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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Ames Test (OECD 471, GLP, K, rel. 1): non mutagenic up to 5000 µg/plate in S. typhimurium TA 1535, TA 1537, TA 98, TA 100 & E.coli WP2uvrA.
- HL/CAT chromosome aberration test (OECD 473, GLP, K, rel. 1): non clastogenic up to cytotoxic concentrations.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 31 to December 07, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspected on August 21, 2007 / Signed on October 15, 2007)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan.
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10% S9: S9-mix from the livers of male Wistar rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day by oral route).
Test concentrations with justification for top dose:
- Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in TA100 or WP2uvrA strain with and without S9-mix using direct plate incorporation method.
- Experiment-1 (Range-finding test): 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix using direct plate incorporation method.
- Experiment-2 (Main test): 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix using direct plate incorporation method.
Limiting factor was the maximum recommended dose.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test material formed a doseable suspension in sterile distilled water at 50 mg/mL but was fully soluble in dimethyl sulphoxide at the same concentration in solubility checks performed in-house. Dimethyl sulphoxide was therefore selected as the vehicle.
- Preparation of test materials: Test material was accurately weighed and approximate half-log dilutions prepared in dimethyl sulphoxide by mixing on a vortex mixer on the day of each experiment.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulphoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
other: 2-Aminoanthracene (2AA)
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applicable
- Exposure duration: Plates were incubated for approximately 48 h at 37 °C.
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): not applicable

NUMBER OF REPLICATIONS: Single plate/dose for preliminary toxicity study; triplicate plates per dose level for mutation study (Experiment 1 and 2).


NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: Growth assessment of the bacterial background lawn.

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using a Domino colony counter.
Rationale for test conditions:
Experiment 1 & 2 - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Evaluation criteria:
- There are several criteria for determining a positive result, such as a dose-related increase in revertant frequency over the dose range tested and/or a reproducible increase at one or more concentrations in at least one bacterial strain with or without
metabolic activation. Biological relevance of the results will be considered first, statistical methods, as recommended by the UKEMS can also be used as an aid to evaluation, however, statistical significance will not be the only determining factor for a positive response.
- A test material will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
- Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test material activity. Results of this type will be reported as equivocal.
Statistics:
- Statistical methods, as recommended by the UKEMS can be used as an aid to evaluation. However statistical significance will not be the only determining factor for a positive response.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
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: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No expected
- Water solubility: The test material was solubilised in dimethyl sulphoxide to improve solubility.
- Precipitation: No
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: Test material exhibited a weak toxic response for TA100 in the absence of S9 and was non-toxic for TA100 in the presence of S9 and WP2uvrA in the presence and absence of S9.

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2006 and 2007 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test material caused a visible reduction in the growth of the bacterial background lawns of several of the Salmonella tester strains at 5000 µg/plate predominantly in the absence of S9.

OTHERS:
- The test material formulation and S9-mix used in this experiment were both shown to be sterile.

Table 7.6.1/2: Test results: Preliminary toxicity test

Metabolic activation

Strain

Dose (µg/plate)

0

0.15

0.5

1.5

5

15

50

150

500

1500

5000

-

TA100

115

125

127

102

108

121

100

111

120

126

67*

+

93

80

79

77

74

76

86

64

76

73

52

-

WP2

uvrA-

22

25

12

18

19

22

15

13

11

20

17

+

22

23

28

20

18

16

20

26

19

20

13

Key: * = Partial absence of bacterial background lawn.

Conclusions:
Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains (TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA- according to the criteria of the Annex VI of the of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA- were exposed to test material diluted in dimethyl sulphoxide both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors) using the plate incorporation method. The dose range for the range-finding test was determined in a preliminary toxicity assay and ranged between 15 and 5000 µg/plate. The experiment was repeated on a separate day using a modified dose range, 50 to 5000 µg/plate based on the results of the range-finding test. Vehicle (dimethyl sulphoxide) and positive control groups were also included in mutagenicity tests.

The vehicle 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 test material caused a visible reduction in the growth of the bacterial background lawns of several of the Salmonella tester strains at 5000 µg/plate predominantly in the absence of S9. The test material was, therefore, tested up to the maximum recommended dose level of 5000 µg/plate. No test material precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test material, either with or without metabolic activation.

