Reaction mass of: N,N'-[1,3-Phenylenebis(methylene)bis[12-hydroxyoctadecanamide], N,N'-hexane-1,6-diylbis(12-hydroxyoctadecanamide) and N,N'-ethane-1,2-diylbis(12-hydroxyoctadecan-1-amide)

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The substance consists of three structurally similar bisamides: N,N'-ethane-1,2-diylbis(12-hydroxyoctadecanamide), N,N'-hexane-1,6-diylbis(12-hydroxyoctadecanamide) and N,N'-[1,3-phenylenebis(methylene)]bis(12-hydroxyoctadecanamide). Experimental data on this substance are not available. The individual constituents and mixtures of them and similar amides are considered suitable read-across substances. In guideline studies on two read-across substances, 12-hydroxyoctadecanoic acid, reaction products with 1,3-benzenedimethanamine and hexamethylenediamine and 1,3-bis[12-hydroxy-octadecamide-N-methylene]-benzene, no mutagenicity was observed in Bacterial Reverse Mutation Assays as well as in chromosome aberration studies. The Bacterial Reverse Mutation Assay of another read-across substance, N,N'-ethane-1,2-diylbis(12-hydroxyoctadecanamide), was also negative. Due to the high structural similarity of the three-constituent substance to these read-across substances the genetic toxicity is expected to be comparable.

Based on the experimental data on read-across substances, the substance does not need to be classified for genetic toxicity according to CLP criteria. Additional testing of the substance is not considered necessary, in vitro as well as in vivo.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 . Dec. 2018 - 11 Apr. 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: Salmonella typhimurium LT2 Strains TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Exp. I: 50, 150, 500, 1500 and 5000 μg/plate (with and without metabolic activation)
Exp. II: 156, 313, 625, 1250, 2500 and 5000 μg/plate (with and without metabolic activation)

Vehicle / solvent:

In a non-GLP pre-test, the solubility of the test item was tested in a concentration of 50 g/L in demineralized water, dimethyl sulfoxide (DMSO), tetrahydrofuran and acetone. The test item is not sufficiently soluble in all solvents. Based on the non-GLP pre-test, acetone was chosen as vehicle, because it showed the best suspension and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants in the tested concentrations.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-Nitro-1,2-phenylene diamine; CAS-No.: 99-56-9

Remarks:

without metabolic activation

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

other: 2-aminoanthracene

Remarks:

with metabolic activation

Evaluation criteria:

The colonies were counted visually and the numbers were recorded. A validated spreadsheet software (Microsoft Excel®) was used to calculate mean values and standard deviations of each treatment, solvent control and positive control. The mean values and standard deviations of each threefold determination was calculated as well as the increase factor f(l) of revertant induction (mean revertants divided by mean spontaneous revertants) of the test item solutions and the positive controls. Additionally, the absolute number of revertants (Rev. Abs.) (mean revertants less mean spontaneous revertants) was given. A substance is considered to have mutagenic potential, if a reproducible increase of revertant colonies per plate exceeding an increase factor of 2 in at least one strain can be observed. A concentration-related increase over the range tested is also taken as a sign of mutagenic activity.

Key result

Species / strain:

other: Salmonella typhimurium LT2 Strains TA97a, TA98, TA100, TA102 and TA1535

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:

All validity criteria are fulfilled. The study can be considered valid. Based on the results of this study it is concluded that Bisamid is not mutagenic in the Salmonella typhimurium strains TA97a, TA98, TA100, TA102 and TA1535 in the absence and presence of metabolic activation under the experimental conditions in this study.

