N-(2-hydroxyethyl)stearamide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Not reported

Deviations:

not applicable

Principles of method if other than guideline:

The study was conducted according to 'Mutation Research 31, 347 - 364 (1975)'.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Not reported

Species / strain / cell type:

other: TA 1535, TA 100, TA 1537, TA 1538 and TA 98

Details on mammalian cell type (if applicable):

Type and identity of media: Agar plates

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Postmitochondrial supernatant fluids from the liver of male rats treated with Aroclor 1254 (S-9 mix)

Test concentrations with justification for top dose:

4, 20, 100, 500 and 2500 µg/plate test material per plate

Vehicle / solvent:

No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

None

Details on test system and experimental conditions:

No data

Evaluation criteria:

No data

Statistics:

Not reported

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Conclusions:

Under the study conditions, test susbtance was considered to be non-mutagenic in an in vitro Ames test.

Executive summary:

A study was conducted to evaluate the in vitro genotoxic toxicity of read across substance, C12-18 and C18-unsatd. MEA, in the Ames test. Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the read across substance using the Ames plate incorporation method at up to five dose levels (4, 20, 100, 500 and 2500 µg/plate test material per plate) both with and without metabolic activation. Under the test conditions, the test substance was found to be non-mutagenic (Wallat, 1981).

Reference 2

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:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

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 µg/L

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

Experiment 2: Tester trains TA1535, TA1537, TA98, TA100: Positive control; Solvent control; 3; 10; 33; 100; 333; 1000 µg/L. Tester strain E.Coli WP2 uvrA: Positive control; Solvent control; 3; 10; 33; 100; 333; 1000; 3330 and 5000 µg/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 5 µg

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

other: ICR-191

Remarks:

TA1537; concentration/plate 2.5 µg

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

2-nitrofluorene

Remarks:

TA98; concentration/plate 10 µg

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Remarks:

without metabolic activation

Positive control substance:

methylmethanesulfonate

Remarks:

TA100; concentration/plate 650 µg

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 15 µg

Positive controls:

yes

Remarks:

with metabolic activation and solvent DMSO

Positive control substance:

other: 2-aminoanthracene

Remarks:

TA1535 2.5 µg S9-mix 5 and 10%; TA1537 2.5 µg S9-mix 5%; TA1537 5 µg S9-mix 10%; TA98 1 µg S9-mix 5 and10%; TA100 1 µg S9-mix 5% ; TA100 2 µg S9-mix 10% ; W2P uvrA 15 µg 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 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: 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.

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:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: 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.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: 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.

Vehicle controls validity:

valid

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:

other: 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.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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.

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.

Table 1: 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.

Table 2: 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.

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 evaluate the in vitro genetic toxicity of the read across substance, C8-18 and C18-unsatd. MEA, 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 read across substance precipitated heavily on the plates at the read across 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 read across 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 read across substance precipitated on the plates at dose levels of 3330 and 5000 μg/plate. Due to the precipitate of the read across 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).

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration 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:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/mL respectively) and 30 U/mL heparin.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

S9 was prepared after inducing metabolizing enzymes by injection of rats with phenobarbitone and β-naphthoflavone.

Test concentrations with justification for top dose:

Dose range finding study:
- At 3 h exposure time: 3, 10, 33, 100 and 333 µg/mL culture medium with and without S9-mix.
- At 24 and 48 h continuous exposure time: 3, 10, 33, 100, 333 and 1000 µg/mL culture medium without S9-mix

Experiment 1 (First cytogenetic assay):
Without and with S9-mix: 33, 100 and 200 µg/mL culture medium (3 h exposure time, 24 h fixation time)

Experiment 2 (Second cytogenetic assay):
- Without S9-mix: 10, 50, 100, 150, 175, 200, 225, 250, 275 and 300 µg/mL culture medium (24 h exposure time, 24 h fixation time)
10, 50, 75, 100, 125, 150, 175 and 200 µg/mL culture medium (48 h exposure time, 48 h fixation time)
- With S9-mix: 50, 100 and 200 µg/mL culture medium (3 h exposure time, 48 h fixation time)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test material was soluble in DMSO

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with metabolic activation: 10 µg/mL 3 h exposure period (24 h fixation time)

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without metabolic activation: 0.1 µg/mL (48 h exposure), 0.2 (24 h exposure) and 0.5 µg/mL (3 h exposure)

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)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h (Experiment 1), 24 and 48 h (Experiment 2 without S9 mix) and 48 h (Experiment 2 with S9 mix)

SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.5 µg/mL medium)
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: Two

NUMBER OF CELLS EVALUATED: 1000 cells

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
- Determination of endoreplication: Yes

Evaluation criteria:

Evaluation criteria
A test substance was considered clastogenic if:
a) A dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations
b) A significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations in the absence of a clear dose-response relationship

A test substance was considered non-clastogenic if:
a) None of the tested concentrations induced a statistically significant increase in the number of cells with chromosome aberrations.