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA- according to the criteria of the Annex VI of the of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

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:
From July 07 to October 08, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on August 19, 2008/ signed on March 4, 2009)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
lymphocytes: humans
Details on mammalian cell type (if applicable):
- Type and identity of media: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 15% foetal calf serum, at 37ºC with 5% CO2 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA) at approximately 90 μg/ml final concentration.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable
- Periodically checked for karyotype stability: not applicable
- Periodically "cleansed" against high spontaneous background: not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
2 % S9 (final concentration); S9 fraction was obtained from the liver homogenates of male Sprague Dawley rats treated with phenobarbitone and β-naphthoflavone
Test concentrations with justification for top dose:
Preliminary Toxicity Test (Cell Growth Inhibition Test): 0, 8.05, 16.11, 32.22, 64.44, 128.88, 257.75, 515.5, 1031 and 2062 μg/mL
Experiment 1: without and with S9 (2 % S9): 0, 64.44, 128.88, 257.75, 515.5, 773.33 and 1031 μg/mL (limited by cytotoxicity)
Experiment 2:
without S9: 0, 16.11, 32.22, 64.44, 128.88, 257.75 and 515.5 μg/mL. (limited by cytotoxicity)
with S9 (1 % S9): 0, 64.44, 128.88, 257.75, 386.65, 515.5 and 773.33 μg/mL. (limited by cytotoxicity)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Formulation preparation: The test material was melted prior to testing; this allowed the test material to be accurately weighed. The test material was dissolved in Dimethyl sulphoxide (DMSO) and serial dilutions prepared. The molecular weight of the test material was given as 206.2, therefore the maximum dose level was 2062 μg/mL, which was equivalent to 10 mM.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9 mix; 0.4 and 0.2 μg/mL for 4(20)-hour and 24-hour cultures respectively
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9 mix; 5 μg/mL
Details on test system and experimental conditions:
TEST SYSTEM: Cultures prepared from the blood of a human volunteer who had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection.

CELL CULTURE: Cells were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 15 % foetal calf serum, at 37 ºC with 5% CO2 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA) at approximately 90 μg/mL final concentration.

DURATION
- Exposure duration: 4 hours (± S9) in Experiment 1, 4 hours (+S9) and 24 hours (-S9) in Experiment 2
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hours (± S9) in Experiment 1 and 2

SPINDLE INHIBITOR (cytogenetic assays): Mitotic activity was arrested by addition of demecolcine (Colcemid 0.1 μg/mL), two hours before the harvest time.
STAIN (for cytogenetic assays): 5 % Giemsa

NUMBER OF REPLICATIONS: Duplicate cultures/dose

NUMBER OF CELLS EVALUATED:
- Total of 2000 lymphocyte cell nuclei was counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
- Where possible the first 100 consecutive well-spread metaphases from each culture were counted. Where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the International System for Chromosome Nomenclature (1985) as described by Scott et al and compatible and equitable to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: If greater than 44 chromosomes are scored and the number is a multiple of the haploid count then the cell is classified as a polyploid cell.
- Determination of endoreplication: If the chromosomes are arranged in closely apposed pairs, ie. 4 chromatids instead of 2, the cell is scored as endoreduplicated (E).
Rationale for test conditions:
Experiment 1 & 2: Mitotic index data was used to estimate test material toxicity and for selection of the dose levels for the main test.
Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells were compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Key result
Species / strain:
lymphocytes: humans
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
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 significant change in pH when the test material was added into media.
Effects of osmolality: Osmolality did not increase by more than 50 mOsm.

RANGE-FINDING/SCREENING STUDIES:
Test material showed some evidence of toxicity in all exposure groups. A red colouration was observed in the parallel blood-free cultures at the end of the exposure period, at and above 128.88 μg/mL, in the 4(20)-hour pulse exposure group without metabolic activation and at and above 1031 μg/mL, in the 4(20)-hour pulse exposure group with metabolic activation. In the continuous exposure group a cloudy precipitate was seen at 2062 μg/mL. Haemolysis was observed at and above 515.5 μg/mL in the 4(20)-hour exposure groups with and without S9, and at and above 1031 μg/mL in the 24-hour exposure group. Microscopic assessment of the slides prepared from the treatment cultures showed that metaphase cells were present up to 515.5 μg/mL in the 4(20)-hour treatment in the presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 257.75 μg/mL.

