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Genetic toxicity in vitro

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Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes:
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
The tests were performed with 250, 500, and 1000 μg/mL. Based on the solubility of the test substance in Pluronic F68.
Vehicle / solvent:
Preliminary range-finding experiments the test substance was found to be sufficiently suspended in Pluronic F68 at the concentration 1000 μg/mL, with minimum precipitation and no cytotoxicity as measured by the cytokinesis-block proliferation index (CBPI).
Untreated negative controls:
yes
Remarks:
NaCl
Negative solvent / vehicle controls:
yes
Remarks:
Pluronic F68
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: vinblastine
Details on test system and experimental conditions:
Experiments 1 and 2 were conducted in the absence of S9, with exposure of the cells to the test substance for 3 h and 24 h, respectively. Experiment 3 involved exposure for 3 h in the presence of S9. All experiments were conducted with duplicate cultures of cells, after growing exponentially for 48 h from culture initiation. Cytochalasin B was added after termination of treatment in Experiments 1 and 3, and added with treatment in Experiment 2. Cell harvesting was done 72 h after culture initiation, slides were prepared, and the cells were stained with 5 % Giemsa. Microscopic examination of 2000 cells determined the percent incidence of micronuclei in binucleated cells (BNCs).
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Under the study conditions, the test substance did not induce a biologically significant, dose-related increase in the percent incidence of micronuclei in binucleated cells (BNCs) at any dose in any of the three experiments, either in the absence or presence of S9 for mammalian metabolism. In addition, the test substance did not produce cytotoxicity as assessed by the cytokinesis-block proliferation index (CBPI) in either the preliminary cytotoxicity screen or in the three experiments as described above.
Executive summary:

A study was conducted to determine the potential of the test substance to induce mutations in human lymphocytes according to in vitro mammalian cell micronucleus test OECD Guideline 487, in compliance with GLP. In the preliminary range-finding test the test substance was found to be sufficiently suspended in Pluronic F68 at the concentration of 1000 μg/mL, with minimum precipitation and no cytotoxicity as measured by the cytokinesis-block proliferation index (CBPI). Based on these results, the tests were performed with 250, 500, and 1000 μg/mL. Experiments 1 and 2 were conducted in the absence of S9, with exposure of the cells to the test substance for 3 h and 24 h, respectively. Experiment 3 involved exposure for 3 h in the presence of S9. All experiments were conducted with duplicate cultures of cells, after growing exponentially for 48 h from culture initiation. Cytochalasin B was added after termination of treatment in Experiments 1 and 3, and added with treatment in Experiment 2. Cell harvesting was done 72 h after culture initiation, slides were prepared, and the cells were stained with 5% Giemsa. Subsequently, microscopic examination of 2000 cells was performed to determine the percent incidence of micronuclei in binucleated cells (BNCs). Under the study conditions, the test substance did not induce a biologically significant, dose-related increase in the percent incidence of micronuclei in binucleated cells (BNCs) at any dose in any of the three experiments, either in the absence or presence of S9 for mammalian metabolism. In addition, the test substance did not produce cytotoxicity as assessed by the cytokinesis-block proliferation index (CBPI) in either the preliminary cytotoxicity screen or in the three experiments as described above (Nestmann, 2016).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
February 17, 2014 to March 7, 2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across study
Justification for type of information:
Refer to the section 13 for details on the read across justification.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes (S9 homogenate)
Test concentrations with justification for top dose:
Range finding test in strain TA100 and E.Coli WP2 uvrA with and without metabolic activation: Positive control; Solvent control; 3; 10; 33; 100; 333; 1000; 3330; 5000 ug/L

Experiment 1: Tester trains TA1535, TA1537, TA98 with and without metabolic activation: Positive control; Solvent control; 3; 10; 33; 100; 333; 1000 ug/L.

