.3, 93.3, 163, 286, 500 µg/mL

Without metabolic activation:
Experiment I: 1.8, 3.2, 5.7, 9.9, 17.4, 30.5, 53.3, 93.3, 163, 286, 500 µg/mL
Experiment II: 1.7, 3.0, 5.2, 9.1, 16.0, 28.0, 49.0, 85.7, 150 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility and relatively low cytotoxicity in accordance to the OECD Guideline 473

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Two independent experiments were performed. In Experiment I the exposure period was 4 hours with and without metabolic activation. In Experiment II the exposure period was 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item. Evaluation of two cultures per dose group.
METHOD OF APPLICATION: in culture medium

DURATION
- Exposure duration: 4 hours (+/- S9 mix) and 22 hours (- S9 mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hours


SPINDLE INHIBITOR (cytogenetic assays): Colcemid
STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: about 1.5


NUMBER OF CELLS EVALUATED: 150 per culture


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

Evaluation criteria:

Evaluation of the cultures was performed (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100x oil immersion objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. 150 well spread metaphases per culture were scored for cytogenetic damage on coded slides.
Only metaphases with characteristic chromosome numbers of 46 ± 2 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05).

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The test material, dissolved in DMSO, was assessed for its potential to induce chromosomal aberrations 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 22 hours without S9 mix. The chromosomes were prepared 22 hours after start of treatment with the test item.
In each experimental group two parallel cultures were analysed. 150 metaphases per culture were evaluated for structural chromosomal aberrations. 1000 cells were counted per culture for determination of the mitotic index.
The highest treatment concentration in this study, 500 µg/mL was chosen with regard to the solubility properties of the test item and with respect to the OECD Guideline for in vitro mammalian cytogenetic tests.
In Experiment I, precipitation of the test item in the culture medium was observed at 17.4 µg/mL and above in the absence and presence of S9 mix. In addition, precipitation occurred in Experiment II, in the absence of S9 mix, at 85.7 µg/mL and above.
No relevant influence on osmolarity or pH was observed.
In this study in the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation.
In Experiment I in the absence and presence of S9 mix, neither statistically significant nor biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test item. In Experiment II in the absence of S9 mix, statistically significant increases in the number of cells carrying structural chromosomal aberrations were observed after continuous treatment with 49.0 and 85.7 µg/mL (2.7 and 3.3 % aberrant cells, excluding gaps). The values exceeded the historical control data range (0.0 – 1.9 % aberrant cells, excluding gaps) and a trend was confirmed via linear regression test.
No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
In both experiments, either EMS (550 or 825 µg/mL) or CPA (10.0 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Summary of results

Exp.	Preparation interval	Test item concentration	Mitotic indices	Aberrant cells(%)
		(µg/mL)	(%of control)	incl. gaps*	excl. gaps*	carrying exchanges

Exposure period 4 hrs without S9 mix

I	22 hrs	Solvent control^1	100.0	1.0	0.7	0.0
		Positive control^2	56.5	9.3	9.0^S	4.7
		5.7	91.1	2.3	2.0	0.3
		9.9	89.2	1.0	1.0	0.0
		17.4^P	99.3	0.3	0.3	0.0

Exposure period 22 hrs without S9 mix

II	22 hrs	Solvent control^1	100.0	0.3	0.3	0.0
		Positive control^3	57.3	23.0	20.7^S	8.7
		28.0	111.7	1.0	1.0	0.0
		49.0	127.3	2.7	2.7^S	0.0
		85.7^P	119.8	3.3	3.3^S	0.0

Exposure period 4 hrs with S9 mix

I	22 hrs	Solvent control^1	100.0	1.3	1.0	0.0
		Positive control^4	61.5	15.0	15.0^S	3.3
		5.7	107.6	1.0	1.0	0.0
		9.9	83.1	1.3	1.3	0.0
		17.4^P	110.4	1.7	1.7	0.3

*  Including cells carrying exchanges

^P   Precipitation occurred at the end of treatment

^S   Aberration frequency statistically significant higher than corresponding control values

¹   DMSO    0.5% (v/v)
²    EMS       825 µg/mL
³    EMS       550 µg/mL
⁴   CPA       10.0 µg/mL

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item induced structural chromosomal aberrations in human lymphocytes in vitro in the absence of S9 mix. Therefore, the test material is considered to be clastogenic in this chromosome aberration test, when tested up to precipitating concentrations.

Executive summary:

The test material, dissolved in DMSO, was assessed for its potential to induce structural chromosomal aberrations in human lymphocytesin vitroin two independent experiments. The following study design was performed:

	Without S9 mix		With S9 mix
	Exp. I	Exp. II	Exp. I
Exposure period	4 hrs	22 hrs	4 hrs
Recovery	18 hrs	-	18 hrs
Preparation interval	22 hrs	22 hrs	22 hrs

In each experimental group two parallel cultures were analyzed. Per culture 150 metaphases were evaluated for structural chromosomal aberrations.

The highest applied concentration in this study (500 µg/mL of the test item) was chosen with regard to the solubility properties of the test item and with respect to the current OECD Guideline 473.

