4-Ethoxy-4'-(trans-4-ethylcyclohexyl)-2,3-difluor-1,1'-biphenyl

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic in the Ames test.

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

In conclusion, treatment of V79 cell cultures with the test item, did not increase the proportion of cells with aberrant chromosomes. The test item was not clastogenic in this in vitro test system.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 03, 2006 - Jun 22, 2006

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)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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):

CELLS USED
- Source of cells: originally received from Hoffmann-La Roche, Pharma, Basel, on May 27, 1997
- Cell cycle length, doubling time or proliferation index: 16 to 17.5 hours
V79 cells have been successfully used in mutagenicity testing for many years. This cell line has a high proliferation rate and cloning efficiency. The cells have a relatively stable karyotype with a modal chromosome number of 22 ± 1, and an aberration rate of about 0-5 % of the metaphases. Since the cell line is not able to metabolize indirect mutagens to reactive forms, the test is performed both in the presence and absence of an external metabolizing system (liver S9 mix of rats pre-treated with Aroclor 1254).

Metabolic activation:

with and without

Metabolic activation system:

S9-mix with S9 from Aroclor 1254 induced rats

Test concentrations with justification for top dose:

In the first experimental series, test material concentrations ranging from 0.50 to 500 µg/mL were tested. The test material precipitated in the culture medium at concentrations ≥158 µg/mL in the absence and presence of S9 mix. No cytotoxic effects were seen. Quality control of the cell preparations did not show a relevant influence of the test material on the structure or spreading of the chromosomes in the concentration range investigated. A change in the pH or the osmotic value of the cell culture medium did not occur in the dose range tested.
For these reasons, cultures treated with the following test material concentrations were evaluated in absence and presence of S9 mix for the occurrence of chromosomal aberrations:
1st series: 15.8, 50.0, and 158 µg/mL
2nd series: 50.0, 88.9, 158, 281 and 500 µg/mL

Vehicle / solvent:

Acetone, 0.1 %

Untreated negative controls:

no

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:

No. of slides per concentration:
Solvent control: 4
Others: 2
No. of metaphases evaluated per slide: 100 (for structural aberrations); 1000 (for polyploidy)

Preparation times: 25 and 35 hours

Exposure times:
- S9 mix: 5, 25 and 35 hours
+ S9 mix: 5 hours

Solvent for the test material: Acetone

Concentrations evaluated:
Test item first series: 15.8, 50.0 and 158 µg/mL
second series: 50.0, 88.9, 158, 281 and 500 µg/mL

Positive controls:
- S9 mix: 250 and 500 µg Ethylmethansulfonat (EMS)/mL
31.6 and 88.9 µg Griseofulvin (GRIS)/mL
+S9 mix: 2.00 µg Cyclophosphamide (CPA)/mL

Rationale for test conditions:

Guideline settings are applied

Evaluation criteria:

Step 1: Evaluation of Slide Quality
Step 2: Evaluation of Chomosomal Aberrations

The decisive parameter for the evaluation of both, the treated and untreated cultures, is the number of aberrant metaphases (excluding gaps) per 100 cells. A basic prerequisite for the acceptance of a test series is (a) the correspondence of the actual negative controls with the historic negative controls of the laboratory and (b) a statistically significant and biologically relevant increase in the number of aberrant metaphases for the respective positive controls in relation to the actual negative controls. The discussion of biological relevance includes, among other things, considerations concerning the type and time dependent appearance of the observed chromosomal aberrations.
A test material is defined as being unambiguously negative or non-clastogenic in this test system if no statistically significant increase in the number of aberrant metaphases per 100 cells, as compared to the actual negative control, occurs at any test material concentration.
A test material is positive or clastogenic in this test system if
• a statistically significant, dose-related increase in the number of aberrant metaphases per 100 cells occurs or
• a statistically significant increase in the number of aberrant metaphases per 100 cells is reproduced at the same test material concentration in independent experiments.
In both cases, however, the number of aberrant metaphases should be above the range defined as the historical negative controls of the laboratory and the biological relevance of the results has to be discussed.
In all other cases, further decisions for testing strategies should be made following the scientific evaluation of all existing data including those of non­ toxicological investigations.

