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Genetic toxicity in vitro

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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:
February 13, 2012 - April 20, 2012
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(2008)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Version / remarks:
(1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: "Kanpoan No. 287 -- Environment Protection Agency", "Eisei No. 127 -- Ministry of Health & Welfare", "Heisei 09/10/31 Kikyoku No. 2 -- Ministry of Economy, Trade & Industry"
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
- Identity: CITRATHAL
- Analytical purity: not applicable (as it is an UVCB substance)
- Batch No.: SC00004113
- Expiration date of the lot/batch: November 16, 2012
Species / strain / cell type:
other: Cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Blood samples
Blood samples were obtained from healthy, non-smoking donors not receiving medication. Blood was collected from a male donor (28 years old) for the first experiment, from a 27 year-old female donor for Experiment IIA and from a 30 year-old male donor for Experiment IIB. All donors had a previously established low incidence of chromosomal aberrations in their peripheral blood lymphocytes.
Blood samples were drawn by venous puncture and collected in heparinized tubes by Dr. V. Theodor (64380 Rossdorf, Germany). The tubes were sent to the laboratory to initiate cell cultures within 24 hours after collection. If necessary, the blood was stored before use at 4°C.
- Culture medium
The culture medium was DMEM:F12 (Dulbecco's modified eagle medium / Ham's F12 medium; mixture 1:1; Life Technologies GmbH (Invitrogen division), 64293 Darmstadt, Germany) already supplemented with 200 mM Glutamax. Additionally, the medium was supplemented with penicillin/streptomycin (100 U/mL / 100 µg/mL) (SEROMED, 12247 Berlin, Germany), the mitogen phytohemagglutinin (PHA, final concentration 3 µg/mL, SEROMED), 10% FBS (fetal bovine serum) provided by PAA Laboratories GmbH (35091 Cölbe, Germany), 10 mM HEPES, and the anticoagulant heparin (25,000 U.S.P.-U/mL, Nattermann, 50829 Köln, Germany).
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.
Test concentrations with justification for top dose:
Highest concentration was determined to be 2264 µg/ mL (10 mM)

Evaluated:

Cytogenetic test I:
Without S9-mix, 4 h exposure: 45.0, 78.8 and 137.9 µg/mL
With S9-mix, 4 h exposure: 45.0, 137.9, 241.4 and 739.3 µg/ mL
Cytogenetic test IIA:
Without S9-mix, 22 hr exposure: 17.4, 30.5 and 53.3 µg/mL
With S9-mix, 4 hr exposure: 45.0, 78.8 and 137.9 µg/mL
Cytogenetic test IIB:
Without S9-mix, 22 hr exposure: 40.0, 50.0, 60.0 and 70.0 µg/mL

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: Ethylmethane sulfonate in nutrient medium (770 µg/mL (exp.I), 660 µg/mL (exp.IIA) and 550 µg/mL (exp.IIB))
Remarks:
without S9
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other: Cyclophosphamide in saline (15 µg/mL (exp.I) and 7.5 µg/mL (exp.II))
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 72 hr
- Exposure duration: 4 hr (with and without S9-mix), 22 (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 22 hr

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

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: no
Evaluation criteria:
A test item is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the historical control data.
- no significant increase of the number of structural chromosome aberrations is observed.

A test item is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of the historical control data and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

The assay can indicate an aneugenic potential of the test item if the number of induced numerical aberrations is not in the range of the historical control data.
Statistics:
Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05). However, both biological and statistical significance should be considered together.
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of osmolality: No
Solvent control: 352, 382 and 384 mOsm/kg
2264 µg/ml: 399 mOsm/kg
500 µg/ml: 374 mOsm/kg
300 µg/ml: 382 mOsm/kg
- Effects on pH: No
Solvent control: 7.7
2264 µg/ml: 7.4
500 µg/ml: 7.7
300 µg/ml: 7.8

- Precipitation: No precipitation was observed up to and including the top dose of 2264 µg/mL (= 0.01 M). Phase separation was observed in all experiments.

