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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The capacity of the substance to induce chromosomal aberration, and gene mutation in bacteria and mammalian cells was investigated using in vitro testing. Results were negative under the conditions of the studies, with and without metabolic activation.

Link to relevant study records

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Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2016 - 2017
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:
histidine for S. typhimurium strains, tryptophan for E. coli strains
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
The maximum concentration was 5000 µg/plate.
Eight concentrations of the test material (1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate) were assayed
Vehicle / solvent:
Tetrahydrofuran
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
Top agar was prepared using 0.6% Bacto agar and 0.5% sodium chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution added to each 100 mL of top agar. Vogel-Bonner Minimal agar plates were purchased from SGL Ltd.

The bacteria used in the test were obtained from:
• University of California, Berkeley, on culture discs, on 04 August 1995.
• British Industrial Biological Research Association, on a nutrient agar plate, on 17 August 1987.

All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen freezer, model SXR 34. Evaluation of the properties of these strains regarding membrane permeability, ampicillin resistance, biotin/tryptophan dependences as well as the number of normal background spontaneous revertant colonies is routinely performed.
Evaluation criteria:
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per plate in the vehicle and untreated controls (negative controls). Acceptable ranges are presented as follows:

TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60

All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.
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
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
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 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
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
Positive controls validity:
valid
Conclusions:
The test material was considered to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and ranged between 0.15 and 5000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix.
Eight test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.


The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains from 150 μg/plate in the absence of S9-mix and 500 μg/plate in the presence S9-mix. Consequently the toxic limit or the maximum recommended dose level (5000 μg/plate) of the test item was employed in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix. In the second mutation test (pre-incubation method), the test item again induced a toxic response with reductions in the growth of bacterial background lawns noted in the absence of S9-mix from 50 μg/plate (TA100, TA98 and TA1537), 150 μg/plate (TA1535) and 1500 μg/plate (WP2uvrA). In the presence of S9-mix toxicity was initially noted from 500 μg/plate (TA100 and TA1537), 1500 μg/plate (TA1535 and TA98) and at 5000 μg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (greasy in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.
There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). Small, statistically significant increases in TA100 revertant colony frequency were observed in the first mutation test at 50 μg/plate (TA100) in the presence of S9-mix only and in the second mutation test at 15 and 50 μg/plate in the presence of S9-mix only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.4 times the concurrent vehicle controls.


The test item was considered to be non-mutagenic under the conditions of this test.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13 February 2017 - 13 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes:
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-naphthoflavone induced rat liver S9
Test concentrations with justification for top dose:
(4 hour -S9): 0, 4, 8, 16, 20, 24, 28, 32, 40 µg/mL; (4 hour +S9): 0, 4, 8, 16, 20, 24, 28, 32, 40 µg/mL; (24 hour -S9): 0, 4, 8, 16, 20, 24, 28, 32, 40 µg/mL

Due to the sensitivity of human lymphocytes to THF, the formulations could only be dosed at 0.25%. The dose range of test item used for the preliminary toxicity test was, therefore, 7.88 µg/mL to 1250 µg/mL. The test item demonstrated marked toxicity in the Preliminary Toxicity Test.
Vehicle / solvent:
Vehicle(s)/solvent(s) used: Tetrahydrofuran (THF). The final concentration of DMSO in the culture medium was 0.5 %. The osmolarity and pH were determined in the solvent control at 1250 µg/mL, the maximum achievable concentration. Justification for choice of solvent/vehicle: The solvent was chosen based upon the test item solubility properties and the relative non-toxicity of THF to the cultured cells.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Demecolcin (DC)
Details on test system and experimental conditions:
TEST SYSTEM: Human Lymphocytes: Blood samples were drawn from healthy non-smoking volunteers (18 – 35) who had been previously screened for suitability and had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. The average generation time (AGT) for these human lymphocytes is considered to be approximately 16 hours, therefore exposure time for 1.5 x AGT is 24 hours
.
Method of Application: in medium

Treatment conditions: Duplicate cultures at each of the following concentrations, 0, 4, 8, 16, 20, 24, 28, 32, 40 µg/mL, both in the absence and in the presence of S9, for each duration of treatment

Duration of treatment with the test substance: 4 hours with S9; 4 hours without S9; 24 hours continuous without S9;

Expression time: Incubation in the presence of cytochalasin B (4.5 µg/mL) (cytokinesis block) was for 24 hours for all treatment conditions.

