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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on a GLP compliant ames test (OECD 471) the test substance is not consicered mutagenic.

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:
Feb 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)
Version / remarks:
1997-07-21
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
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.
Target gene:
trp / his
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

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The genotixic/mutagnic potential of N-benzylethylenediamine was tested different in vitro tests:

Ames test (OECD 471) [Envigo, 2019]

This study was performed to investigate the potential of N-benzylethylenediamine to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and the Escherichia coli strain WP2 uvrA. The assay was performed in two independent experiments both with and without liver microsomal activation. The test item was tested up to 5000 µg/plate in both experiments. 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, 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). Appropriate reference mutagens, used as positive controls, induced a distinct increase in revertant colonies and thus, showed the sensitivity of the test system and the activity of the metabolic activation system. 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.

Ames test (OECD 471) [BASF, 2019]

The test substance N-benzylethylenediamine was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay. A biologically relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100 and TA 98 or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test using the tester strains TA 1535, TA 1537, TA 98 and E. coli WP2 uvrA both with and without S9 mix. Using tester strain TA 100 with metabolic activation no relevant increase in the number of his+ revertants was observed in standard plate test. A relevant, reproducible and dose-dependent increase in the number of his+ revertants exceeding a factor of 2 compared to the concurrent vehicle control was observed using TA 100 without metabolic activation: increase at concentrations of 1000 and 2500 µg/plate in the 1st experiment and at concentrations of 1000 up to 5000 μg/plate in the 2nd experiment. The test substance would be considered positive. However, the reanalysis of the substance batch used in this test after study conduct showed a decrease of the purity from 99.713% to 93.754% under unexpected formation of an impurity. It has to be considered that N-benzylethylenediamine could undergo degradation to benzaldehyde and ethylene diamine. Benzaldehyde can then easily react with N-benzylethylenediamine under formation of a dibenzylated mono-imine. Further analytical investigation regarding the structure of the impurity confirmed that postulation. Therefore, the positive result of this study was observed using a test substance batch, which was not stable under the given storage conditions. Thus, it cannot be excluded that the test substance instability is the cause of the positive response. Under the experimental conditions of this study, it is concluded that the mutagenic potential of N-benzylethylenediamine cannot be addressed in the Salmonella typhimurium/Escherichia coli reverse mutation assay due to the substance instability. The test was repeated with a fresh batch of N-benzylethylenediamine (see Ames test Envigo, 2019).

HPRT test (OECD 476) [BASF, 2019]

N-benzylethylenediamine was tested to assess the potency of the test substance or its metabolite(s) to induce gene mutations in CHO cells. After four experiments, showing inconsistend results a subsequently homogeneity testing was carried out using CLSM (confocal laser-scanning microscopy). The homogeneity testing showed test substance precipitation in the cell culture medium used as stock solution. Thus, the dilutions prepared thereof were not correctly prepared, which invalidates the performed experiments. The test substance precipitation was not macroscopically visible. The study was terminated after the 4th experiment.

Discussion

N-benzylethylenediamine was tested different in vitro tests. An Ames test conducted at BASF (BASF, 2019) with a positive response is considered invalid, since the test substance was shown to be instable under formation of an impurity. It could not be excluded that the test substance instability or the impurity is the cause of the positive response. The repetition of the test at Envigo (Envigo, 2019) showed the substance to be not mutagenic. A HPRT test conducted with the registered substance showed inconsitent results because of a precipitation of the test substance in the stock solution. The results of that test were consindered not reliable.

In conclusion, based on the curreently available date N-benzylethylenediamine is not considered to cause genotoxic/mutagenic effects.

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 1272/2008. As a result the substance is not considered to be classified for genetic toxicity under Regulation (EC) No. 1272/2008.