INFORMATION ON CYTOTOXICITY:
- In experiment I in the absence and presence of S9-mix and in experiment IIB in the absence of S9-mix, cytoxicity was observed at the highest evaluated concentration (44.4 and 42.4% of control, respectively). In experiment IIA in the absence and presence of S9-mix, concentrations showing clear cytoxicity were not evaluable for cytogenetic damage.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.

OTHER:
Either with or without metabolic activation, no clastogenicity was observed at the concentrations evaluated. However, in Experiment I in the absence of S9 mix, one statistically significant increase was observed after treatment with 78.8 µg/mL (2.0 % aberrant cells, excluding gaps). The value is in the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) and no dose-related response was seen; therefore considered as being biologically irrelevant. In Experiment IIA in the presence of S9 mix one single value (3.5 % aberrant cells, excluding gaps) at concentration 78.8 µg/mL slightly exceeded the laboratory’s historical control range (0.0 – 3.0 % aberrant cells, excluding gaps). Since the value was not statistically significant, compared to the respective solvent control, and no dose-related response was observed, the finding was regarded as biologically irrelevant.

No evidence of an increase in polyploid metaphases was noticed after treatment with the test item as compared to the control cultures.
Conclusions:
A chromosome aberration study with Citrathal was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that Citrathal is not clastogenic in human lymphocytes.
Executive summary:

A chromosome aberration study with Citrathal was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments up to cytotoxic concentrations with and without S9 -mix.

Either with or without metabolic activation, no clastogenicity was observed at the concentrations evaluated. However, in Experiment I in the absence of S9 mix, one statistically significant increase was observed after treatment with 78.8 µg/mL (2.0 % aberrant cells, excluding gaps). The value is in the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) and no dose-related response was seen; therefore considered as being biologically irrelevant. In Experiment IIA in the presence of S9 mix one single value (3.5 % aberrant cells, excluding gaps) at concentration 78.8 µg/mL slightly exceeded the laboratory’s historical control range (0.0 – 3.0 % aberrant cells, excluding gaps). Since the value was not statistically significant compared to the respective solvent control and no dose-related response was observed, the finding was regarded as biologically irrelevant.

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

It is concluded that Citrathal is not clastogenic in human lymphocytes when tested up to cytotoxic concentrations with and without S9 -mix.

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:
May 29, 2001 - June 11, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(1997)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(2000)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
- Name of test material (as cited in study report): CITRATHAL R
- Description: Colourless to pale yellow liquid
- Analytical purity: not applicable (as it is an UVCB substance)
- Composition of test material, percentage of components: as defined in section 1.2
- Lot/batch No.: 9000416199
- Expiration date of the lot/batch: August 27, 2001
- Storage condition of test material: In the original container, at room temperature (range of 17-20 'C), away from direct sunlight
Target gene:
- S. typhimurium: Histidine gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
- Experiment 1 (plate incorporation)
Preliminary test (without and with S9), TA98 and TA100: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Main study, TA1535, TA1537 and TA102:
Without and with S9-mix: 10, 33, 100, 333, 1000 and 2500 µg/plate

- Experiment 2 (pre-incubation)
Preliminary test (without and with S9), TA98 and TA100: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Main study, TA1535, TA1537 and TA102:
Without and with S9-mix: 10, 33, 100, 333, 1000 and 2500 µg/plate
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent/vehicle: because of its solubility properties and its relative non-toxicity to the bacteria
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: see section "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Experiment 1: in agar (plate incorporation)
- Experiment 2: pre-incubation

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted, simultaneously.