Cell harvest/slide preparation: After centrifugation and removal of the supernate the cells were resuspended hypotonic solution ((0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v) and stored at approximately 4 ºC. The lymphocytes were re-suspended in fresh fixative, resuspended and dropped onto clean, wet microscope slides. and left to air dry. Each slide was air dried and labelled with the appropriate identification prior to .staining in 5% Giemsa.

NUMBER OF CELLS EVALUATED: at least 1000 binucelate cells per culture; 2000 per treatment concentration.

DETERMINATION OF CYTOTOXICITY- at least 500 cells per per culture were counted and cytotoxicity is characterized by the percentages of reduction in the CBPI in comparison with the controls (% cytostasis).

OTHER EXAMINATIONS:Micronuclei in mononucleated cells were recorded when observed; only cells containing clearly visible cytoplasm were included in the analysis.
Evaluation criteria:
Providing that all of the acceptability criteria are fulfilled, a test substance is considered to be positive if, the p value calculated from the frequency of binucleate cells was less than 0.05 and there was a dose related, reproducible increase in the frequency of binucleate cells with micronuclei.
Statistics:
Statistical significance was confirmed using the Chi-squared test (Hoffman et al, 2003).
Key result
Species / strain:
lymphocytes: See remarks
Remarks:
Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: no effects on pH were observed. - Effects of osmolality: no effects on osmolality were observed. -

Results summary: All vehicle (Tetrahydrofuran) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item demonstrated marked toxicity in the Preliminary Toxicity Test.

The CBPI data confirm the qualitative observations that dose-related inhibition of CBPI was observed. The 4-hour exposure group in the absence of S9 demonstrated 44% cytostasis at 28 µg/mL, the lowest precipitating dose level in this exposure group. In the presence of S9, 11% cytostasis was demonstrated at the lowest precipitating dose level (28 µg/mL). The 24-hour exposure group demonstrated dose related increases in cytostasis with 26%, 50% and 73% cytostasis at 16 µg/mL, 20 µg/mL and 24 µg/ml, respectively. The maximum dose level selected for analysis of binucleate cells was the lowest precipitating dose level (28 µg/mL) for the 4-hour exposure groups and was 20 µg/mL, where optimim toxicity was achieved, for the 24-hour exposure group.

The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, in the absence of metabolic activation. In the presence of metabolic activation there was a statistically significant increase in the frequency of binucleate cells at 20 µg/mL but this was mainly due to a high number of micronuclei being scored in the ‘B’ replicate and is considered to be of no biological relevance.

Tabulated below are the results for the experimental groups evaluated for cytogenetic damage.

 

Exposure period 4 hrs without S9 mix

Exp.

Preparation

interval

Test item

concentration

in µg/mL

Proliferation

index

CBPI

Cytostasis

in %1

Micronucleated

cells

in %2

I

24 hrs (Cytochalasin B treatment)

Vehicle control

1.20

0

0.85

Positive control

1.17

15

4.35***

16

1.24

0

0.90

20

1.14

31

1.10

24

1.13

33

1.40

28P

1.11

44

1.35

 

Exposure period 4 hrs with S9 mix

Preparation

interval

Test item

concentration

in µg/mL

Proliferation

index

CBPI

Cytostasis

in %1

Micronucleated

cells

in %2

 

24 hrs (Cytochalasin B treatment)

Vehicle control

1.28

0

0.85

 

Positive control

1.11

60

3.25***

 

16

1.28

0

1.20

 

20

1.30

0

1.65*

 

24

1.28

0

0.80

 

28P

1.25

11

0.75

 

Exposure period 24 hrs without S9 mix

Preparation

interval

Test item

concentration

in µg/mL

Proliferation

index

CBPI

Cytostasis

in %1

Micronucleated

cells

in %2

 

24 hrs (Cytochalasin B treatment)

Vehicle control

1.74

0

0.65

 

Positive control

1.56

29

4.95***

 

4

1.70

5

0.35

 

8

1.65

12

0.90

 

16

1.55

26

0.70

 

20

1.37

50

0.85

 

1 For the positive control groups and the test item treatment groups, the values are related tothe solvent controls.

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

P Precipitation occurred at the end of treatment.

