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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are valid in vitro studies, on the analog substance, which assess mutagenicity in bacterial and mammalan cells as well as an in vitro micronuleus study on the analog substance, which are negative. The in vitro studies with 1999-85-5 are all also negative, but are K3 for various deficiencies.

Link to relevant study records

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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:
11 June 2012 - 17 July 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable.
Species / strain / cell type:
S. typhimurium TA 102
Details on mammalian cell type (if applicable):
Not applicable.
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
see Executive summary
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide
- Justification for choice: test item was soluble in the vehicle at 100 mg/mL.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide, 9-aminoacridine, 2-nitrofluorene, mitomycin C (-S9 mix); 2-anthramine, benzo(a)pyrene (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar
see Executive summary

DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 to 72 hours.

DETERMINATION OF CYTOTOXICITY
- Method: decrease in number of revertant colonies and/or thinning of the bacterial lawn
Evaluation criteria:
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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
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
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
negative

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or in the absence of a rat liver metabolizing system.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutation in Salmonella typhimurium.

 

The study was performed according to the international guidelines (OECD No. 471 and Commission Directive No. B.13/14) and in compliance with the principles of Good Laboratory Practice.

 

Methods

A preliminary toxicity test was performed to define the dose-levels of m/p DIOL to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver post‑mitochondrial fraction (S9 fraction) of rats induced with Aroclor 1254.

 

Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the pre-incubation method (60 minutes, 37°C).

 

Five strains of bacteria Salmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose‑level). After 48 to 72 hours of incubation at, the revertant colonies were scored.

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

The test item was dissolved in dimethylsulfoxide (DMSO).

 

Results

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered to be valid.

 

Since the test item was found freely soluble and non-cytotoxic in the preliminary test, the highest dose-level was 5000 µg/plate, according to the criteria specified in the international guidelines.

 

The selected treatment-levels were: 312.5, 625, 1250, 2500 and 5000 µg/plate for both mutagenicity experiments, with and without S9 mix.

 

No precipitate was observed in the Petri plates when scoring the revertants at any dose-levels.

 

No noteworthy toxicity (decrease in the number of revertants or thinning of the bacterial lawn) was noted towards any of the tested strains, either with or without S9 mix.

 

No noteworthy increase in the number of revertants, which could be considered as biologically relevant, was noted in any of the five strains used, either with or without S9 mix.

 

Conclusion

The test item did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium, either in the presence or in the absence of a rat liver metabolizing system.

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:
11 June 2012 - 03 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium
pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
see Executive summary
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide

The test item was suspended in the vehicle at the concentration of 500 mg/mL for the preliminary toxicity test, for the first experiment with and without S9 mix as well as for the second experiment with S9 mix. It was dissolved at 150 mg/mL for the second experiment without S9 mix.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: methylmethanesulfonate (-S9 mix); cyclophosphamide (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 and 24 hours
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 11-12 days

SELECTION AGENT (mutation assays): trifluorothymidine

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth.
Evaluation criteria:
IWGT recommendations were followed for the determination of a positive result, which should fulfill the following criteria:
- at least at one dose-level the mutation frequency minus the mutation frequency of the vehicle control (IMF) equals or exceeds the Global Evaluation Factor (GEF) of 126 x 10-6,
- a dose-related trend is demonstrated by a statistically significant trend test.

Unless an effect is considered as clearly positive, the reproducibility of a positive effect should be confirmed.

Noteworthy increases in the mutation frequency observed only at high levels of cytotoxicity (Adj. RTG lower than 10%), but with no evidence of mutagenicity at dose-levels with Adj. RTG between 10 and 20%, are not considered as positive results.