Experiment 2: Tester trains TA1535, TA1537, TA98, TA100: Positive control; Solvent control; 3; 10; 33; 100; 333; 1000 ug/L. Tester strain E.Coli WP2 uvrA: Positive control; Solvent control; 3; 10; 33; 100; 333; 1000; 3330 and 5000 ug/L
Vehicle / solvent:
Ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
Negative solvent / vehicle controls:
yes
Remarks:
Saline
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
sodium azide
Remarks:
TA1535; concentration/plate 5µg
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
other: ICR-191
Remarks:
TA1537; concentration/plate 2.5µg
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
2-nitrofluorene
Remarks:
TA98; concentration/plate 10µg
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
methylmethanesulfonate
Remarks:
TA100; concentration/plate 650µg
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
without metabolic activation
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
WP2 uvrA; concentration/plate 15µg
Positive controls:
yes
Remarks:
with metabolic activation and solvent DMSO
Positive control substance:
other: 2-aminoanthracene
Remarks:
TA1535 2.5µg S9-mix 5 and 10%; TA1537 2.5µg S9-mix 5%; TA1537 5µg S9-mix 10%; TA98 1µg S9-mix 5 and10%; TA100 1µg S9-mix 5% ; TA100 2µg S9-mix 10% ; W2P uvrA 15µg S9-mix 5 and 10%
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)
NUMBER OF REPLICATIONS: Triplicates
DETERMINATION OF CYTOTOXICITY: Reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.
Evaluation criteria:
A Salmonella typhimurium reverse mutation assay and/or Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:

a)The negative control data (number of spontaneous revertants per plate) should be within the laboratory historical range for each tester strain

b)The positive control chemicals should produce responses in all tester strains, which are within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be at least three times the concurrent vehicle control group mean c)The selected dose range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extendto 5 mg/plate.

No formal hypothesis testing was done.

A test substance is considered negative (not mutagenic) in the test if:

a)The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2 uvrA is not greater than three (3) times the concurrent vehicle control.

b)The negative response should be reproducible in at least one independently repeated experiment.


A test substance is considered positive (mutagenic) in the test if:

a)The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2 uvrA is greater than three (3) times the concurrent vehicle control.

b)In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.


Key result
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:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested

Dose range finding test/Experiment 1 The test substance was tested in the tester strains TA100 and WP2 uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the test substance was tested in the first mutation assay at a concentration range of 3 to 666 µg/plate in the absence of S9-mix and at a concentration range of 10 to 1000 µg/plate in the presence of 5% (v/v) S9-mix in the tester strains TA1535, TA1537 and TA98. The results are shown in Table 3 and Table 4 of the study report.  Precipitate Dose range finding test: Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at concentrations of 3330 and 5000 µg/plate. First mutation experiment: Precipitation of the test substance on the plates was observed at the start of the incubation period at concentrations of 666 and 1000 µg/plate and no precipitate was observed at the end of the incubation period. Except in tester strain TA98 where no precipitate was observed at the start or at the end of the incubation period. Toxicity To determine the toxicity of the test substance, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.   In tester strain WP2 uvrA, the bacterial background lawn was not reduced at any of the concentrations tested. No biologically relevant decrease in the number of revertants was observed up to the dose level of 3330 μg/plate. Since the test substance precipitated heavily on the plates at the test substance concentration of 5000 μg/plate, the number of revertants of this dose level could not be determined. The reduction of the bacterial background lawn and the reduction in the number of revertants in the other tester strains are presented in Table 1.

Table1: Toxicity of Amides, C8-18(even numbered) and C18 unsatd., N-(hydroxyethyl) in the dose range finding/first experiment

(Reduction of the bacterial background lawn and in the number of revertant colonies)

Strain

Without S9-mix

With S9-mix

                 Dose          Bacterial              Revertant

                (μg/plate)    background lawn  colonies

Dose       Bacterial                Revertant

(µg/plate) background lawn  colonies

TA1535

333          moderate                  -2

666          extreme          microcolonies

 333          slight                         -1

 666          slight                         -2

1000        moderate                   -2

TA1537

333          moderate                  -1

666          extreme          microcolonies

 333          slight                         -1

 666          slight                         -2

1000        moderate             extreme

TA98

666           normal               extreme

1000         normal                       -2

TA100

 333         slight                 moderate

1000         extreme          microcolonies

3330         absent              complete

5000         absent                      -3

1000         moderate          extreme

3330         absent             complete

5000         absent                      -3

-1  No reduction in the number of revertant colonies

-2  Reduction in the number of revertant colonies, but not less than the minimal value of the historical control data range.