Statistics:

Fisher’s Exact Test

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

Conclusions:

In conclusion, treatment of V79 cell cultures with the test item, did not increase the proportion of cells with aberrant chromosomes. The test item was not clastogenic in this in vitro test system.

Executive summary:

The test item was investigated in two experimental series for induction of chromosomal aberrations in V79 Chinese hamster cells in vitro according to OECD 473. This also included examinations on whether or not the test material may have the potential to induce numerical aberrations, i.e. an increase in number of polyploid cells.

The following experimental conditions were selected in the absence and presence of an exogenous metabolizing system (S9-mix from livers of rats pretreated with Aroclor 1254):

No. of slides per concentration:

Solvent control: 4

Others: 2

No. of metaphases evaluated per slide: 100 (for structural aberrations); 1000 (for polyploidy)

Preparation times: 25 and 35hours

Exposure times: - S9 mix: 5, 25 and 35 hours; + S9 mix: 5 hours

Solvent for the test material: Acetone

Concentrations evaluated: Test item first series: 15.8, 50.0 and 158 µg/mL and second series: 50.0, 88.9, 158, 281 and 500 µg/mL

Positive controls:- S9 mix: 250 and 500 µg Ethylmethansulfonat (EMS)/mL, 31.6 and 88.9 µg Griseofulvin (GRIS)/mL and +S9 mix: 2.00 µg Cyclophosphamide (CPA)/mL

The positive control test materials, EMS and CPA, induced the expected clear increase in the proportion of cells with chromosomal aberrations. Griseofulvin induced a clear increase in the number polyploid cells.

The test item precipitated in the culture medium at concentrations of 158 µg/mL in the absence and presence of S9-mix. No cytotoxic effects were observed.

The mean values of aberrant metaphases (gaps excluded) in the negative controls of both experiments (in the absence and presence of S9 mix) ranged from 0.75 to 2.25 %. Treatment with the test item did not result in any biologically or statistically relevant increase in the number of aberrant metaphases in two independent experimental series at concentrations ranging from 15.8 to 500 µg/mL, except a slight but statistically significant increase at 281 µg/mL in the second experiment in the presence of S9 after 5+20 hour treatment. However, this effect did not occur at 500 µg/mL and is thus not concentration dependent. Furthermore, the mean number of aberrant metaphases was well within the range of historical control values of this laboratory. Therefore, this effect is considered incidental and of no biological relevance.

In the first experimental series an increase in the number of polypoid cells was observed in cultures treated with the test item in the presence of S9 at 158 µg/mL (5+20 hour treatment). However, in the second experiment there was no increase in the number of polypoid cells at test item concentrations ranging from 50.0 to 500 µg/mL after 5+20 hour treatment or 5+30 hour treatment. Thus, this effect was only observed in 1 of 3 experiments. Moreover, the detection of numerical aberrations is not the primary endpoint of this assay, therefore these observations are of questionable biological relevance.

In conclusion, treatment of V79 cell cultures with the test item did not increase the proportion of cells with aberrant chromosomes. The test item was not clastogenic in this in vitro test system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct 08, 2001 - April 03, 2002

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

GLP compliance:

yes

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

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):

uvrA pkM101

Additional strain / cell type characteristics:

other: mutations in the tryptophan operon

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:
1. Series: 5.00, 15.8, 50.0, 158, 500, 1580 and 5000 μg per plate (S9 10 %)
1. Series: 5.00, 15.8, 50.0, 158 and 500 μg per plate (S9 30 %)

Vehicle / solvent:

acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

cumene hydroperoxide

other: daunomycin

Remarks:

without S9

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

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 the Actual
(Solvent Control) 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:

See above

Statistics:

n.a.

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 applicable

True negative controls validity:

not applicable

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 applicable

True negative controls validity:

not applicable

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

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 applicable

True negative controls validity:

not applicable

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 applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

True negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

With and without addition of S9 mix as the external metabolizing system, the test material was not mutagenic in the Ames test.

Executive summary:

The purpose of this assay, conducted according to OECD 471, was to provide information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man.

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 pKM101. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed.

The test material was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred starting from concentrations of 500 µg/plate. Toxicity to the bacteria was not observed.

Each treatment with the test materials used as positive controls (see listed controls above) 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.