Executive summary:

Three valid experiments were performed. The study procedures described in this report were based on the most recent OECD and EC guidelines. The test item Bisamid was tested in the Salmonella typhimurium reverse mutation assay with five strains of Salmonella typhimurium (TA97a, TA98, TA100, TA102 and TA1535). The test was performed in three experiments in the presence and absence of metabolic activation, with +S9 standing for presence of metabolic activation, and –S9 standing for absence of metabolic activation. Experiment 1a: In the experiment 1a, the test item (suspended in acetone) was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA97a, TA98, TA100, TA102 and TA1535 using the plate incorporation method. The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that none of the tested concentrations showed a significant increase in the number of revertants in all tested strains, in the presence and the absence of metabolic activation. Due to contamination of bacteria strain TA97a, the evaluation of the bacteria strain TA97a was not possible, therefore a repetition of the experiment for this bacteria strain was performed (Experiment 1b). Experiment 1b: Based on the contamination of the bacteria strain TA97a in experiment 1a, the experiment was repeated under the same conditions with this bacteria strain. The test item (suspended in acetone) was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix for the bacteria strain TA97a. The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiment showed that none of the tested concentrations showed an increase in the number of revertants in the tested strain, in the presence and the absence of metabolic activation.

Experiment 2: Based on the first experiments, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in all bacteria strains using the pre-incubation method. The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was not reduced at any of the concentrations and no relevant decrease in the number of revertants was observed in all bacteria strains. The test item showed no signs of toxicity towards the bacteria strains in both the absence and presence of metabolic activation. The results of this experiments showed that the test item caused no increase in the number of revertants in all bacteria strains compared to the solvent control, in both the absence and presence of metabolic activation. The test item did not induce a dose-related increase in the number of revertant colonies in all strains, in the presence and absence of metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Version / remarks:

EEC Directive 92/69, B14

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

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9-homogenate

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 3 ... 333 μg/plate
Concentration range in the main test (without metabolic activation): 3 ... 333 μg/plate

Concentration of 333 μg/plate of the test substance results in precipitation.

Vehicle / solvent:

Solvent: DMSO

Details on test system and experimental conditions:

Concentration of the test substance resulting in precipitation: 333 μg/plate

Species / strain:

bacteria, other: TA1537, TA1535, TA98, TA100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 5000 µg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

bacteria, other: TA1537, TA1535, TA98, TA100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 5000 µg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

bacteria, other: TA1537, TA1535, TA98, TA100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 333 μg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

bacteria, other: TA1537, TA1535, TA98, TA100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 333 μg/plate

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

Observations: None.

Remarks on result:

other: preliminary test

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Executive summary:

The genetic toxicity of 1,3-bis[12-hydroxy-octadecamide-N-methylene]-benzene is assessed in a guideline study (bacterial reverse mutation assay, Ames test) according to EU Method B.14. Under the conditions of the test, no genetic toxicity is observed on Salmonella typhimurium strains TA 100, TA 1535, TA 1537 and TA 98 in the presence or absence of metabolic activation using Aroclor 1254-induced rats liver S9 homogenates. Therefore, we conclude that the test substance has no mutagenic effects.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

EEC Directive 92/69 B.10

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cytogenicity (B10)

Species / strain / cell type:

lymphocytes: Peripheral human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9-homogenate.

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 1 ... 10 μg/ml
Concentration range in the main test (without metabolic activation): 1 ... 10 μg/ml

At a concentration of 10 μg/ml the test substance precipitated in the culture medium. Therefore, a concentration of 10 μg/ml was used as the highest concentration.

Vehicle / solvent:

DMSO

Details on test system and experimental conditions:

Exposure period (with metabolic activation): 3 h
Exposure period (without metabolic activation): 24 h

Expression time:
With S9 mix 3 h treatment
Without S9 mix 24 and 48 h treatment

Fixation time:
With S9 mix treatment cells were incubated for another 20-22 h (24 h fixation) or 44-46 h (48 h fixation).
Cells treated without S9 were worked up immediately after treatment (24h and 48h fixation time).

