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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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
Reliability:
1 (reliable without restriction)
Cross-reference
Reason / purpose:
read-across source
Reference
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2013
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Justification for read-across is detailed in the report attached to the IUCLID section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine Kinase locus (TK)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Informatio on cell cultures:
Prior to mutagenicity testing the amount of spontaneous mutants is reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with:
Hypoxanthine 5.0 * 10-3 M
Aminopterin 2.0 * 10-5 M
Thymidine 1.6 * 10-3 M
Glycin 5.0 * 10-3 M
The incubation of the cells in HAT-medium is followed by a recovery period of 2 days in RPMI 1640 medium containing:
Hypoxanthine 1.0* 10-4 M
Thymidine 1.6 * 10-3 M
After this incubation the L5178Y cells are returned to normal RPMI 1640 medium (complete culture medium)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix
Test concentrations with justification for top dose:
A pre-test was performed in order to determine the concentration range of the mutagenicity experiments. Both, pH value and osmolarity were determined at the maximal concentration of the test item and in the solvent control without metabolic activation.

According to the results of the pre-test at least four adequate concentrations were chosen for the mutation experiment.

Experiment I
Exposure period 4h (S9 mix -): 187.5, 375, 750, 1500, 3000, 6000 µg/mL
Exposure period 4h (S9 mix +): 187.5, 375, 750, 1500, 3000, 6000 µg/mL

Experiment II
Exposure period 24h (S9 mix -): 375, 750, 1500, 3000, 4500, 6000 µg/mL
Exposure period 4h (S9 mix +): 375, 750, 1500, 3000, 4500, 6000 µg/mL

Following the expression phase of 48 hours the cultures at the lowest concentrations (187.5, 375 ) in experiment I and II were not continued since a minimum of only four concentrations is required by the guidelines.
Vehicle / solvent:
Water
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Local tap water deionised at Harlan CCR
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
On the day of the experiment (immediately before treatment), the test item was diluted with deionised water (10 %). The final concentration of deionised water in the culture medium was 10% (v/v).

The osmolarity and the pH-value were determined in culture medium of the solvent control and of the maximum concentration in the pre-experiment without metabolic activation:

Solvent control DIRECT ORANGE 39 (C.I. 40215)
6000 µg/mL
Osmolarity [mOsm] 270 304
pH-value 7.51 7.70


Experimental Performance
In the mutation experiment 1*10E7 (3x10E6 during 24 h exposure) cells/flask (80 cm2 flasks) suspended in 10 mL RPMI medium with 3 % horse serum (15 % horse serum during 24 h exposure) were exposed to various concentrations of the test item either in the presence or absence of metabolic activation. Positive and solvent controls were performed in parallel. After 4 h (24 h in the second experiment) the test item was removed by centrifugation (425 x g, 10 min) and the cells were washed twice with "saline G". Subsequently the cells were resuspended in 30 mL complete culture medium and incubated for an expression and growth period of 48 h.

The cell density was determined each day and adjusted to 3*10E5 cells/mL, if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number according to the method of Clive and Spector.

After the expression period the cultures were selected. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4*10E3 cells in selective medium (see below) with TFT (Serva, 69042 Heidelberg, Germany). The viability (cloning efficiency) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT). The plates were incubated at 370°+- 1.5 °C in 4.5 % CO2/95.5 % water saturated air for 10 - 15 days. Then the plates were evaluated. The relative total growth (RTG) is calculated by the RSG multiplied by the viability.

Complete Culture Medium
RPMI 1640 medium supplemented with 15 % horse serum (HS) (3 % HS during 4 hour treatment), 1% of 100 U/100 µg/mL Penicillin/Streptomycin, 220 µg/mL Sodium-Pyruvate, and 0.5 – 0.75 % Amphotericin used as antifungal agent.

Selective Medium
RPMI 1640 (complete culture medium) by addition of 5 µg/mL TFT.

Saline G Solution: The "saline G" solution was composed as follows (per litre):
NaCl8000 mg; KCl 400 mg; Glucose 1100 mg; Na2HPO4x7H2ONa2HPO4x2H2O 192 mg; KH2PO4 150 mg; pH: 7.2

Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 106 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
A test item is considered equivocal in this assay if the threshold is reproducibly exceeded but the increase of the mutation frequency is not dose dependent.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 106 cells above the corresponding solvent control.
A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.
Statistics:
A linear regression (least squares) will be performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT(R)11 (SYSTAT Software, Inc.) statistics software. The number of mutant colonies obtained for the groups treated with the test item will be compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance will be considered together
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
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:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
6000 µg/mL (24h)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test medium was checked for precipitation visible to the naked eye at the end of the 4 hours treatment just before the test item was removed. No precipitation meeting the criteria mentioned above was noted in the pre-experiment and in both main experiments.