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered as positive if a biologically relevant and dose related increase in the number of revertants is induced. A test substance producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any one of the test points is considered non-mutagenic in this system. A biologically relevant response is described as follows: A test substance is considered mutagenic if in strains TA98, TA100 and TA102 the number of reversions will be at least twice as high and in strains TA1535 and TA1537 at least three times higher as compared to the spontaneous reversion rate. Also, a dose-dependent and reproducible increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test substance, regardless whether the highest dose will induce the above described enhancement factors or not.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
(although not under both conditions in all strains)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- Direct plate assay: In tester strain TA98, toxicity was observed at dose levels of 2500 μg/plate and above in the absence and presence of S9-mix. In tester strain TA100, toxicity was observed at dose levels of 1000 μg/plate and above in the absence of S9-mix and at 2500 μg/plate and above in the presence of S9-mix.
- Pre-incubation assay: In tester strain TA98, toxicity was observed at dose levels of 1000 μg/plate and above in the absence of S9-mix and at 2500 μg/plate and above in the presence of S9-mix. In tester strain TA100, toxicity was observed at dose levels of 1000 μg/plate and above in the absence and presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA (without metabolic activation)
- The negative, solvent and strain-specific positive control values were within the laboratory historical control data ranges, indicating that the test conditions were adequate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
TA1535: No toxicity or mutagenicity was observed up to and including the top dose of 2500 µg/plate
TA1537: without S9: 1000 µg/plate and above in experiment 2 and without S9 (Experiment 1) and
with S9 (both experiments): No toxicity or mutagenicity was observed up to and including the top dose of 2500 µg/plate
TA98 and TA100: see above
TA102: without and with S9: at 2500 µg/plate
Conclusions:
In the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay performed according to OECD 471 (1997).
Executive summary:

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in two independent experiments, a direct plate assay and a pre-incubation assay, both in the absence and presence of S9-mix. The two experiments were performed simultaneously. Strains TA98 and TA100 were tested up to 5000 µg/plate in both experiments and the dose levels for the tester strains TA1535, TA1537 and TA102 were based on the observed cytotoxicity in TA98 and TA100 and were up to 2500 µg/plate in both experiments. Cytotoxicity was observed in the TA98, TA100 and TA102 strains. Adequate negative and positive controls were included. In both experiments, the substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100 and TA102), both in the absence and presence of S9-metabolic activation. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental part of this study was conducted between 20 September 2016 and 7 November 2016.
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 (incl. QA statement)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
Identification: Citrathal
Appearance: Yellow liquid
Batch: SC00016331
Purity/Composition: UVCB
Test item storage: In refrigerator (2-8°C) protected from light
Stable under storage conditions until: 15 January 2017 (expiry date)
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
Evaluation of Toxicity.
3 and 24 hour treatment without S9: 3.75, 7.5, 15, 30, 40, 45, 55 and 60 µg/mL
3 treatment with S9: 3.75, 7.5, 15, 30, 60, 85 and 100 µg/mL

Evalution of Mutagenicity.
24-hour treatment without S9: 0.94, 1.88, 3.75, 7.5, 15, 30, 35, 40, 45, 50, 55 and 60 µg/mL
Vehicle / solvent:
Dimethyl sulfoxide (DMSO)
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
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:
not applicable
Positive controls validity:
valid
Conclusions:
The test item did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells.
Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No.490 "Genetic Toxicity: In Vitro Mammalian Cell Gene Mutation Tests using the Thimidine Kinase Gene" adopted 28 July 2015.

Method/Experimental

This report describes the effects of Citrathal on the induction of forward mutations at the thymidine-kinase locus (TK-locus) in L5178Y mouse lymphoma cells. The test was performed in the absence of S9-mix with 3 and 24-hour treatment periods and in the presence of S9-mix with a 3 hour treatment period (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

The study procedures described in this report were based on the most recent OECD guideline.

Batch SC00016331 of Citrathal was a yellow liquid. The test item was dissolved in dimethyl sulfoxide.

In the first experiment, effect of Citrathal was evaluated up to the dose levels of 55 and 100 μg/ml in the absence and presence of S9-mix, respectively giving Relative Total Growth

values of 21 and 13%. In the absence of S9-mix, above the dose level of 55 μg/ml the RTG was below the acceptable limit of 10%. The incubation time was 3 hours.

In the absence and presence of S9-mix, none of the tested concentrations reached a mutation frequency of MF(controls) + 126.

In the second experiment, Citrathal was tested up to concentrations of 50 μg/ml in the absence of S9-mix. The incubation time was 24 hours. The RTG was 11%.

In the prolonged treatment period, none of the tested concentrations reached a mutation frequency of MF(controls) + 126.