* The number of micronucleated cells is statistically significantly (p¿0.05) higher than corresponding control values.

*** The number of micronucleated cells is statistically significantly (p¿0.01) higher than corresponding control values.

Conclusions:
The test material was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro with and without metabolic activation under the conditions of the test.
Executive summary:

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at four dose levels, together with vehicle and positive controls.
Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the absence and in the presence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.
The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by toxicity up to the lowest precipitating dose level.
All vehicle (Tetrahydrofuran) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.
The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item demonstrated marked toxicity in the Preliminary Toxicity Test. In the main test the test item did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in the absence of a metabolizing system. In the presence of S9 the test item did not induce a toxicologically significant increase in the frequency of binucleate cells with micronuclei. The dose range selected included the lowest precipitating dose level for both the 4-hour exposure groups and a dose level where optimum toxicity was achieved for the 24-hour exposure group.
The test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
TK +/-, locus of the L5178Y mouse lymphoma cell line.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
2% S9 Mix
Test concentrations with justification for top dose:
The test item is a UVCB compound; therefore the maximum recommended dose level was set at 5000 µg/mL with no correction for purity. However the maximum achievable concentration was set at 1250 µg/mL due to the toxicity of THF to L5178Y cells.
Vehicle / solvent:
Solvent tetrahydrofuran used as vehicle controls.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK.

Cell Culture: The stocks of cells are stored in liquid nitrogen at approximately -196°C. Cells were routinely cultured in RPMI 1640 medium. The cells have a generation time of approximately 12 hours and were subcultured accordingly. Master stocks of cells were tested and found to be free of mycoplasma.

Cell Cleansing: Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours.

Test Item Preparation: the test item was accurately weighed and formulated in THF prior to serial dilutions being prepared. The test item is a UVCB compound; therefore the maximum recommended dose level was set at 5000 µg/mL with no correction for purity. However the maximum achievable concentration was set at 1250 µg/mL due to the toxicity of THF to L5178Y cells. There was no marked change in pH when the test item was dosed into media and the osmolality did not increase by more than 50 mOsm.
Evaluation criteria:
A mutation assay is considered acceptable if it meets the following criteria:
1.The majority of the plates, for both viability (%V) and 5-TFT resistance, are analysable for each experiment.
2. The absolute viability (%V) at the time of mutant selection of the solvent controls is 65 to 120%.
3. The total suspension growth of the solvent control following 4-hour exposure, calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold increase in cell number, should be in the range of 8 to 32. Following 24-hour exposure the total suspension growth should be in the range of 32 to 180.
4. The in-house vehicle control mutant frequency is in the range of 50 – 170 x 10-6 cells.
5. Positive controls meets at least one of the following two acceptance criteria developed by the IWGT workgroup:
The positive control should demonstrate an absolute increase in total MF.
The positive control has an increase in the small colony MF of at least 150 x 10-6 above that seen in the concurrent untreated/solvent control.
6. The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures.
7. For non-toxic test materials the upper test material concentrations are 10 mM, 2 mg/mL or 2-µL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCBs) the upper dose level may need to be higher and the maximum concentration is 5 mg/mL.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
The test material is considered to be non-mutagenic with and without metabolic activation under the conditions of the test.
Executive summary:

One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at 8 dose levels in duplicate, together with vehicle (tetrahydrofuran; THF), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-hour exposure group in the absence of metabolic activation.
The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The test item exhibited marked toxicity in all three exposure groups and precipitate was also noted.
The maximum dose level used was predominantly limited by test item induced toxicity, however precipitate was also considered. No precipitate of the test item was observed throughout the main test. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. Optimum levels of toxicity were considered to have been achieved in both 4-hour exposure groups and very near to optimum toxicity was achieved in the 24-hour exposure group. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.
The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.
The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6, consequently it is considered to be non-mutagenic in this assay.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In accordance with REACH, it is not required to investigate the in vivo genotoxicity of the substance since in vitro testing returned negative results.