A relevant cytotoxic effect as indicated by a relative total growth of 50% or below at in both parallel cultures was solely observed in experiment II following 24 hours treatment at the maximum concentration of 6000 µg/mL without metabolic activation.

No substantial and reproducible increase of the mutation frequency was noted in both experiments with and without metabolic activation. The threshold of 126 above the corresponding solvent control was not reached.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in all experimental groups.

In this study the range of the solvent controls was from 51 up to 88 mutant colonies per 106 cells; the range of the groups treated with the test item was from 33 up to 148 mutant colonies per 106 cells. The viability of the solvent control of the first experiment, culture II without metabolic activation slightly exceeded the upper limit of the acceptance criteria (121 versus 120%, c.f. table 8, column 8). This deviation was judged as irrelevant as it was very minor and the viability of the parallel culture remained within the acceptable range.

MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 µg/mL and 4.5 µg/mL in both main experiments) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity at with at least one of the concentrations of the controls. The positive controls remained within the range of the historical positive control data throughout the study.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Summary of Results, Experiment I and II

       relative  mutant    relative  mutant  
   conc. ug/mL  S9 mix  total growth  colonies 106 cells  treshold  total growth   colonies 106cells treshold 
column   1  3  4  5  6  7  8
experiment I / 4 h treatment            culture I        culture II
 Solvent control with water    -  100.0  88  214  100.0  86  212
 Pos. control with MMS  19.5  -  38.8  312  214  19.4  346  212
 Test item  187.5  -        culture was not continued        culture was not continued
 Test item  375.0  -  132.3  68  214  78.1  81  212
 Test item  750.0  -  160.3  59  214  106.8  62  212
 Test item  1500.0  -  119.2  58  214  45.2  142  212
 Test item  3000.0  -  73.2  81  214  60.8  77  212
 Test item  6000.0  -  92.6  90  214  29.6  127  212
                 
  Solvent control with water    +  100.0  59  185  100.0  54  180
 Pos. control with CPA  3.0  +  52.7  216  185  46.5  231  180
 Pos. control with CPA  4.5  +  18.8  417  185  17.9  481  180
 Test item  187.5  +         culture was not continued         culture was not continued
  Test item  375.0  +  92.4  50  185  137.1  39  180
  Test item  750.0  +  91.0  46  185  150.7  33  180
  Test item  1500.0  +  106.0  66  185  108.7  35  180
  Test item  3000.0  +  92.0  56  185  91.4  41  180
  Test item  6000  +  98.2  73  185  110.7  37  180
 Experiment II / 24 h treatment            culture I        culture II
 Solv. control with water    -  100.0  51  177  100.0  51  177
 Pos. control with MMS  13.0  -  23.2  461  177  19.0  439  177
  Test item  375.0  -        culture was not continued        culture was not continued
  Test item  750.0  -  125.1  47  177  115.2  66  177
  Test item  1500.0  -  57.0  92  177  50.7  148  177
  Test item  3000.0  -  59.8  57  177  52.4  77  177
  Test item 4500.0  -  50.6  75  177  33.5  77  177
  Test item  6000.0  -  22.5  112  177  18.5  76  177
 Experiment II / 4h treatment            culture I        culture II
 Solvent control with water    +  100.0  68  194  100.0  81  207
 Pos. control with CPA  3.0  +  23.8  379  194  24.0  495  207
 Pos. Control with CPA  4.5  +  6.7  648  194  11.3  527  207
 Test item  375.0  +         culture was not continued         culture was not continued
  Test item  750.0  +  98.0  51  194  109.0  99  207
 Test item   1500.0  +  135.3  76  194  188.0  93  207
  Test item  3000.0  +  223.6  59  194  208.5  79  207
  Test item  4500.0  +  151.5  47  194  182.3  63  207
  Test item  6000  +  116.2  52  194  144.4  89  207
                 

threshold = number of mutant colonies per 106cells of each solvent control plus 126

#   culture was not continued since a minimum of only four concentrations is required by the guidelines

Conclusions:
negative

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Therefore, the substance is considered to be non-mutagenic in this mouse lymphoma assay.

Executive summary:

The study was performed to investigate the potential of the substance to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. The first experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The treatment period of the second experiment was 4 hours with and 24 hours without metabolic activation.