-3  Due to the amount of precipitate no colony determination was possible

Experiment 2 To obtain more information about the possible mutagenicity of the test substance, a second mutation experiment was performed in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the following dose range was selected for the second mutation assay: TA1535, TA1537, TA98, TA100: Without S9-mix: 3, 10, 33, 100, 333 and 666 μg/plate and with S9-mix: 10, 33, 100, 333, and 1000 μg/plate WP2 uvrA: Without and with S9-mix: 10, 33, 100, 333, 1000 and 3300 µg/plate The results are shown in Table 5 of the study report. Precipitate Precipitation of the test substance on the plates was only observed in tester strain WP2 uvrA at the start of the incubation period at concentrations of 1000 and 3330 µg/plate in the absence of S9-mix and at 3330 µg/plate in the presence of S9-mix. At the end of the incubation period, precipitation on the plates was only observed at 3330 µg/plate in the absence of S9-mix. Toxicity In tester strain WP2 uvrA, no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. The reduction of the bacterial background lawn and the reduction in the number of revertants in the other tester strains is presented in Table 2. Table2: Toxicity of the test substance in second experiment (Reduction of the bacterial background lawn and in the number of revertant colonies)

Strain

Without S9-mix

With S9-mix

                 Dose          Bacterial              Revertant

                (μg/plate)    background lawn  colonies

Dose       Bacterial                Revertant

(µg/plate) background lawn  colonies

TA1535

333          moderate                  -2

666          extreme             absent

1000        moderate                   -1

TA1537

333          slight                  moderate

666          extreme           microcolonies

1000        normal                       -2

TA98

666           moderate          extreme

1000         normal             moderate

TA100

 100          slight                 moderate

 333         moderate           extreme

1000         extreme          microcolonies

 333         normal             moderate

 666         moderate         extreme

1000         moderate         extreme

-1  No reduction in the number of revertant colonies

-2  Reduction in the number of revertant colonies, but not less than the minimal value of the historical control data range.

Experiment 3 In the first experiment in tester strain TA98 and in the second experiment in the tester strains TA1537 and WP2 uvrA no toxicity or precipitate on the plates was observed in the presence of S9-mix. Therefore a third mutation experiment was performed with these strains and tester strain TA98 in the presence of S9-mix at a concentration range of 333 to 5000 µg/plate. The results are shown in Table 6 of the study report.   Precipitate Precipitation of the test substance on the plates was observed at the start and at the end of the incubation period at concentrations of 3330 and 5000 µg/plate. Toxicity Due to precipitate of the test substance on the plates the bacterial background could not be determined at the dose levels of 3330 and 5000 μg/plate, except at tester strain WP2 uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA98 in the presence of 5 and 10 %(v/v) S9-mix. Mutagenicity In the third mutation assay, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

Conclusions:
Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay.
Executive summary:

A study was conducted to determine the mutagenic potential of the read across substance, amides, C8-18 (even numbered) and C18-unsatd., N-(hydroxyethyl), according to OECD Guideline 471, in compliance with GLP. The substance was tested in the Salmonella typhimuriumreverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2 uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor). An additional experiment was performed with the strains TA1537, TA98 and WP2 uvrA in the presence of S9-mix. In the dose range finding test, the substance was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2 uvrA. The substance precipitated on the plates at dose levels of 3330 and 5000 μg/plate. In tester strain TA100, toxicity was observed at dose levels of 333 μg/plate and upwards in the absence of S9-mix and at dose levels of 1000 μg/plate and upwards in the presence of S9-mix. In tester strain WP2 uvrA, the bacterial background lawn was not reduced at any of the concentrations tested. No biologically relevant decrease in the number of revertants was observed up to the dose level of 3330 μg/plate. Since the test substance precipitated heavily on the plates at the test substance concentration of 5000 μg/plate, the number of revertant colonies of this dose level could not be determined. Based on the results of the dose range finding test, the substance was tested in the first mutation assay at a concentration range of 3 to 666 μg/plate in the absence of S9-mix and at a concentration range of 10 to 1000 μg/plate in the presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. The test substance did not precipitate on the plates at this dose level. Toxicity was observed in all tester strains, except in TA98 in the presence of S9-mix. In an independent repeat of the assay with additional parameters, the substance was tested at a concentration range of 3 to 666 μg/plate in the absence of S9-mix and at a concentration range of 10 to 1000 μg/plate in the presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98 and TA100 and at 10 to 3330 μg/plate in tester strain WP2 uvrA in the absence and presence of 10% (v/v) S9-mix. Precipitate on the plates was only observed at the dose level of 3330 μg/plate in the absence of S9-mix. Toxicity was observed in all tester strains, except in TA1537 and TA98 in the presence of S9-mix and in WP2 uvrA in the absence and presence of S9-mix. Since in the first experiment in tester strain TA98 and in the second experiment in the tester strains TA1537, TA98 and WP2 uvrA no toxicity or precipitate on the plates was observed, a third mutation experiment was performed with these strains in the presence of S9-mix (5% %(v/v) S9-mix and 10 %(v/v) S9-mix, for experiment 1 and 2, respectively). The substance was tested up to 5000 μg/plate. The test substance precipitated on the plates at dose levels of 3330 and 5000 μg/plate. Due to the precipitate of the test substance on the plates the bacterial background could not be determined at the dose levels of 3330 and 5000 μg/plate, except at tester strain WP2 uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA98 in the presence of 5 and 10 %(v/v) S9-mix. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2 uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay (Verspeek-Rip, 2014).

