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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The carboxylic acid component of the registered substance was tested to determine the genotoxicity of the substance using the Ames test (OECD 471), mammalian gene cell mutation (OECD 476) and an in vitro micronculeus test (OECD 487). The test substance was found to be negative for genotoxicity in all studies, with and without metabolic activation.

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: genome mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The following read-across hypothesis is proposed for the registered substance:
The registered substance 6-[(p-tosyl)amino]hexanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1) (target substance) is manufactured directly from 6-[(p-Tosyl)amino]hexanoic acid (source substance) by simple neutralisation with triethanolamine (TEA). Other than ionization of the carboxylic acid group, the 6-[(p-Tosyl)-amino]hexanoic acid remains chemically unchanged upon salt formation. In water, the acid and amine components of the target substance dissociate completely to the source substance and TEA and these two components behave essentially as independent substances. Moreover, it is hypothesised here that TEA is non-hazardous substance and the acid component of the salt that will have a more significant impact on the outcome of any (eco)toxicological or environmental tests. Hence, the toxicity data on the source substance will accurately represent the toxicity of the target substance.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Details are attached in Section 13 of the dataset

3. ANALOGUE APPROACH JUSTIFICATION
Details are attached in section 13 of the dataset

4. DATA MATRIX
Details are attached in section 13 of the dataset
Reason / purpose for cross-reference:
read-across source
Deviations:
no
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
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
At the highest test item concentration (5000 μg/plate) in experiment 2.
Vehicle controls validity:
valid
Untreated 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
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
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
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
Positive controls validity:
valid
Remarks on result:
not determinable
Remarks:
The test item showed no increase in the number of revertants in all bacterial strains in all three valid experiments.

No significant increase in the mutant frequency was observed for the 5 tested strains, neither in the standard plate incorporation nor in the preincubation assays with or without metabolic activation.

Conclusions:
Based on the results of this study it is concluded that the source substance is not mutagenic in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in the presence and absence of metabolic activation under the experimental conditions in this study. In the cytotoxicity test, the test item shows no cytotoxicity towards all tested bacterial strains in the highest test item concentration (5000 μg/plate).
Executive summary:

The genetic toxicity of the source test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 471. The source test substance was dissolved in DMSO and tested with the 5 strains of Salmonella typhimurium: TA100, TA98, TA1537, TA102 and TA1535.


Three experiements were conducted:


In experiment 1b, the test item dissolved in DMSO was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA98, TA100, TA102, TA1535 and TA1537 using the plate incorporation method. The test item showed no precipitates on the plates at any of the concentrations. The bacterial background lawn was visible and not affected at any of the test item concentrations. The results of this experiment showed that none of the tested concentrations showed a significant decrease or increase in the number of revertants in all bacterial strains, in the presence and the absence of metabolic activation.


Experiment 2 was performed by using the pre-incubation method.
The test item showed no precipitates on the plates at any of the test item concentrations. At the highest test item concentration (5000 μg/plate), the bacterial background lawn for TA1537 (-S9) was reduced and a decrease in the number of revertants could be observed. TA1537 (+S9) showed also a decrease in the number of revertants at 5000 μg/plate. The number of revertants for all other strains was not relevantly affected.
At the second highest concentration the number of revertants for TA1537 (+/-S9) was slightly reduced, but the bacterial background lawn was not affected.
In the lower concentrations, the bacterial strains showed no significant decrease or increase in the number of revertants in all valid bacterial strains, in the presence and the absence of metabolic activation. For TA102 (-S9) experiment 2 was invalid due to a not mutagenic positive control. To achieve a valid experiment for TA102 (-S9), experiment 2 was repeated under the same conditions.


Experiment 2b was performed with the following concentrations:
TA102 (-S9): 5000; 2500; 1250; 625; 313, 156 μg/plate
The test item showed no precipitates on the plates and the bacterial background lawn was not affected at any of the test item concentrations.
The results of this experiments showed that the test item caused no significant decrease or increase in the number of revertants in strain TA102 (-S9) compared to the solvent control.


The test substance was the carboxylic acid component of the registered substance. Read-across between the tosyl salt carboxylic acid (6-[(p-Tosyl)amino]hexanoic acid) and the registered substance is considered justified as the registered substance is manufactured directly from 6-[(p-Tosyl)amino]hexanoic acid by simple neutralisation with triethanolamine (TEA). Other than ionization of the carboxylic acid group, the 6-[(p-Tosyl)amino]hexanoic acid remains chemically unchanged upon salt formation. In water, the acid and amine components of 6-[(p-Tosyl)amino]hexanoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) dissociate completely and behave essentially as independent substances. Since TEA can be considered non-hazardous, it is the acid component of the salt that will have a more significant impact on the outcome of any (eco)toxicological or environmental tests. The pKa of the carboxylic acid group in 6-[(p-Tosyl)amino]hexanoic acid (pKa = 4.90) is the same in the free acid as it is in the TEA salt. As a result, 6-[(p-Tosyl)amino]hexanoic acid will respond to changes of pH in the same way whether it is in the salt form or as the parent carboxylic acid and hence it’s bioavailability will be the same (further justifications to support the read-across hypothesis are attached in Section 13).

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

Additional information

Additional information from genetic toxicity in vitro:

The carboxylic acid component of the registered substance was tested to determine the genotoxicity of the substance using the Ames test (OECD 471), mammalian gene cell mutation (OECD 476) and an in vitro micronculeus test (OECD 487). The test substance was found to be negative for genotoxicity in all studies, with and without metabolic activation. Read-across between the tosyl salt carboxylic acid (6-[(p-Tosyl)amino]hexanoic acid) and the registered substance is considered justified as the registered substance is manufactured directly from 6-[(p-Tosyl)amino]hexanoic acid by simple neutralisation with triethanolamine (TEA). Other than ionization of the carboxylic acid group, the 6-[(p-Tosyl)amino]hexanoic acid remains chemically unchanged upon salt formation. In water, the acid and amine components of 6-[(p-Tosyl)amino]hexanoic acid, compound with 2,2’,2’’-nitrilotriethanol (1:1) dissociate completely and behave essentially as independent substances. Since TEA can be considered non-hazardous, it is the acid component of the salt that will have a more significant impact on the outcome of any (eco)toxicological or environmental tests. The pKa of the carboxylic acid group in 6-[(p-Tosyl)amino]hexanoic acid (pKa = 4.90) is the same in the free acid as it is in the TEA salt. As a result, 6-[(p-Tosyl)amino]hexanoic acid will respond to changes of pH in the same way whether it is in the salt form or as the parent carboxylic acid and hence it’s bioavailability will be the same. In addition, tests on the tirethanolamine component of the registered substance found this component to also be negative for genotoxicity. It can therefore be concluded that the registered substance (tosyl salt) is negative for genotoxicity and does not need to be classified as a genetic toxin in accordance with EU CLP criteria.

Justification for selection of genetic toxicity endpoint

A study has been conducted by a GLP accredited laboratory using OECD Testing Guideline 471. The study was conducted on the parent carboxylic acid of the tosyl salt (the registered substance) which is the component of the tosyl salt which will have a more significant impact on the environment based on the limited toxicity of triethanolamine. The test substance was found to be negative for genotoxic effects, both in the presence and absence of metabolic activation.

Justification for classification or non-classification

The carboxylic acid component of the registered substance was tested to determine the genotoxicity of the substance using the Ames test (OECD 471), mammalian gene cell mutation (OECD 476) and an in vitro micronculeus test (OECD 487). The test substance was found to be negative for genotoxicity in all studies, with and without metabolic activation.

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