N-benzylethylenediamine

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Feb 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997-07-21

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

N-benzylethylenediamine

EC Number:

223-984-5

EC Name:

N-benzylethylenediamine

Cas Number:

4152-09-4

Molecular formula:

C9H14N2

IUPAC Name:

N-benzylethane-1,2-diamine

Test material form:

liquid

Details on test material:

Batch No.of test material: R1640 Fr. 16/199/17-18 Redes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Batch No.of test material: R1640 Fr. 16/199/17-18 Redes
- Purity: 99.86 %
- Physical state / color: Liquid / colorless, clear
- Expiry date: 06 Feb 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In the refrigerator between 2 and 8°C, under light exclusion and under Nitrogen

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Test item preparation: On the day of the experiment, the test item N-benzylethylenediamine was dissolved in deionised water. The solvent was chosen because of its solubility properties and its relative nontoxicity to the bacteria.
All formulations were prepared freshly before treatment and used within two hours of preparation.
The pH value of the stock solution was adjusted to eight with HCl 2M.

Method

Target gene:

trp / his

Species / strain

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Due to the limited capacity for metabolic activation of potential mutagens in in vitro methods an exogenous metabolic activation system is necessary.
Phenobarbital/β-naphthoflavone induced rat liver S9 were used as the metabolic activation system.
The protein concentration of the S9 preparation was 34.4 mg/mL in both experiments.

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment met the acceptance criteria, thus, it is reported as experiment I.
Since the second experiment was performed before the results of experiment I were available, the same concentrations as in experiment I were tested.The test item was tested at the following concentrations in both experiments:
3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Vehicle / solvent:

- Vehicle/solvent used: deionised water

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene (2-AA); 4-nitro-o-phenylenediamine (NOPD)

Remarks:

positive control: 2-aminoanthracene was tested with S9 mix; other without S9 mix

Details on test system and experimental conditions:

Pre-experiment for Toxicity
To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates.
The following concentrations were tested in the pre-experiment / experiment II:
3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants (below a factor of 0.5) or a clearing of the bacterial background lawn. The pre-experiment is reported as experiment I, since the acceptance criteria were met: plates were scorable (> 0 revertant colonies) at five dose groups or more.

Dose Selection
According to the current OECD Guideline No. 471 the maximum concentration should be 5000 μg/plate, unless limited by toxicity or solubility of the test item.
In the pre-experiment the concentration range of the test item was 3 – 5000 μg/plate. The pre-experiment met the acceptance criteria, thus, it is reported as experiment I.
Since the second experiment was performed before the results of experiment I were available, the same concentrations as in experiment I were tested.

Evaluation criteria:

The Salmonella typhimurium and Escherichia coli reverse mutation assay is considered acceptable if it meets the following criteria:
• regular background growth in the negative and solvent controls;
• the spontaneous reversion rates in the negative and solvent controls are in the range of our historical control data;
• the positive control substances produced an increase above the threshold of two-fold (strains TA 98, TA 100, and WP2 uvrA) or three-fold (strains TA 1535 and TA 1537) of the revertant colony count of the corresponding solvent control;
• a minimum of five analysable dose levels should be present with at least three dose levels showing no signs of toxic effects, evident as a reduction in the number of revertants below the indication factor of 0.5.
Evaluation of Results
A test item is considered as a mutagen if a biologically relevant increase in the number of revertants of two-fold or above (strains TA 98, TA 100, and WP2 uvrA) or of three-fold or above (strains TA 1535 and TA 1537) the spontaneous mutation rate of the corresponding solvent control is observed.
A dose dependent increase is considered biologically relevant if the threshold is reached or exceeded at more than one concentration.
An increase of revertant colonies equal or above the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment. However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Test results 4

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

True negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item occurred up to the highest investigated dose.
The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.
No toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.
No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with N-benzylethylenediamine at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). A dose dependent increase in revertant colony numbers, which were slightly above the historical control data, was observed in strain WP2 uvrA with and without S9 mix in both experiments. This effect was observed in the concentrations ranging from 1000 to 5000 μg/plate. Since this deviation is rather small and the threshold of twice the number of the corresponding solvent control is neither reached nor exceeded in any case, it can be judged as biologically irrelevant with no impact on the outcome of the study.

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.
Therefore, N-benzylethylenediamine is considered to be non-mutagenic in this Salmonella typhimurium and Escherichia coli reverse mutation assay.