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
Not reported
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Read across study
Justification for type of information:
Refer to the section 13 for details on the read across justification.
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: Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) (Invitrogen Corporation) containing penicillin/streptomycin (50 U/mL and 50 µg/mL, respectively) , 1 mM sodium pyruvate and 2 mM L-glutamin.
Growth medium: Basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
Exposure medium: For 3 h exposure cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat inactivated horse serum (R5-medium) and for 24 h exposure: Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat inactivated horse serum (R10-medium).
Selective medium: Selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20) and 5 µg/mL trifluorothymidine (TFT).
Non-selective medium: Non-selective medium consisted of basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
First mutagenicity test:
Without S9-mix: 0.3, 1, 3, 10, 30, 40, 50, 60, 70, 80, 90 and 100 µg/mL exposure medium
With 8% (v/v) S9-mix: 0.3, 1, 3, 10, 30, 50, 100, 150, 200, 250, 300 and 350 µg/mL exposure medium
Second mutagenicity test:
Without S9-mix: 0.3,1,3,10,15,20,22.5,25,27.5,30,32.5,35,40,45 and 50 µg/mL exposure medium
With 12% (v/v) S9-mix: 0.3, 1, 3, 10, 30, 50, 100, 125, 150, 175, 200, 225 and 250 µg/mL exposure medium
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without metabolic activation: 15 and 5 µg/mL for a 3 and 24 h treatment period
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation: 7.5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 3h (Experiment 1), 24 and 48 h (Experiment 2 without S9 mix) and 3h (Experiment 2 with S9 mix)
- Expression time (cells in growth medium): For expression of the mutant phenotype, the remaining cells were cultured for 2 d after the treatment period. During this culture period at least 4 x 106 cells (if possible) were subcultured every day in order to maintain log phase growth. Two days after the end of the treatment with the test substance the cells were plated for determination of the cloning efficiency (CE day 2) and the mutation frequency (MF).


SELECTION AGENT (mutation assays): Trifluorothymidine 0.5 mg/mL


NUMBER OF REPLICATIONS: Two


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency
Evaluation criteria:
A test substance is considered positive (mutagenic) in the mutation assay if:
a) It induces a MF of more then MF(controls) + 126 in a dose-dependent manner; or
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
An observed increase should be biologically relevant and will be compared with the historical control data range.
A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Statistics:
No data
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the absence of S9-mix, no toxicity in the relative suspension growth was observed up to concentrations of 33 µg/mL compared to the relative suspension growth of the solvent control. No cell survival was observed at test substance concentrations of 100 µg/mL and above. In the presence of S9-mix, no toxicity in the relative suspension growth was observed up to concentrations of 100 µg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test substance concentration of 333 µg/mL.


COMPARISON WITH HISTORICAL CONTROL DATA: The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.


Evaluation of the mutagenicity: No significant increase in the mutation frequency at the TK locus was observed after treatment with Ninol CMF-E either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test material-treated cultures were comparable to the numbers of small and large colonies of the solvent controls.


ADDITIONAL INFORMATION ON CYTOTOXICITY: First mutagenicity test: Evaluation of toxicity: In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 74% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 77% compared to the total growth of the solvent controls.
Second mutagenicity test: In the absence of S9-mix, the relative total growth of the highest test substance was reduced by 95% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 72% compared to the total growth of the solvent controls.