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

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 November 2016 until 30 December 2016

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 using the Hprt and xprt genes)

Version / remarks:

2016-07-29

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008-05-30

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 (hypoxanthine-guanine phosphoribosyl transferase)

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:

4 hours treatment without S9 mix: 1.0; 3.1; 9.3; 27.8 (p); 83.3 (p) µg/mL
4 hours treatment with S9 mix: 1.0, 3.1; 9.3, 27.8 (p); 83.3 (p) µg/mL

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

Vehicle / solvent:

DMSO (1.0 % (v/v))

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

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 2.0 µg/mL and 250 µg/mL were used. The highest concentration was based on the solubility properties of the test item in DMSO.
No relevant toxic effects were observed after 4 hours treatment up to the maximum 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 without metabolic activation and at 15.6 µg/mL and above with 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 the data of the pre-experiment. Again, the maximum concentration was 250 µg/mL. The individual concentrations were spaced by a factor of 3.
To overcome problems with possible deviations in toxicity the main experiment was started with more than four concentrations.

Evaluation criteria:

A test item is classified as positive if it induces a concentration-related increase of the mutant frequency exceeding the historical solvent control range.
A test item producing no concentration-related increase of the mutant frequency above the historical solvent control range is considered to be non-mutagenic in this system.
A mutagenic response is described as follows:
The test item is classified as mutagenic if it induces with at least one of the concentrations in both parallel cultures a mutation frequency that exceeds the historical negative and solvent control data range (95 % confidence interval limits).
The increase should be significant and dose dependent as indicated by statistical analysis (linear regression, least squares).

Statistics:

A linear regression (least squares, calculated using a validated excel spreadsheet) was performed to assess a possible dose dependent increase of mutant frequencies. The numbers of mutant colonies generated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05.
A t-Test 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:

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 7.31 in the solvent control versus pH 7.34 at 250.0 µg/mL)
- Effects of osmolality: No relevant increase (445 mOsm in the solvent control versus 461mOsm at 250.0 µg/mL)
- Evaporation from medium: Not examined
- Precipitation: determined at 27.8 and 83.3 µg/mL
- Other confounding effects: None

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.

 

	conc. µg/mL	S9 Mix	relavtive CE I	relative cell density	rel. adj. CE I	mutant colonies/10^6 cells	95 % conf. interval
Culture I
Solvent control (DMSO)		-	100.0	100.0	100.0	40.6	1.7 - 30.2
Positive control (EMS)	300.0	-	99.0	90.2	89.3	361.0	1.7 - 30.2
Test Item	1.0	-	130.4	81.6	106.3	26.5	1.7 - 30.2
Test Item	3.1	-	97.2	96.8	94.1	32.2	1.7 - 30.2
Test Item	9.3	-	88.6	91.1	80.7	22.1	1.7 - 30.2
Test Item	27.8 (p)	-	88.9	101.8	90.5	46.5	1.7 - 30.2
Test Item	83.3 (p)	-	117.7	59.8	70.4	21.7	1.7 - 30.2
Test Item	250.0 (p)	-	121.2	60.4	73.1	not continued#
Solvent control (DMSO)		+	100.0	100.0	100.0	18.9	2.0 - 29.4
Positive control (DMBA)	2.3	+	79.5	97.6	77.7	115.0	2.0 - 29.4
Test Item	1.0	+	92.2	95.9	88.5	37.5	2.0 - 29.4
Test Item	3.1	+	83.0	84.0	69.7	11.4	2.0 - 29.4
Test Item	9.3	+	90.0	95.3	85.8	26.5	2.0 - 29.4
Test Item	27.8 (p)	+	89.9	89.5	80.4	19.7	2.0 - 29.4
Test Item	83.3 (p)	+	89.3	93.4	83.4	34.4	2.0 - 29.4
Test Item	250.0 (p)	+	91.3	76.5	69.8	not continued#