The maximum test item concentration of 6000 µg/mL used in the pre-experiment and in the main experiments with and without metabolic activation was equal to approximately 5000 µg/mL of the pure substance. The test item was dissolved in deionised water.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Physical appearance: solid
Expiry date : June 1995
Storage: room temperature

Method

Target gene:
Thymidine Kinase locus (TK)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Informatio on cell cultures:
Prior to mutagenicity testing the amount of spontaneous mutants is reduced by growing the cells for one day in RPMI 1640-HAT medium supplemented with:
Hypoxanthine 5.0 * 10-3 M
Aminopterin 2.0 * 10-5 M
Thymidine 1.6 * 10-3 M
Glycin 5.0 * 10-3 M
The incubation of the cells in HAT-medium is followed by a recovery period of 2 days in RPMI 1640 medium containing:
Hypoxanthine 1.0* 10-4 M
Thymidine 1.6 * 10-3 M
After this incubation the L5178Y cells are returned to normal RPMI 1640 medium (complete culture medium)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Mammalian Microsomal Fraction S9 Mix
Test concentrations with justification for top dose:
A pre-test was performed in order to determine the concentration range of the mutagenicity experiments. Both, pH value and osmolarity were determined at the maximal concentration of the test item and in the solvent control without metabolic activation.

According to the results of the pre-test at least four adequate concentrations were chosen for the mutation experiment.

Experiment I
Exposure period 4h (S9 mix -): 187.5, 375, 750, 1500, 3000, 6000 µg/mL
Exposure period 4h (S9 mix +): 187.5, 375, 750, 1500, 3000, 6000 µg/mL

Experiment II
Exposure period 24h (S9 mix -): 375, 750, 1500, 3000, 4500, 6000 µg/mL
Exposure period 4h (S9 mix +): 375, 750, 1500, 3000, 4500, 6000 µg/mL

Following the expression phase of 48 hours the cultures at the lowest concentrations (187.5, 375 ) in experiment I and II were not continued since a minimum of only four concentrations is required by the guidelines.
Vehicle / solvent:
Water
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Local tap water deionised at Harlan CCR
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
On the day of the experiment (immediately before treatment), the test item was diluted with deionised water (10 %). The final concentration of deionised water in the culture medium was 10% (v/v).

The osmolarity and the pH-value were determined in culture medium of the solvent control and of the maximum concentration in the pre-experiment without metabolic activation:

Solvent control DIRECT ORANGE 39 (C.I. 40215)
6000 µg/mL
Osmolarity [mOsm] 270 304
pH-value 7.51 7.70


Experimental Performance
In the mutation experiment 1*10E7 (3x10E6 during 24 h exposure) cells/flask (80 cm2 flasks) suspended in 10 mL RPMI medium with 3 % horse serum (15 % horse serum during 24 h exposure) were exposed to various concentrations of the test item either in the presence or absence of metabolic activation. Positive and solvent controls were performed in parallel. After 4 h (24 h in the second experiment) the test item was removed by centrifugation (425 x g, 10 min) and the cells were washed twice with "saline G". Subsequently the cells were resuspended in 30 mL complete culture medium and incubated for an expression and growth period of 48 h.

The cell density was determined each day and adjusted to 3*10E5 cells/mL, if necessary. The relative suspension growth (RSG) of the treated cell cultures was calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number according to the method of Clive and Spector.

After the expression period the cultures were selected. Cells from each experimental group were seeded into 2 microtiter plates so that each well contained approximately 4*10E3 cells in selective medium (see below) with TFT (Serva, 69042 Heidelberg, Germany). The viability (cloning efficiency) was determined by seeding about 2 cells per well into microtiter plates (same medium without TFT). The plates were incubated at 370°+- 1.5 °C in 4.5 % CO2/95.5 % water saturated air for 10 - 15 days. Then the plates were evaluated. The relative total growth (RTG) is calculated by the RSG multiplied by the viability.

Complete Culture Medium
RPMI 1640 medium supplemented with 15 % horse serum (HS) (3 % HS during 4 hour treatment), 1% of 100 U/100 µg/mL Penicillin/Streptomycin, 220 µg/mL Sodium-Pyruvate, and 0.5 – 0.75 % Amphotericin used as antifungal agent.

Selective Medium
RPMI 1640 (complete culture medium) by addition of 5 µg/mL TFT.

Saline G Solution: The "saline G" solution was composed as follows (per litre):
NaCl8000 mg; KCl 400 mg; Glucose 1100 mg; Na2HPO4x7H2ONa2HPO4x2H2O 192 mg; KH2PO4 150 mg; pH: 7.2

Evaluation criteria:
A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 106 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
A test item is considered equivocal in this assay if the threshold is reproducibly exceeded but the increase of the mutation frequency is not dose dependent.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.
A test item is classified as non-mutagenic if the induced mutation frequency does not reproducibly exceed a threshold of 126 colonies per 106 cells above the corresponding solvent control.
A test item not meeting the conditions for a classification as mutagenic or non-mutagenic will be considered equivocal in this assay and may be considered for further investigation.
Statistics:
A linear regression (least squares) will be performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT(R)11 (SYSTAT Software, Inc.) statistics software. The number of mutant colonies obtained for the groups treated with the test item will be compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological relevance and statistical significance will be considered together

Results and discussion

Test resultsopen allclose all
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
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:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
6000 µg/mL (24h)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The test medium was checked for precipitation visible to the naked eye at the end of the 4 hours treatment just before the test item was removed. No precipitation meeting the criteria mentioned above was noted in the pre-experiment and in both main experiments.