Statistics:

Statistics
One sided, Chi-square test to calculate dose-related statistically significant increase in the number of cells with chromosome aberrations

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:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
At the 24 and 48 h exposure time, test material was tested beyond the limit of solubility to obtain adequate toxicity data. - Precipitate of the test material was seen at 333 µg/mL

COMPARISON WITH HISTORICAL CONTROL DATA:
Yes, test data were within the laboratory historical control data range

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Increased number of polyploid cells in the absence of S9-mix in a dose dependent manner in the first cytogenetic assay indicating potential to inhibit mitotic processes and to induce numerical chromosome aberrations.

The doses selected for scoring of chromosome aberrations:

Without S9-mix: 50, 100 and 150 µg/mL culture medium (24 h exposure time, 24 h fixation time).

50, 100 and 125 µg/mL culture medium (48 h exposure time, 48 h fixation time)

With S9-mix:50, 100 and 200 µg/mL culture medium (3 h exposure time, 48 h fixation time)

Conclusions:

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

Executive summary:

A study was conducted to evaluate the in vitro genetic toxicity of the read across substance, C8-18 and C18-unsatd. MEA, according to OECD Guideline 473 and EU Method B. 10, in compliance with GLP. Peripheral human lymphocytes were treated with the read across substance (experiment 1: 33, 100 and 200 µg/mL without and with S9-mix; experiment 2: 10 – 300 µg/mL without S9-mix, 50, 100 and 200 µg/mL with S9-mix) for either 3, 24 or 48 h. The frequency of cells with aberrations in the vehicle control group was within the historical control data range. Both of the positive control substances induced significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The read across substance did not induce any significant or biologically relevant increases in the frequency of cells with chromosome aberrations in the presence or absence of metabolic activation, in either independent repeat experiment. No effects on the number of polyploid cells were observed both in the absence and presence of S9-mix. The substance did not disturb the mitotic processes, cell cycle progression and did not induce numerical chromosome aberrations. Under the study conditions, the test substance was considered to be non-clastogenic in cultured human lymphocytes in vitro (Verspeek-Rip, 2009).

Reference 4

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:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the category justification.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 was prepared after inducing metabolizing enzymes by injection of rats with phenobarbitone and β-naphthoflavone.

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 Migrated to IUCLID6: 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 Migrated to IUCLID6: 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 in vitro genetic toxicity of the read across substance, C8-18 and C18-unsatd. MEA, 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 conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In vitro

A study was conducted to evaluate the in vitro genetic toxicity of the read across substance, C8-18 and C18-unsatd. MEA, 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 read across substance precipitated heavily on the plates at the read across 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 read across 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 read across substance precipitated on the plates at dose levels of 3330 and 5000 μg/plate. Due to the precipitate of the read across 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).

A study was conducted to evaluate the in vitro genotoxic toxicity of read across substance, C12-18 and C18-unsatd. MEA, in the Ames test. Salmonella typhimurium strains TA1535, TA1537, TA1538, TA98 and TA100 were treated with the read across substance using the Ames plate incorporation method at up to five dose levels (4, 20, 100, 500 and 2500 µg/plate test material per plate) both with and without metabolic activation. Under the test conditions, the test substance was found to be non-mutagenic (Wallat, 1981). Based on the results of the read across study, the test substance is also expected to have similar mutagenicity potential in an in vitro Ames test.

A study was conducted to evaluate the in vitro genetic toxicity of the read across substance, C8-18 and C18-unsatd. MEA, according to OECD Guideline 473 and EU Method B. 10, in compliance with GLP. Peripheral human lymphocytes were treated with the read across substance (experiment 1: 33, 100 and 200 µg/mL without and with S9-mix; experiment 2: 10 – 300 µg/mL without S9-mix, 50, 100 and 200 µg/mL with S9-mix) for either 3, 24 or 48 h. The frequency of cells with aberrations in the vehicle control group was within the historical control data range. Both of the positive control substances induced significant increases in the frequency of aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The read across substance did not induce any significant or biologically relevant increases in the frequency of cells with chromosome aberrations in the presence or absence of metabolic activation, in either independent repeat experiment. No effects on the number of polyploid cells were observed both in the absence and presence of S9-mix. The substance did not disturb the mitotic processes, cell cycle progression and did not induce numerical chromosome aberrations. Under the study conditions, the test substance was considered to be non-clastogenic in cultured human lymphocytes in vitro (Verspeek-Rip, 2009).

A study was conducted to evaluate the in vitro genetic toxicity of the read across substance, C8-18 and C18-unsatd. MEA, 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).

Also, after discussion with ECHA in the frame of a Dossier Improvement Action Plan (DIAP), in vitro genotoxicity testing according to OECD Guidelines 471, 473 and 476 are planned with C16-18 MEA in order to obtain information on the substance itself and further support the read across approach proposed for the FAA category members. 

Justification for classification or non-classification

Based on the results of in vitro testing on the read across substances, C12-18 and C18-unsatd. MEA and C8-18 and C18-unsatd. MEA, no classification for genetic toxicity is warranted according to CLP (EC 1272/2008) criteria.