 

	conc. µg/mL	S9 Mix	relavtive CE I	relative cell density	rel. adj. CE I	mutant colonies/10^6cells	95 % conf. interval
Culture II
Solvent control (DMSO)		-	100.0	100.0	100.0	13.3	1.7 - 30.2
Positive control (EMS)	300.0	-	113.8	85.9	97.7	340.9	1.7 - 30.2
Test Item	1.0	-	94.7	105.1	99.5	12.0	1.7 - 30.2
Test Item	3.1	-	68.2	94.9	64.8	17.3	1.7 - 30.2
Test Item	9.3	-	90.1	71.5	64.4	36.2	1.7 - 30.2
Test Item	27.8 (p)	-	54.9	105.1	57.6	18.1	1.7 - 30.2
Test Item	83.3 (p)	-	72.3	74.5	53.9	17.3	1.7 - 30.2
Test Item	250.0 (p)	-	62.1	104.9	65.1	not continued#
Solvent control (DMSO)		+	100.0	100.0	100.0	14.0	2.0 - 29.4
Positive control (DMBA)	2.3	+	87.1	100.4	87.4	108.7	2.0 - 29.4
Test Item	1.0	+	90.8	107.4	97.5	16.1	2.0 - 29.4
Test Item	3.1	+	89.0	86.4	76.9	33.4	2.0 - 29.4
Test Item	9.3	+	88.1	102.3	90.2	21.9	2.0 - 29.4
Test Item	27.8 (p)	+	92.4	99.3	91.7	23.8	2.0 - 29.4
Test Item	83.3 (p)	+	95.7	101.1	96.7	15.2	2.0 - 29.4
Test Item	250.0 (p)	+	87.3	100.7	88.0	not continued#

CE = Cloning efficiency

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:

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

Executive summary:

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation.

No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50 % in both cultures occurred up to the maximum concentration with and without metabolic activation.

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

The 95 % confidence interval was exceeded at several concentrations in the presence and absence of metabolic activation. A t-test run at any experimental point above the 95 % confidence interval was not significant. Furthermore, the mean values of the mutation frequeny of both parallel cultures remained within the 95 % confidence interval at all soluble concentrations.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In the main experiment with and without S9 mix the range of the solvent controls was from 13.3 up to 40.6 mutants per 10⁶ cells; the range of the groups treated with the test item was from 11.4 up to 46.5 mutants per 10⁶ cells. The highest solvent control value of 40.6 mutants per 10⁶cells exceeded the 95 % confidence interval but remained within the historical range of solvent controls. The mutation frequency of the parallel culture and the mean of both cultures (40.6 and 13.3, equal to a mean of 27.0) was fully acceptable.

EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

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 purpose of this assay, conducted according to OECD 471, was to provide information on possible health hazards for the test material and serve as a rational basis for risk assessment to the genotoxic potential of the test item in man.

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 pKM101. The plate incorporation test with and without addition of liver S9 mix from Aroclor 1254-pretreated rats was used. Two independent experimental series were performed.

The test material was dissolved in acetone and tested at concentrations ranging from 5 to 5000 µg/plate. Precipitation of the test material on the agar plates occurred starting from concentrations of 500 µg/plate. Toxicity to the bacteria was not observed.

Each treatment with the test materials used as positive controls (see listed controls above) 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.

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

 

OECD 476

Key study

The test item was assessed for its potential to induce gene mutations at the HPRT locus using V79 cells of the Chinese hamster. The treatment period was 4 hours with and without metabolic activation.

No relevant cytotoxic effect indicated by an adjusted cloning efficiency I below 50 % in both cultures occurred up to the maximum concentration with and without metabolic activation.

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

The 95 % confidence interval was exceeded at several concentrations in the presence and absence of metabolic activation. A t-test run at any experimental point above the 95 % confidence interval was not significant. Furthermore, the mean values of the mutation frequeny of both parallel cultures remained within the 95 % confidence interval at all soluble concentrations.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in any of the experimental groups.

In the main experiment with and without S9 mix the range of the solvent controls was from 13.3 up to 40.6 mutants per 10⁶ cells; the range of the groups treated with the test item was from 11.4 up to 46.5 mutants per 10⁶ cells. The highest solvent control value of 40.6 mutants per 10⁶cells exceeded the 95 % confidence interval but remained within the historical range of solvent controls. The mutation frequency of the parallel culture and the mean of both cultures (40.6 and 13.3, equal to a mean of 27.0) was fully acceptable.