A relevant cytotoxic effect as indicated by a relative total growth of 50% or below at in both parallel cultures was solely observed in experiment II following 24 hours treatment at the maximum concentration of 6000 µg/mL without metabolic activation.

No substantial and reproducible increase of the mutation frequency was noted in both experiments with and without metabolic activation. The threshold of 126 above the corresponding solvent control was not reached.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in all experimental groups.

In this study the range of the solvent controls was from 51 up to 88 mutant colonies per 106 cells; the range of the groups treated with the test item was from 33 up to 148 mutant colonies per 106 cells. The viability of the solvent control of the first experiment, culture II without metabolic activation slightly exceeded the upper limit of the acceptance criteria (121 versus 120%, c.f. table 8, column 8). This deviation was judged as irrelevant as it was very minor and the viability of the parallel culture remained within the acceptable range.

MMS (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and CPA (3.0 µg/mL and 4.5 µg/mL in both main experiments) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity at with at least one of the concentrations of the controls. The positive controls remained within the range of the historical positive control data throughout the study.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of Results, Experiment I and II

       relative  mutant    relative  mutant  
   conc. ug/mL  S9 mix  total growth  colonies 106 cells  treshold  total growth   colonies 106cells treshold 
column   1  3  4  5  6  7  8
experiment I / 4 h treatment            culture I        culture II
 Solvent control with water    -  100.0  88  214  100.0  86  212
 Pos. control with MMS  19.5  -  38.8  312  214  19.4  346  212
 Test item  187.5  -        culture was not continued        culture was not continued
 Test item  375.0  -  132.3  68  214  78.1  81  212
 Test item  750.0  -  160.3  59  214  106.8  62  212
 Test item  1500.0  -  119.2  58  214  45.2  142  212
 Test item  3000.0  -  73.2  81  214  60.8  77  212
 Test item  6000.0  -  92.6  90  214  29.6  127  212
                 
  Solvent control with water    +  100.0  59  185  100.0  54  180
 Pos. control with CPA  3.0  +  52.7  216  185  46.5  231  180
 Pos. control with CPA  4.5  +  18.8  417  185  17.9  481  180
 Test item  187.5  +         culture was not continued         culture was not continued
  Test item  375.0  +  92.4  50  185  137.1  39  180
  Test item  750.0  +  91.0  46  185  150.7  33  180
  Test item  1500.0  +  106.0  66  185  108.7  35  180
  Test item  3000.0  +  92.0  56  185  91.4  41  180
  Test item  6000  +  98.2  73  185  110.7  37  180
 Experiment II / 24 h treatment            culture I        culture II
 Solv. control with water    -  100.0  51  177  100.0  51  177
 Pos. control with MMS  13.0  -  23.2  461  177  19.0  439  177
  Test item  375.0  -        culture was not continued        culture was not continued
  Test item  750.0  -  125.1  47  177  115.2  66  177
  Test item  1500.0  -  57.0  92  177  50.7  148  177
  Test item  3000.0  -  59.8  57  177  52.4  77  177
  Test item 4500.0  -  50.6  75  177  33.5  77  177
  Test item  6000.0  -  22.5  112  177  18.5  76  177
 Experiment II / 4h treatment            culture I        culture II
 Solvent control with water    +  100.0  68  194  100.0  81  207
 Pos. control with CPA  3.0  +  23.8  379  194  24.0  495  207
 Pos. Control with CPA  4.5  +  6.7  648  194  11.3  527  207
 Test item  375.0  +         culture was not continued         culture was not continued
  Test item  750.0  +  98.0  51  194  109.0  99  207
 Test item   1500.0  +  135.3  76  194  188.0  93  207
  Test item  3000.0  +  223.6  59  194  208.5  79  207
  Test item  4500.0  +  151.5  47  194  182.3  63  207
  Test item  6000  +  116.2  52  194  144.4  89  207
                 

threshold = number of mutant colonies per 106cells of each solvent control plus 126

#   culture was not continued since a minimum of only four concentrations is required by the guidelines

Applicant's summary and conclusion

Conclusions:
negative

In conclusion it can be stated that during the mutagenicity test described and under the experimental conditions reported the test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.
Therefore, the substance is considered to be non-mutagenic in this mouse lymphoma assay.

Executive summary:

The study was performed to investigate the potential of the substance to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. The first experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The treatment period of the second experiment was 4 hours with and 24 hours without metabolic activation.

The maximum test item concentration of 6000 µg/mL used in the pre-experiment and in the main experiments with and without metabolic activation was equal to approximately 5000 µg/mL of the pure substance. The test item was dissolved in deionised water.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive and valid.