A test item may be considered as non-mutagenic when there is no culture showing an Adj. RTG value between 10 and 20% if (Moore et al., 2002):
- there is at least one negative data point between 20 and 25% Adj. RTG and no evidence of mutagenicity in a series of data points between 100 and 20% Adj. RTG,
- there is no evidence of mutagenicity in a series of data points between 100 and 25% and there is also a negative data point between 10 and 1% Adj. RTG.
Species / strain:
mouse lymphoma L5178Y cells
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
Remarks on result:
other: strain/cell type: mouse lymphoma L5178Y cells
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
negative

The test item was not mutagenic to mammalian cells in the presence or absence of metabolic activation.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce mutations at the TK (Thymidine Kinase) locus in L5178Y TK+/-mouse lymphoma cells.

 

The study was performed according to international guidelines (OECD guideline No. 476 and Council Regulation (EC) No. 440/2008 Annex, Part B.17) and in compliance with the principles of Good Laboratory Practice.

 

Methods

After a preliminary toxicity test, the test item was tested in two independent experiments, with and without a metabolic activation system (S9 mix) prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254.

Cultures of 20 mL at 5 x 105cells/mL (3-hour treatment) or cultures of 50 mL at 2 x 105cells/mL (24-hour treatment) were exposed to the test or control items, in the presence or absence of S9 mix (final concentration of S9 fraction 2%). During the treatment period, the cells were maintained as suspension culture in RPMI 1640 culture medium supplemented by heat inactivated horse serum at 5% (3-hour treatment) or 10% (24-hour treatment) in a 37°C, 5% CO2humidified incubator. For the 24-hour treatment, flasks were gently shaken at least once.

 

Cytotoxicity was measured by assessment of Adjusted Relative Total Growth (Adj. RTG), Adjusted Relative Suspension Growth (Adj. RSG) andCloning Efficiency following the expression time (CE2).

The number of mutant clones (differentiating small and large colonies) was evaluated after expression of the mutant phenotype.

 

The vehicle used to prepare the dose formulations was dimethylsulfoxide (DMSO).

 

Results

The Cloning Efficiencies (CE2), the mutation frequencies and the suspension growths of the vehicle controls were as specified in the acceptance criteria. Moreover, the induced mutation frequencies obtained for the positive controls met the acceptance criteria specified in the study plan. The study was therefore considered to be valid.

 

Since the test item was found freely soluble but cytotoxic in the preliminary test, the selection of the highest dose-level for the main experiments was based on the level of toxicity, according to the criteria specified in the international guidelines (decrease in Adj. RTG).

 

Experiments without S9 mix

Using a treatment volume of 1% in culture medium, the selected dose-levels were:

. 312.5, 625, 1250, 2500, 3750 and 5000 µg/mL for the first experiment (3-hour treatment),

. 46.88, 93.75, 187.5, 375, 75, 1125 and 1500 µg/mL for the second experiment (24‑hour treatment).

 

A precipitate was noted in the culture medium at dose-levels ≥ 2500 µg/mL at the end of the 3‑hour treatment period. No precipitate was noted in the culture medium at the end of the 24‑hour treatment period up to the highest tested dose-level of 1500 µg/mL.


Cytotoxicity

Following the 3-hour treatment, a slight to severe toxicity was induced at dose-levels ≥ 1250 µg/mL, as shown by a 29 to 100% decrease in Adj. RTG.

Following the 24-hour treatment, a moderate to severe toxicity was induced at dose‑levels ≥ 375 µg/mL, as shown by a 43 to 86% decrease in Adj. RTG.

 

Mutagenicity

Following the 3- or 24-hour treatments, no noteworthy increases in the mutation frequency, which could be considered as biologically relevant, were induced.

Experiments with S9 mix

Using a treatment volume of 1% in culture medium, the selected dose-levels were:

. 156.3, 312.5, 625, 1250, 2500 and 5000 µg/mL for the first experiment,

. 312.5, 625, 1250, 2500, 3750 and 5000 µg/mL for the second experiment.

 

At the end of the treatment periods, a precipitate was noted in the culture medium at 5000 µg/mL and at dose-levels ≥ 2500 µg/mL in the first and second experiments, respectively.

 

Cytotoxicity

A slight to severe toxicity was induced at dose-levels ≥ 625 µg/mL in the first and second experiments as shown by a 28 to 99% and by a 37 to 94% decrease in Adj. RTG, respectively.