The growth rate over the two-day expression period for cultures treated with DMSO was between 13 and 23 (3 h treatment) and 37 and 38 (24 h treatment). Mutation frequencies in cultures treated with positive control chemicals were increased by 8.2 and 16-fold for MMS in the absence of S9-mix, and by 10- and 13-fold for CP in the presence of S9-mix, in the first and second experiment respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate for the detection of a mutagenic response and that the metabolic activation system (S9-mix) functioned properly. In addition the observed mutation frequencies of the positive control substances were within the acceptability criteria of this assay.

Conclusions:
Under the study conditions, the test substance was not mutagenic in the TK mutation test system both with and without metabolic activation.

Executive summary:

A study was conducted to evaluate the mutagenic potential of the read across substance, amides, C8-18 (even numbered) and C18-unsatd., N-(hydroxyethyl), in L5178Y mouse lymphoma cells according to OECD Guideline 476 and EU method B.17, in compliance with GLP. The test was performed in two independent experiments with L5178Y mouse lymphoma cells, in the absence and presence of S9-mix. In the first experiment, the substance was tested up to concentrations of 60 and 200 µg/mL in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 h. Test material was tested up to cytotoxic levels of 74 and 77% in the absence and presence of S9-mix, respectively. In the second experiment, test material was tested up to concentrations of 45 and 225 µg/mL in the absence and presence of 12% (v/v) S9-mix with incubation times of 24 and 3 h, respectively. The substance was tested up to the cytotoxic level of 95% (absence of S9-mix) and up to 72% (presence of S9-mix), but failed to induce a significant increase in the frequency of mutations. The spontaneous mutation frequencies in the solvent-treated control cultures were within historical control data range and therefore within the acceptability criteria of the assay. Mutation frequencies in positive control cultures were elevated 8.2- and 16-fold for MMS (absence of S9-mix), and 10- and 13-fold for CP (presence of S9-mix). Negative results were confirmed in an independent repeat experiment with extended exposures. Under the study conditions, the test substance was not mutagenic in the TK mutation test system both with and without metabolic activation (Verspeek-Rip, 2009).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 97
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Species / strain / cell type:
S. typhimurium TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
The test substance was found to be uniformly suspended in Pluronic F68 at the concentration of 50 mg/mL. It was shown to precipitate when incorporated into agar plates at the levels of 4000 and 5000 but not at 3000 μg/plate, chosen as the highest test dose as recommended by OECD (1997). The doses of 3000, 2000, 1000, 500, and 250 μg/plate were selected to evaluate toxicity in strain TA100 with the presence and absence of S9. Slight cytotoxicity was found at 3000 μg/plate (-S9), the maximum dose for both the plate incorporation test and preincubation assay.
Main test selected range was: 30, 90, 300, 900, and 3000 μg/plate.
Untreated negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene, 2-aminofluorene, danthron
Details on test system and experimental conditions:
The tester strains were obtained from Moltox of Boone, North Carolina. Their identity and characteristics were verified prior to testing.

Media:
Test strains were grown overnight in Oxoid nutrient broth No. 2, combined with test chemical and S9 in soft overlay agar containing 0.6 % agar, 0.5 % NaCl, and 0.05 mmol/L histidine–biotin, then plated on agar (2 %) containing Vogel–Bonner minimal medium E and 2 % glucose, as previously described.

Test method:
Both plate incorporation and preincubation methods were employed, both in the absence and presence of S9. For preincubation, the treatment mixtures were incubated at 37 °C for 20 min prior to plating. Test plates in triplicate were incubated at 37°C for 68–72 h at which time revertant colonies were counted and the plates were examined for extent of background growth as an indicator of cytotoxicity.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 97
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Results from triplicate cultures in Experiment 1 using the plate incorporation test, and in Experiment 2 using the preincubation assay show that the test substance did not induce a dose-dependent or 2-fold increase in revertant colonies at doses of 30, 90, 300, 900, and 3000 μg/plate, without and with S9 for metabolic activation. Neither were there any cytotoxic effects. All negative and positive control values were within expected and normal ranges.
Conclusions:
Under the study conditions, the test substance was not mutagenic in the Ames test.
Executive summary:

A study was conducted to determine the mutagenic potential of the test substance according to a study design based on OECD Guideline 471, in compliance with GLP. The test substance was examined using five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in three experiments in the presence and absence of S9-mix (rat liver). The test substance was found to be uniformly suspended in Pluronic F68 at the concentration of 50 mg/mL. It was shown to precipitate when incorporated into agar plates at the levels of 4000 and 5000 but not at 3000 μg/plate, chosen as the highest test dose. The doses of 3000, 2000, 1000, 500, and 250 μg/plate were selected to evaluate toxicity in strain TA100 with the presence and absence of S9. Slight cytotoxicity was found at 3000 μg/plate (-S9). This concentration level was established to be the maximum dose tested in both the plate incorporation test and preincubation assay. Selected range for both assays was: 30, 90, 300, 900, and 3000 μg/plate. The treatment mixtures were incubated at 37°C for 20 min prior to plating. Test plates in triplicate were incubated at 37°C for 68–72 h. After incubation time revertant colonies were counted and the plates were examined for extent of background growth as an indicator of cytotoxicity. Negative, including blank sterility controls, and positive controls were included in the testing. Results show that the test substance did not induce a dose-dependent or 2-fold increase in revertant colonies at the top dose level of 3000 μg/plate, without and with S9 for metabolic activation. Neither were there any cytotoxic effects. All negative and positive control values were within expected and normal ranges. Under the study conditions, the substance was not mutagenic in the Ames test (Nestmann, 2017).

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Genetic toxicity in vitro, Ames test

A study was conducted to determine the mutagenic potential of the test substance according to a study design based on OECD Guideline 471, in compliance with GLP. The test substance was examined using five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in three experiments in the presence and absence of S9-mix (rat liver). The test substance was found to be uniformly suspended in Pluronic F68 at the concentration of 50 mg/mL. It was shown to precipitate when incorporated into agar plates at the levels of 4000 and 5000 but not at 3000 μg/plate, chosen as the highest test dose. The doses of 3000, 2000, 1000, 500, and 250 μg/plate were selected to evaluate toxicity in strain TA100 with the presence and absence of S9. Slight cytotoxicity was found at 3000 μg/plate (-S9). This concentration level was established to be the maximum dose tested in both the plate incorporation test and preincubation assay. Selected range for both assays was: 30, 90, 300, 900, and 3000 μg/plate. The treatment mixtures were incubated at 37°C for 20 min prior to plating. Test plates in triplicate were incubated at 37°C for 68–72 h. After incubation time revertant colonies were counted and the plates were examined for extent of background growth as an indicator of cytotoxicity. Negative, including blank sterility controls, and positive controls were included in the testing. Results show that the test substance did not induce a dose-dependent or 2-fold increase in revertant colonies at the top dose level of 3000 μg/plate, without and with S9 for metabolic activation. Neither were there any cytotoxic effects. All negative and positive control values were within expected and normal ranges. Under the study conditions, the substance was not mutagenic in the Ames test (Nestmann, 2017).

 

Read across study, Ames test

A study was conducted to determine the mutagenic potential of the read across substance, amides, C8-18 (even numbered) and C18-unsatd., N-(hydroxyethyl), according to OECD Guideline 471, in compliance with GLP. The substance was tested in the Salmonella typhimurium reverse mutation assay with four histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2 uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by Aroclor). An additional experiment was performed with the strains TA1537, TA98 and WP2 uvrA in the presence of S9-mix. In the dose range finding test, the substance was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2 uvrA. The substance precipitated on the plates at dose levels of 3330 and 5000 μg/plate. In tester strain TA100, toxicity was observed at dose levels of 333 μg/plate and upwards in the absence of S9-mix and at dose levels of 1000 μg/plate and upwards in the presence of S9-mix. In tester strain WP2 uvrA, the bacterial background lawn was not reduced at any of the concentrations tested. No biologically relevant decrease in the number of revertants was observed up to the dose level of 3330 μg/plate. Since the test substance precipitated heavily on the plates at the test substance concentration of 5000 μg/plate, the number of revertant colonies of this dose level could not be determined. Based on the results of the dose range finding test, the substance was tested in the first mutation assay at a concentration range of 3 to 666 μg/plate in the absence of S9-mix and at a concentration range of 10 to 1000 μg/plate in the presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. The test substance did not precipitate on the plates at this dose level. Toxicity was observed in all tester strains, except in TA98 in the presence of S9-mix. In an independent repeat of the assay with additional parameters, the substance was tested at a concentration range of 3 to 666 μg/plate in the absence of S9-mix and at a concentration range of 10 to 1000 μg/plate in the presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98 and TA100 and at 10 to 3330 μg/plate in tester strain WP2 uvrA in the absence and presence of 10% (v/v) S9-mix. Precipitate on the plates was only observed at the dose level of 3330 μg/plate in the absence of S9-mix. Toxicity was observed in all tester strains, except in TA1537 and TA98 in the presence of S9-mix and in WP2 uvrA in the absence and presence of S9-mix. Since in the first experiment in tester strain TA98 and in the second experiment in the tester strains TA1537, TA98 and WP2 uvrA no toxicity or precipitate on the plates was observed, a third mutation experiment was performed with these strains in the presence of S9-mix (5%%(v/v) S9-mix and 10%(v/v) S9-mix, for experiment 1 and 2, respectively). The substance was tested up to 5000 μg/plate. The test substance precipitated on the plates at dose levels of 3330 and 5000 μg/plate. Due to the precipitate of the test substance on the plates the bacterial background could not be determined at the dose levels of 3330 and 5000 μg/plate, except at tester strain WP2 uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in tester strain TA98 in the presence of 5 and 10% (v/v) S9-mix. The substance did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2 uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, the test substance was not mutagenic in the Salmonella typhimurium and Escherichia coli reverse mutation assay (Verspeek-Rip, 2014).

