N,N'-bis(1,3-dimethylbutylidene)ethylenediamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AMES: negative (+/- S9)

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2021-09-14 to 2021-09-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

updated 2020-06-26

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Salmonella typhimurium: histidine (his);
Escherichia coli WP2 uvrA: (trp)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Remarks:

TA1535, TA100: detection of base pair mutations TA1537, TA98: detection of frame shift mutations Escherichia coli WP2 uvrA: detection of base pair substitutions

Additional strain / cell type characteristics:

other: S.typh: deep rough mutation (rfa) and deletion in the uvrB gene; E.coli: pKM101 plasmid R-factor and deletion in the uvrA gene

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver was provided by Trinova Biochem GmbH
- method of preparation of S9 mix: according to Ames et al. (1975)
- volume of S9 mix and S9 in the final culture medium: 500 µL
- quality controls of S9: yes

Test concentrations with justification for top dose:

5000, 1600, 500, 160, 50, and 16 μg/plate;
Selection of the concentrations was done on the basis of a solubility test and a concentration range finding test. Recommended maximum test concentration of 5000 μg/plate according to OECD TG 471 was used as top dose.

Vehicle / solvent:

- Vehicle used: DMSO (for NPD (4-Nitro-1,2-phenylenediamine), 9AA (9-Aminoacridine), 2AA (2-Aminoanthracene)) and ultrapure water (for SAZ (Sodium azide) and MMS (Methyl methanesulfonate))

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO and ultrapure water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene (2AA); 4-Nitro-1,2-phenylenediamine (NPD)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 10xE9 cells/mL
- Test substance added in agar (plate incorporation) in experiment I and preincubation in experiment II

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period: 20 min (experiment II)
- Exposure duration: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: background growth inhibition

METHODS FOR MEASUREMENTS OF GENOTOXICITY
The colony numbers on the untreated, vehicle and positive controls and the test item treated plates were determined (counted manually, evaluated by unaided eye).

TEST VALIDITY
The tests (initial and confirmatory mutation experiments) are considered to be valid if:
- All of the Salmonella tester strains demonstrate the presence of the deep rough mutation (rfa) and the deletion in the uvrB gene.
- The Salmonella typhimurium TA98 and TA100 tester strains demonstrate the presence of the pKM101 plasmid R-factor.
- The Escherichia coli WP2 uvrA culture demonstrates the deletion in the uvrA gene.
- The bacterial cultures demonstrate the characteristic mean number of spontaneous revertants in the vehicle controls.
- The tester strain culture titer is in the 10E9 cells/mL order.
- The batch of S9 used in this study shows the appropriate biological activity.
- The reference mutagens show the expected increase (at least a 3.0-fold increase) in induced revertant colonies over the mean value of the respective vehicle control.
- There are at least five analysable concentrations (at each tester strain) (a minimum of three non-toxic dose levels is required to evaluate assay data).

Rationale for test conditions:

according to OECD TG 471

Evaluation criteria:

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain Salmonella typhimurium TA100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain Salmonella typhimurium TA98, TA1535, TA1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

Statistics:

According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 1600 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

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

Remarks:

at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not examined

Positive controls validity:

valid

TEST VALIDITY

Valid tests were performed since the tester strains (used in this study) demonstrated the specific phenotype characteristics, agreed with the corresponding historical control data ranges and showed the adequate strain culture titer. Each batch of the S9 fractions used in this test had the appropriate biological activity (according to the provided Certificate) and was active in the applied system (2AA treatments).
The reference mutagen treatments (positive controls) showed the expected, biological relevant increases (at least a 3.0-fold increase) in induced revertant colonies in all experimental phases, in all tester strains. Each of the positive controls showed the expected increase in induced revertant colonies over the mean value of the respective vehicle control in all experimental phases and additionally the number of revertants fell in the corresponding historical control ranges, thereby meeting the criteria for the positive control in all experimental phases, in all tester strains1.
The revertant colony numbers of vehicle control (dimethyl sulfoxide (DMSO)) plates with and without S9 Mix demonstrated the characteristic mean number of spontaneous revertants that was in line with the corresponding historical control data ranges in all experimental phases, in all tester strains.
The revertant colony numbers of the parallel investigated untreated and ultrapure water (ASTM Type I) control plates were in line with the dimethyl sulfoxide (DMSO) control plates in both experimental phases. Nearly all of the slightly higher or lower revertant counts of these controls remained in the corresponding historical control data ranges; however, in the confirmatory mutation test, in the case of Salmonella typhimurium TA100 in water control, the revertant colony numbers were above the corresponding historical control data range (actual value: 124; historical control data range: 62-117), without any effect on the results and final conclusion of the study.
Seven concentration levels were investigated in the informatory toxicity test and six in the main mutation experiments.
The number of analysable concentrations (at least five) and the non-precipitated, non-toxic concentration levels (at least three) fulfilled the validity criterion of the study.
In summary, the actual values of untreated, vehicle and positive controls were in line with the criteria for validity of the assay.
All criteria for the validity of these experiments have therefore been met.

Conclusions:

The reported data of this mutagenicity assay shows that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base pair substitution in the genome of the tester strains used. Therefore, the test substance is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

Five bacterial strains were used to investigate the mutagenic potential of the test sbstance in two independent experiments, in a plate incorporation test (experiment I, initial mutation test) and in a pre-incubation test (experiment II, confirmatory mutation test). Each assay was conducted with and without metabolic activation (±S9). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently.
In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled. No substantial increases in revertant colony numbers were observed in any of the five test strains following treatment with the test sbstance at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values were observed in both independently performed main experiments; however, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.
In the initial and confirmatory mutation tests an inhibitory effect of the test item on the bacterial cell growth was observed. The inhibition in terms of cytotoxicity was indicated by decreased revertant colony counts (revertants below the historical control data ranges and/or corresponding vehicle control data ranges) and/or affected background lawn development (reduced or slightly reduced background lawn).
The 500 μg/plate was found to be the lowest cytotoxic concentration, observed in the confirmatory mutation test in the case of Salmonella typhimurium TA100 and TA1537 strains, in the absence of exogenous metabolic activation (−S9).
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9) throughout the study.
The reported data of this mutagenicity assay shows that under the experimental conditions reported the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test sbstance is considered to be non-mutagenic in this bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

AMES (ToxiCoop, 2021)

Five bacterial strains were used to investigate the mutagenic potential of the test sbstance in two independent experiments, in a plate incorporation test (experiment I, initial mutation test) and in a pre-incubation test (experiment II, confirmatory mutation test). Each assay was conducted with and without metabolic activation (±S9). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently.
In the performed experiments all of the validity criteria, regarding the investigated strains, negative (vehicle) and positive controls, S9 activity and number of investigated analysable concentration levels were fulfilled. No substantial increases in revertant colony numbers were observed in any of the five test strains following treatment with the test sbstance at any concentration level, either in the presence or absence of metabolic activation (S9 Mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values were observed in both independently performed main experiments; however, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments.
In the initial and confirmatory mutation tests an inhibitory effect of the test item on the bacterial cell growth was observed. The inhibition in terms of cytotoxicity was indicated by decreased revertant colony counts (revertants below the historical control data ranges and/or corresponding vehicle control data ranges) and/or affected background lawn development (reduced or slightly reduced background lawn).
The 500 μg/plate was found to be the lowest cytotoxic concentration, observed in the confirmatory mutation test in the case of Salmonella typhimurium TA100 and TA1537 strains, in the absence of exogenous metabolic activation (−S9).
No precipitation of the test item was observed on the plates in the examined bacterial strains at any examined concentration level (±S9) throughout the study.
The reported data of this mutagenicity assay shows that under the experimental conditions reported the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, the test sbstance is considered to be non-mutagenic in this bacterial reverse mutation assay.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The test item is not considered a mutagen. The substance does not need to be classified for this endpoint according to to Regulation (EC) No 1272/2008 (CLP), as amended for the 17th time in Regulation (EU) 2021/849.