 

Mutagenicity

No noteworthy increases in the mutation frequency, which could be considered as biologically relevant, were induced in any of the experiments.

 

Conclusion

The test item did not show any mutagenic activity in the mouse lymphoma assay, in the presence or in the absence of a rat metabolizing system.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: other: chromosome aberration and aneuploidy
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 August 2012 - 18 October 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.
Qualifier:
according to
Guideline:
other: OECD Guideline 487 (In vitro micronucleus)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
Not applicable (not a gene mutation assay).
Species / strain / cell type:
other: mouse lymphoma L5178Y TK+/- cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640 medium containing 10% inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
rat liver S9 mix
Test concentrations with justification for top dose:
Experiments without S9 mix
- 12.5, 25, 50, 75, 100 and 150 µg/mL for the first experiment,
- 31.3, 62.5, 125, 250, 375, 500 and 750 µg/mL for the second experiment,
- 62.5, 125, 250, 500, 750, 1000, 1500 and 3000 µg/mL for the third experiment.

Experiments with S9 mix
- 75, 150, 300, 450, 600 and 900 µg/mL for the first experiment,
- 62.5, 125, 250, 500, 750, 1000 and 1500 µg/mL for the second experiment.
Vehicle / solvent:
- Vehicle used: dimethylsulfoxide
- Justification for choice: using a test item concentration of 500 mg/mL in the vehicle and a treatment volume of 1% in culture medium, the highest recommended dose-level of 5000 µg/mL was achievable.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: mitomycin C, colchicine (-S9 mix); cyclophosphamide (+S9 mix)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
Without S9 mix:
- 3 h treatment + 24 h recovery in the first experiment,
- 24 h treatment + 20 h recovery in the second and the third experiments.

With S9 mix:
- 3 h treatment + 24 h recovery in both experiments.

NUMBER OF CELLS EVALUATED: 2000/dose.

DETERMINATION OF CYTOTOXICITY
- Method: population doubling
Evaluation criteria:
The biological relevance of the results should be considered first. Statistical methods are used as an aid in evaluating the test results but should not be the only determinant of a positive response. A result is considered as positive if at least a 2.5-fold increase in the number of micronucleated cells in comparison to the concurrent control is observed, with a statistically significant difference, at one or more concentrations. Concentration-related increases in the frequency of micronucleated cells and comparison to the vehicle control historical data will also be taken into account.
Species / strain:
other: mouse lymphoma L5178Y TK/- cells
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results:
negative

The test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the presence or in the absence of a rat liver metabolizing system.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cells, in L5178Y TK+/-mouse lymphoma cells.

 

Methods

After a preliminary toxicity test, the test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

 

 

First experiment

Second experiment

Without S9 mix

3 h treatment + 24 h recovery

24 h treatment + 20 h recovery

With S9 mix

3 h treatment + 24 h recovery

3 h treatment + 24 h recovery

 

Since the highest analyzable dose-level did not exhibit about 55% toxicity, either in the first or in the second experiment without S9 mix, a third experiment was undertaken without metabolic activation, using a 24-h treatment + 20-h recovery period.

 

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells and quality of the cells on the slides has also been taken into account.

 

The test item was dissolved in dimethylsulfoxide (DMSO).

 

Results

With two exceptions which were not considered to impact the validity of the results, the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria, and positive controls showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

Since the test item was cytotoxic in the preliminary test, the highest dose-level selected for the main experiment was based on the level of toxicity, according to the criteria specified in the international guidelines.

 

Experiments without S9 mix

The dose-levels used for treatment were as follows:

.           12.5, 25, 50, 75, 100 and 150 µg/mL for the first experiment,

.           31.3, 62.5, 125, 250, 375, 500 and 750 µg/mL for the second experiment,

.           62.5, 125, 250, 500, 750, 1000, 1500 and 3000 µg/mL for the third experiment.

 

No precipitate was observed in the culture medium at the end of treatment.