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

in vitro gene mutation in bacteria
Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with the test item using both the Ames plate incorporation and pre-incubation methods at eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system (10% liver S9 in standard co-factors). The dose range for Experiment 1 was predetermined and was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was amended following the results of Experiment 1 and ranged between 0.15 and 5000 μg/plate, depending on bacterial strain type and presence or absence of S9-mix.
Eight test item concentrations were selected in Experiment 2 in order to achieve both four non-toxic dose levels and the potential toxic limit of the test item following the change in test methodology.
The vehicle (tetrahydrofuran) control plates gave counts of revertant colonies within the normal range. All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The maximum dose level of the test item in the first experiment was selected as the maximum recommended dose level of 5000 μg/plate. In the first mutation test (plate incorporation method), the test item caused a visible reduction in the growth of the bacterial background lawns of all of the tester strains from 150 μg/plate in the absence of S9-mix and 500 μg/plate in the presence S9-mix. Consequently the toxic limit or the maximum recommended dose level (5000 μg/plate) of the test item was employed in the second mutation test, depending on bacterial strain type and presence or absence of S9-mix. In the second mutation test (pre-incubation method), the test item again induced a toxic response with reductions in the growth of bacterial background lawns noted in the absence of S9-mix from 50 μg/plate (TA100, TA98 and TA1537), 150 μg/plate (TA1535) and 1500 μg/plate (WP2uvrA). In the presence of S9-mix toxicity was initially noted from 500 μg/plate (TA100 and TA1537), 1500 μg/plate (TA1535 and TA98) and at 5000 μg/plate (WP2uvrA). The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (greasy in appearance) was noted at and above 1500 μg/plate, this observation did not prevent the scoring of revertant colonies.
There were no toxicologically significant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no toxicologically significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9-mix) in Experiment 2 (pre-incubation method). Small, statistically significant increases in TA100 revertant colony frequency were observed in the first mutation test at 50 μg/plate (TA100) in the presence of S9-mix only and in the second mutation test at 15 and 50 μg/plate in the presence of S9-mix only. These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for the tester strain and the maximum fold increase was only 1.4 times the concurrent vehicle controls.
The test item was considered to be non-mutagenic under the conditions of this test.


 


in vitro micronucleus
Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at four dose levels, together with vehicle and positive controls.
Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the absence and in the presence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.
The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by toxicity up to the lowest precipitating dose level.
All vehicle (Tetrahydrofuran) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.
The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item demonstrated marked toxicity in the Preliminary Toxicity Test. In the main test the test item did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in the absence of a metabolizing system. In the presence of S9 the test item did not induce a toxicologically significant increase in the frequency of binucleate cells with micronuclei. The dose range selected included the lowest precipitating dose level for both the 4-hour exposure groups and a dose level where optimum toxicity was achieved for the 24-hour exposure group.
The test item was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.


 


in vitro gene mutation in mammalian cells
One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at 8 dose levels in duplicate, together with vehicle (tetrahydrofuran; THF), and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24-hour exposure group in the absence of metabolic activation.
The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The test item exhibited marked toxicity in all three exposure groups and precipitate was also noted.
The maximum dose level used was predominantly limited by test item induced toxicity, however precipitate was also considered. No precipitate of the test item was observed throughout the main test. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. Optimum levels of toxicity were considered to have been achieved in both 4-hour exposure groups and very near to optimum toxicity was achieved in the 24-hour exposure group. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.
The test item did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups.
The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the Global Evaluation Factor (GEF) of 126 x 10-6, consequently it is considered to be non-mutagenic in this assay.

Justification for classification or non-classification

In vitro genotoxicity testing was conducted on the substance in accordance with REACH and returned negative results. Therefore, it does not meet the criteria for classification in accordance with Regulation (EC) No 1272/2008.