The mutation frequency found in the solvent control cultures was within the range of the acceptability criteria of this assay and within the 95% control limits of the distribution of the

historical concurrent solvent control database, except in the first experiment in which the mutation frequency of one of the solvent control cultures in the absence of S9-mix was not

within the range of the acceptability criteria. Since the mutation frequency was just above the upper limit of the acceptability criteria range and clear negative results were obtained, this deviation in the mutation frequency had no effect on the validity of the results of the first mutation experiment.

Positive control chemicals, methyl methanesulfonate and cyclophosphamide, both produced significant increases in the mutation frequency. In addition, the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Citrathal did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with

modification in the duration of treatment. In the presence of S9-mix, Citrathal did not induce a significant increase in the mutation frequency.

Conclusion

It is concluded that Citrathal is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

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

Additional information

Ames test (OECD 471 (1997))

The mutagenic activity of the substance was evaluated in accordance with OECD 471 (1997) and according to GLP principles. The test was performed in two independent experiments, a direct plate assay and a pre-incubation assay, both in the absence and presence of S9-mix. The two experiments were performed simultaneously. Strains TA98 and TA100 were tested up to 5000 µg/plate in both experiments and the dose levels for the tester strains TA1535, TA1537 and TA102 were based on the observed cytotoxicity in TA98 and TA100 and were up to 2500 µg/plate in both experiments. Cytotoxicity was observed in the TA98, TA100 and TA102 strains. Adequate negative and positive controls were included. In both experiments, the substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the five S. typhimurium tester strains (TA1535, TA1537, TA98, TA100 and TA102), both in the absence and presence of S9-metabolic activation. Based on the results of this study it is concluded that the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Chromosome aberration study (OECD 473 (1997))

A chromosome aberration study with Citrathal was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments up to cytotoxic concentrations with and without S9 -mix.

Either with or without metabolic activation, no clastogenicity was observed at the concentrations evaluated. However, in Experiment I in the absence of S9 mix, one statistically significant increase was observed after treatment with 78.8 µg/mL (2.0 % aberrant cells, excluding gaps). The value is in the range of the laboratory historical solvent control data (0.0 – 3.0 % aberrant cells, excluding gaps) and no dose-related response was seen; therefore considered as being biologically irrelevant. In Experiment IIA in the presence of S9 mix one single value (3.5 % aberrant cells, excluding gaps) at concentration 78.8 µg/mL slightly exceeded the laboratory’s historical control range (0.0 – 3.0 % aberrant cells, excluding gaps). Since the value was not statistically significant compared to the respective solvent control and no dose-related response was observed, the finding was regarded as biologically irrelevant.

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

It is concluded that Citrathal is not clastogenic in human lymphocytes when tested up to cytotoxic concentrations with and without S9 -mix.


Short description of key information:
The substance is not mutagenic in the Salmonella typhimurium reverse mutation assay performed according to OECD 471 (1997). A chromosome aberration study with Citrathal was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that Citrathal is not clastogenic in human lymphocytes.

Endpoint Conclusion:

Based on the available data, obtained by performing in vitro studies, the substance does not need to be classified in accordance with the CLP Regulation.

Justification for classification or non-classification

A mutation is a permanent change in the amount or structure of the genetic material in a cell. The term “mutation” applies to both heritable genetic changes that may be manifested at the phenotypic level and to the underlying DNA modifications when known, including specific base pair changes and chromosomal translocations. The term “mutagenic” and “mutagen” are used for agents giving rise to an increased occurrence of mutations in populations of cells or organisms.

The more generic terms “genotoxic” and “genotoxicity” apply to agents or processes which alter the structure, information content or segregation of DNA, including those which cause

DNA damage by interfering with normal replication processes, or which in a non-physiological manner temporarily alter its replication. Genotoxicity test results are usually taken as indicators for mutagenic effects.

This hazard class is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity testsin vitroand in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.

To arrive at a classification, test results are considered from experiments determining mutagenic and genotoxic effects in germ and/or somatic cells of exposed animals and in in vitro tests.

The system is hazard based, classifying substances on the basis of their intrinsic ability to induce mutations in germs cells, and does not give a quantitative assessment of the risk.

Three in vitro studies were performed on the target substance. The test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. The test

item did not induce any toxicologically significant increases in the mutant frequency and was non-clastogenic to human lymphocytes in vitro.

The test substance is therefore not classified for genotoxicity.