 

Read across study, mouse lymphoma assay

A study was conducted to evaluate the mutagenic potential of the read across substance, amides, C8-18 (even numbered) and C18-unsatd., N-(hydroxyethyl), in L5178Y mouse lymphoma cells according to OECD Guideline 476 and EU method B.17, in compliance with GLP. The test was performed in two independent experiments with L5178Y mouse lymphoma cells, in the absence and presence of S9-mix. In the first experiment, the substance was tested up to concentrations of 60 and 200 µg/mL in the absence and presence of 8% (v/v) S9-mix. The incubation time was 3 h. Test material was tested up to cytotoxic levels of 74 and 77% in the absence and presence of S9-mix, respectively. In the second experiment, test material was tested up to concentrations of 45 and 225 µg/mL in the absence and presence of 12% (v/v) S9-mix with incubation times of 24 and 3 h, respectively. The substance was tested up to the cytotoxic level of 95% (absence of S9-mix) and up to 72% (presence of S9-mix), but failed to induce a significant increase in the frequency of mutations. The spontaneous mutation frequencies in the solvent-treated control cultures were within historical control data range and therefore within the acceptability criteria of the assay. Mutation frequencies in positive control cultures were elevated 8.2- and 16-fold for MMS (absence of S9-mix), and 10- and 13-fold for CP (presence of S9-mix). Negative results were confirmed in an independent repeat experiment with extended exposures. Under the study conditions, the test substance was not mutagenic in the TK mutation test system both with and without metabolic activation (Verspeek-Rip, 2009).

 

Genetic toxicityin vitro, mammalian cells

A study was conducted to determine the potential of the test substance to induce mutations in human lymphocytes according to in vitro mammalian cell micronucleus test OECD Guideline 487, in compliance with GLP. In the preliminary range-finding test the test substance was found to be sufficiently suspended in Pluronic F68 at the concentration of 1000 μg/mL, with minimum precipitation and no cytotoxicity as measured by the cytokinesis-block proliferation index (CBPI). Based on these results, the tests were performed with 250, 500, and 1000 μg/mL. Experiments 1 and 2 were conducted in the absence of S9, with exposure of the cells to the test substance for 3 h and 24 h, respectively. Experiment 3 involved exposure for 3 h in the presence of S9. All experiments were conducted with duplicate cultures of cells, after growing exponentially for 48 h from culture initiation. Cytochalasin B was added after termination of treatment in Experiments 1 and 3, and added with treatment in Experiment 2. Cell harvesting was done 72 h after culture initiation, slides were prepared, and the cells were stained with 5% Giemsa. Subsequently, microscopic examination of 2000 cells was performed to determine the percent incidence of micronuclei in binucleated cells (BNCs). Under the study conditions, the test substance did not induce a biologically significant, dose-related increase in the percent incidence of micronuclei in binucleated cells (BNCs) at any dose in any of the three experiments, either in the absence or presence of S9 for mammalian metabolism. In addition, the test substance did not produce cytotoxicity as assessed by the cytokinesis-block proliferation index (CBPI) in either the preliminary cytotoxicity screen or in the three experiments as described above (Nestmann, 2016).

Justification for classification or non-classification

Based on the results of in vitro testing, the test substance does not require classification for mutagenicity according to CLP (EC 1272/2008) criteria.