COMPARISON WITH HISTORICAL CONTROL DATA: All vehicle and solvent controls were in the range of historical laboratory control data.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
EXPERIMENT 1: The results of the mitotic indices (MI) from the cultures after their respective treatments showed 32% and 76% growth inhibition respectively at 257.75 and 515.5 μg/mL in the absence of S9. In the presence of S9 the data show 26% and 67% growth inhibition respectively was achieved at 257.75 and 515.5 μg/mL. However due to excessive toxicity the dose level 515.5 μg/mL was not suitable for metaphase analysis in either exposure group. A precipitate of the test material was not seen at the end of the treatment period in either exposure group; however haemolysis was seen in both exposure groups at and above 515.5 μg/mL, at the end of the treatment period. The maximum dose level selected for metaphase analysis was based on toxicity and was the highest dose level with acceptable levels of toxicity (257.75 μg/mL).
EXPERIMENT 2: The results of the mitotic indices (MI) from the cultures after their respective treatments showed 31% growth inhibition at 257.75 μg/mL in the absence of S9 and 24% and 70% growth inhibition respectively was achieved at 386.65 and 515.5 μg/mL in the presence of S9. A precipitate of the test material was not seen at the end of the treatment period in either exposure group; however haemolysis was seen in both exposure groups, in the continuous exposure group at and above 64.44 μg/mL and at and above 257.75 μg/mL in the presence of S9. The maximum dose level selected for metaphase analysis was 257.75 μg/mL for the 24-hour exposure group, and 515.5 μg/mL for the 4(20)-hour exposure group in the presence of S9.

The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation. The test item did not induce a significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

None

Conclusions:
The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

In an in vitro chromosome aberration test performed according to OECD Guideline 473 and in compliance with GLP, human primary lymphocytes were exposed to test item diluted in DMSO at the following concentrations:

Preliminary Toxicity Test (Cell Growth Inhibition Test):

0, 8.05, 16.11, 32.22, 64.44, 128.88, 257.75, 515.5, 1031 and 2062 μg/mL; 4 h exposure time with and without metabolic activation followed by a 20 h recovery period, and a continuous exposure of 24 hours without metabolic activation

Experiment 1: 4 hour treatment followed by 24 hour harvest after the start of treatment

without and with S9 (2 % S9): 0, 64.44, 128.88, 257.75, 515.5, 773.33 and 1031 μg/mL

Experiment 2:

without S9: 0, 16.11, 32.22, 64.44, 128.88, 257.75 and 515.5 μg/mL; 24 hour treatment followed by harvest at the end of treatment

with S9 (1 % S9): 0, 64.44, 128.88, 257.75, 386.65, 515.5 and 773.33 μg/mL; 4 hour treatment followed by 24 hour harvest after the start of treatment

Mitotic activity was arrested by addition of colcemid at 0.1 µg/mL for each culture, two hours before the harvest. The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations. Vehicle and positive controls were also included in this test.

All vehicle (solvent) control groups had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that induced approximately 50% mitotic inhibition.

Under the test conditions, the test item was considered to be non-clastogenic to human lymphocytes in vitro.

This study is considered as acceptable and satisfies the requirement for in vitro mammalian chromosome aberration assay.

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

Additional information

Table 7.6/1: Summary of genotoxicity tests

 

Test n°

Test / Guideline

Reliability

Focus

Strains tested

Metabolic activation

Test concentration

Statement

1

 

Harlan, 2009

Ames Test

(OECD 471)

K, rel. 1

Gene mutation

TA 1535,

TA 1537,

TA 98,

TA 100,

E. coli WP2

-S9

+S9

Up to limit concentration

-S9 : non mutagenic

+S9 : non mutagenic

2

 

Harlan, 2009

HL/CAT (OECD 473)

K, rel. 1

Gene mutation

Human Lymphocytes

-S9

+S9

Up to cytotoxic concentrations

-S9 : non mutagenic

+S9 : non mutagenic

 

Gene mutation Assays (Tests n° 1):

A Bacterial Reverse mutation Assay (Ames test)was performed according to OECD guideline No. 471 with the substance (See Table 7.6/1). No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains under the test condition, with any dose of the substance, either in the presence or absence of metabolic activation. The substance does not induce gene mutations in bacteria whereas all positive control chemicals (with and without metabolic activation) induced significant increase of colonies.The substance is therefore considered as non-mutagenic according to the Ames test.

  

Chromosomal aberration (Test n°2)

The clastogenic potential of the substance was determined using an in vitro chromosome aberration test in human lymphocytes (OECD guideline No. 473), which measures the potential of a substance to increase the incidence of structural chromosome aberrations in cultured human lymphocytes.

None of the dose levels up to the cytotoxicity limit with the substance, either in the presence or absence of metabolic activation, induced significant increases in the frequency of cells with aberrations in either of three experiments. The substance does not induce structural aberrations in the chromosomes of human lymphocytes under activation and non-activation conditions, whereas both positive control chemicals (with and without metabolic activation) induced significant increases in the frequency of aberrant cells.The substance is therefore considered as negative for inducing chromosomal mutations in human lymphocyte cells under activation and non-activation conditions used in this assay.

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self classification:

Based on the available data, no additional classification is proposed regarding genetic toxicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.