Dose selection of the cytogenetic experiment was performed considering the toxicity data and the occurrence of test item precipitation in accordance with OECD Guideline 473.

In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest evaluated concentration, which showed precipitation.

In Experiment I in the absence and presence of S9 mix, neither statistically significant nor biologically relevant increases in the number of cells carrying structural chromosomal aberrations were observed after treatment with the test item. In Experiment II in the absence of S9 mix, statistically significant increases in the number of cells carrying structural chromosomal aberrations were observed after continuous treatment with 49.0 and 85.7 µg/mL (2.7 and 3.3 % aberrant cells, excluding gaps). The values exceeded the historical control data range (0.0 – 1.9 % aberrant cells, excluding gaps) and a trend was confirmed via linear regression test.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases in cells with structural chromosome aberrations

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-12-18 until 2019-02-05

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:

mammalian cell gene mutation assay

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:

Phenobarbital/Beta-Naphtoflavone induced Rat liver S9

Test concentrations with justification for top dose:

0.9; 1.9; 3.8; 7.5; 15.0; 30.0; 60.0 (p); 120.0 (p) µg/mL with and without metabolic activation (4 hours treatment)

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 0.9 and 1.9 µg/mL with and without metabolic activation were not evaluated for mutagenicity as a minimum of only four concentrations is required by the guidelines.
The cultures at 120.0 µg/mL with and without metabolic activation were not continued to avoid analysis of too many precipitating concentrations.

Vehicle / solvent:

Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: solubility properties of the test substance in DMSO and aqueous media.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

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 concen¬trations 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.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not effected (pH 7.53 in the solvent control versus pH 7.48 at 500.0 µg/mL)
- Effects of osmolality: No relevant increase (395 mOsm in the solvent control versus 394 mOsm at 500.0 µg/mL)
- Evaporation from medium: Not examined
- Precipitation: determined at 60.0 µg/mL and above
- 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 31.3 µg/mL and above in the absence of metabolic activation, and at 62.5 µg/mL and above in the presence 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 cytotoxic effect indicated by the mean adjusted cloning efficiency I below 50% was observed neither in absence nor presence of metabolic activation in the main experiment.

Summary of Results (mean values of culture I and II)

				relative	relative	rel. adjusted	mutant	95%
	conc.	P	S9	cloning	cell	cloning	colonies/	confidence
	µg/mL		mix	efficiency I	density	efficiency I	10exp. 6 cells	interval
				%	%	%
Solvent control with DMSO			-	100.0	100.0	100.0	18.4	2.8 - 30.9
Positive control (EMS)	300.0		-	93.9	82.1	77.1	120.0	2.8 - 30.9
Test item	0.9		-	95.3	84.8	81.2	#	#
Test item	1.9		-	96.9	88.1	85.7	#	#
Test item	3.8		-	94.6	100.8	94.9	17.3	2.8 - 30.9
Test item	7.5		-	92.9	91.1	85.1	12.9	2.8 - 30.9
Test item	15.0		-	93.5	81.1	75.5	13.6	2.8 - 30.9
Test item	30.0		-	93.6	83.7	78.0	18.0	2.8 - 30.9
Test item	60.0	P	-	94.6	81.0	76.6	11.1	2.8 - 30.9
Test item	120.0	P	-	##	##	##	##	##
Solvent control with DMSO			+	100.0	100.0	100.0	19.9	3.1 - 30.7
Positive control (DMBA)	2.3		+	98.7	124.3	122.6	95.0	3.1 - 30.7
Test item	0.9		+	94.9	86.2	81.3	#	#
Test item	1.9		+	83.5	98.6	81.8	#	#
Test item	3.8		+	89.5	86.6	76.5	9.5	3.1 - 30.7
Test item	7.5		+	95.7	67.1	62.5	19.0	3.1 - 30.7
Test item	15.0		+	91.6	67.1	60.9	16.4	3.1 - 30.7
Test item	30.0		+	90.5	82.3	73.8	13.3	3.1 - 30.7
Test item	60.0	P	+	88.2	70.9	62.4	15.4	3.1 - 30.7
Test item	120.0	P	+	##	##	##	##	##

P = precipitation visible at the end of treatment

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

##  culture was not continued to avoid too many concentrations with precipitation

Results of Linear Regression Analysis calculated for the mean mutant frequencies of culture I and II

experimental group	p-value *
without S9 mix	0.239
with S9 mix	0.786

A t-test was not performed since the 95% confidence interval was not exceeded at any experimental point.

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 material is considered to be non-mutagenic in this HPRT assay.

Executive summary:

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

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 (120.0 µg/mL) was limited by precipitation observed in the pre-experiment.

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

No cytotoxic effects indicated by the mean adjusted cloning efficiency I 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 material is considered to be non-mutagenic in this HPRT assay.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Justification for classification or non-classification

The provided information is insufficient for classification for this endpoint according to the EU Regulation (EC) No 1272/2008 on Classification,Labelling and Packaging of Substances and Mixtures.