Species / strain:

lymphocytes: Peripheral human lymphocytes

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 10 μg/ml

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Species / strain:

lymphocytes: Peripheral human lymphocytes

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 10 μg/ml

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

not specified

Remarks on result:

other: preliminary test

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Executive summary:

The genetic toxicity of 1,3-bis[12-hydroxy-octadecamide-N-methylene]-benzene is assessed in a guideline study (chromosome aberration study in mammalian cells) according to EU Method B.10. Under the conditions of the test, no genetic toxicity is observed on peripheral human lymphocytes in the presence or absence of metabolic activation using Aroclor 1254-induced rats liver S9 homogenates. Therefore, we conclude that the test substance has no mutagenic effects.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Annex V (Ames test)

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 pKM 101

Remarks:

strain CM891

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S-9

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 5 ... 5000 μg/plate
Concentration range in the main test (without metabolic activation): 5 ... 5000 μg/plate

Vehicle / solvent:

Solvent: Purified water with 0.15% agar to assist suspension

Species / strain:

bacteria, other: as specified above

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

bacteria, other: as specified above

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 5000 μg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

Observations: No signs of toxicity were observed towards the tester strains in either mutation test.

Remarks on result:

other: main test

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Executive summary:

The genetic toxicity of 12-hydroxyoctadecanoic acid, reaction products with 1,3-benzenedimethanamine and hexamethylenediamine is assessed in a guideline study (bacterial reverse mutation assay, Ames test) according to Annex V. Under the conditions of the test, the test substance does not exhibit genetic toxicity on Salmonella typhimurium strains TA 100, TA 1535, TA 1537, TA 98 and Escherichia coli strain CM891 in the presence or absence of metabolic activation using Aroclor 1254-induced rats liver S9 homogenates. Therefore, we conclude that the test substance has no mutagenic effects.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Annex V (In vitro cytogenetics)

GLP compliance:

yes

Type of assay:

other: in vitro mammalian cytogenicity (B10)

Species / strain / cell type:

mammalian cell line, other: Human lymphocytes in whole blood culture

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 1250 ... 5000 μg/ml
Concentration range in the main test (with metabolic activation): 2500 ... 5000 μg/ml
Concentration range in the main test (without metabolic activation): 1250 ... 5000 μg/ml
Concentration range in the main test (without metabolic activation): 625 ... 2000 μg/ml

Vehicle / solvent:

Culture medium

Details on test system and experimental conditions:

Exposure period (with metabolic activation): 3 hours
Exposure period (without metabolic activation): 3 hours

Fixation time:
All cultures were harvested after 20 hours.

Species / strain:

mammalian cell line, other: Human lymphocytes in whole blood culture

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

5000 μg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

mammalian cell line, other: Human lymphocytes in whole blood culture

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

2000 µg/ml

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Observations:
In both the absence and presence of S-9 mix, the test substance caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations, at any dose level, when compared with the solvent control, in either test. Positive and negative controls produced appropriate results.

Remarks on result:

other: main test

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Executive summary:

The genetic toxicity of 12-hydroxyoctadecanoic acid, reaction products with 1,3-benzenedimethanamine and hexamethylenediamine is assessed in a guideline study (chromosome aberration study in mammalian cells) according to EU Method B.10. In the presence of metabolic activation (Aroclor 1254 -induced rats liver S9), the test substance does not exhibit genetic toxicity on human lymphocytes in whole blood culture up to 5000 µg/mL. In the absence of metabolic activation, no genetic toxicity is observed for a test substance concentration of up to 2000 µg/mL. Therefore, we conclude that the test substance has no mutagenic effects.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

There are no in vivo genetic toxicity data on the substance and suitable read-across substances available. Nevertheless, the available information from in vitro studies is considered sufficient for assessment. Therefore, testing of the genetic toxicity of the substance in vivo is not necessary.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Predictions of the genetic toxicity of the three individual constituents by the (Q)SAR software tool ChemProp 6.7 were disregarded because the three constituents were outside of the applicability domain of the used models.

Justification for classification or non-classification

Based on the experimental data on read-across substances, the substance does not need to be classified for genetic toxicity according to CLP criteria. Additional testing of the substance is not considered necessary, in vitro as well as in vivo.