Cytotoxicity

Following the 3-hour treatment + 24-hour recovery period (first experiment), only slight decreases in the PD were noted at 25 and 100 µg/mL (26 and 34% decrease, respectively).

Following the 24-hour treatment + 20-hour recovery period in the second experiment, no noteworthy decrease in the PD was induced at any of the tested dose-levels.

Following the 24-hour treatment + 20-hour recovery period in the third experiment, a moderate to severe toxicity was noted at dose-levels ≥ 500 µg/mL, as shown by a 47 to 100% decrease in the PD.

 

Micronucleus analysis

The dose-levels selected for micronucleus analysis were as follows:

.           50, 100 and 150 µg/mL for the first experiment, the latter being the highest tested dose-level,

.           250, 500 and 750 µg/mL for the second experiment, the latter being the highest tested dose‑level,

.           125, 250 and 500 µg/mL for the third experiment, the latter inducing a 47% decrease in the PD and higher dose-levels being too cytotoxic.

 

No increase in the frequency of micronucleated cells was noted in the first and third experiments.

 

Slight increases in the frequency of micronucleated cells were observed in the second experiment. At 500 µg/mL, the increase slightly exceeded the positive threshold of 2.5-fold the vehicle control value (3.5-fold). Since it was not statistically significant in comparison to the vehicle control, not dose-related and not reproducible (no similar effect was observed in the third experiment), this increase, which did not meet the criteria for a positive response, was not considered to be biologically relevant.

 

Experiments with S9 mix

The dose-levels used for treatment were as follows:

.           75, 150, 300, 450, 600 and 900 µg/mL for the first experiment,

.           62.5, 125, 250, 500, 750, 1000 and 1500 µg/mL for the second experiment.

 

A precipitate was observed in the culture medium at dose-levels ≥ 750 µg/mL, at the end of the3‑hour treatment + 24-hour recovery period of the second experiment.

 

Cytotoxicity

In the first experiment, a moderate toxicity was noted at 900 µg/mL as shown by a 43% decrease in the PD.

In the second experiment, no noteworthy decrease in the PD was noted at any of the tested dose‑levels. A precipitate was observed from 750 µg/mL.

 

Micronucleus analysis

The dose-levels selected for micronucleus analysis were as follows:

.           300, 600 and 900 µg/mL for the first experiment, the latter inducing a 43% decrease in the PD and being the highest tested dose-level,

.           250, 500 and 750 µg/mL for the second experiment, the latter corresponding to the lowest dose-level showing precipitate in the culture medium at the end of the treatment period.

 

No noteworthy increase in the frequency of micronucleated cells was noted after the 3-hour treatment in either experiment.


Conclusion

The test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/-mouse lymphoma cells, either in the presence or in the absence of a rat liver metabolizing system.

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

Additional information

Additional information from genetic toxicity in vitro:

Two bacterial reverse mutation test are available on the registered substance which are negative. However both studies have defieicencies. A valid bacterial reverse mutation test and valid mammalian cell gene mutation test are available on the analog substance which are negative. Therefore, taken as a weight of evidence, the registered substance is not considered mutagenic.

A chromosomal aberration study is available on the registered substance which is negative. However this study has deficiencies. A valid in vitro micronucleus study is available on the anlog substance which is negative. Therefore, taken as a weight of evidence, the registerd substance in not considered a cytogenetic toxicant.

Justification for selection of genetic toxicity endpoint

There are no valid studies, on the registered substance, to assess genetic toxicity.  However, studies conducted on the regisered substance were all negative.  There are valid studies available, to assess genetic toxicity, on the analog substance, 27138-01-8, which are are negative.  Therefore, taken as a weight of evidence, the registered substance is not considered mutagenic.

Justification for classification or non-classification

There are no valid studies, on the registered substance, to assess genetic toxicity. However, studies conducted on the regisered substance were all negative. There are valid studies available, to assess genetic toxicity, on the analog substance, 27138-01-8, which are are negative. Therefore, taken as a weight of evidence, the registered substance is not considered mutagenic.