EMS (300 µg/mL) and DMBA (2.3 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

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

 

Supporting study

The objective of the study conducted according to OECD 476 was to evaluate the mutagenic potential of the test item in mammalian cells (V79). Cell cultures were exposed to the test item or positive controls in the presence (exposure period 3 h) and the absence (exposure period 24 and 3 h) of the external metabolizing system (S9 mix). Various concentrations of the test item ranging from 0.158 to 158 µg/mL were tested with and without S9 mix. At the end to the exposure period precipitation of the test item was observed at a concentration of 158 µg/mL. No clear signs of cytotoxicity were observed up to this concentration. Hence, solubility of the test item was the limiting factor for the selection of the highest concentration tested. Negative (solvent) and positive controls were included in each experiment. Mutant frequencies in negative control cultures fell within normal ranges and clear increase in mutant were induced by the positive control chemicals n-methyl-n’-nitro-n-nitrosoguanidine (MNNG, without S9 mix) and DMBA (7,12-dimethylbenz[a]antracene, with S9 mix). Therefore, the study was accepted as valid. In the absence and presence of S9 mix the test item did not increase the mutant frequency of V79 cells as compared to the actual solvent controls. According to the predetermined criteria for the evaluation of results the test item was clearly negative in the test system. In conclusion, the test item was not mutagenic in the V79 mammalian cell gene mutation test up to the limit of solubility under conditions where the positive controls exerted potent mutagenic effects.

 

OECD 473

The test item was investigated in two experimental series for induction of chromosomal aberrations in V79 Chinese hamster cells in vitro according to OECD 473. This also included examinations on whether or not the test material may have the potential to induce numerical aberrations, i.e. an increase in number of polyploid cells.

The following experimental conditions were selected in the absence and presence of an exogenous metabolizing system (S9-mix from livers of rats pretreated with Aroclor 1254):

No. of slides per concentration:

Solvent control: 4

Others: 2

No. of metaphases evaluated per slide: 100 (for structural aberrations); 1000 (for polyploidy)

Preparation times: 25 and 35hours

Exposure times: - S9 mix: 5, 25 and 35 hours; + S9 mix: 5 hours

Solvent for the test material: Acetone

Concentrations evaluated: Test item first series: 15.8, 50.0 and 158 µg/mL and second series: 50.0, 88.9, 158, 281 and 500 µg/mL

Positive controls:- S9 mix: 250 and 500 µg Ethylmethansulfonat (EMS)/mL, 31.6 and 88.9 µg Griseofulvin (GRIS)/mL and +S9 mix: 2.00 µg Cyclophosphamide (CPA)/mL

The positive control test materials, EMS and CPA, induced the expected clear increase in the proportion of cells with chromosomal aberrations. Griseofulvin induced a clear increase in the number polyploid cells.

The test item precipitated in the culture medium at concentrations of 158 µg/mL in the absence and presence of S9-mix. No cytotoxic effects were observed.

The mean values of aberrant metaphases (gaps excluded) in the negative controls of both experiments (in the absence and presence of S9 mix) ranged from 0.75 to 2.25 %. Treatment with the test item did not result in any biologically or statistically relevant increase in the number of aberrant metaphases in two independent experimental series at concentrations ranging from 15.8 to 500 µg/mL, except a slight but statistically significant increase at 281 µg/mL in the second experiment in the presence of S9 after 5+20 hour treatment. However, this effect did not occur at 500 µg/mL and is thus not concentration dependent. Furthermore, the mean number of aberrant metaphases was well within the range of historical control values of this laboratory. Therefore, this effect is considered incidental and of no biological relevance.

In the first experimental series an increase in the number of polypoid cells was observed in cultures treated with the test item in the presence of S9 at 158 µg/mL (5+20 hour treatment). However, in the second experiment there was no increase in the number of polypoid cells at test item concentrations ranging from 50.0 to 500 µg/mL after 5+20 hour treatment or 5+30 hour treatment. Thus, this effect was only observed in 1 of 3 experiments. Moreover, the detection of numerical aberrations is not the primary endpoint of this assay, therefore these observations are of questionable biological relevance.

In conclusion, treatment of V79 cell cultures with the test item did not increase the proportion of cells with aberrant chromosomes. The test item was not clastogenic in this in vitro test system.

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 on genetic toxicity in vitro, the test item doesnot require classification according to Regulation (EC) No 1272/2008 (CLP).