Registration Dossier

Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: genome mutation
Type of information:
experimental study
Remarks:
Source data for the read-across to the test substance
Adequacy of study:
key study
Study period:
2020
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted by a GLP accredited laboratory using OECD Testing Guideline 471. The study was conducted on 6-[(p-Tosyl)amino]hexanoic acid, which is the carboxylic acid component of the registered substance i.e. tosyl salt.
Justification for type of information:
The test substance was the carboxylic acid component of the registered substance (tosyl salt). Readacross 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-[(pTosyl)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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
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

Test material

Constituent 1
Chemical structure
Reference substance name:
6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid
EC Number:
278-934-5
EC Name:
6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid
Cas Number:
78521-39-8
Molecular formula:
C13H19NO4S
Constituent 2
Reference substance name:
6-[(p-tosyl)amino] hexanoic acid
IUPAC Name:
6-[(p-tosyl)amino] hexanoic acid
Constituent 3
Reference substance name:
Tosyl carboxylic acid
IUPAC Name:
Tosyl carboxylic acid
Test material form:
solid: particulate/powder
Details on test material:
Full name of test item: 6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid
Name in the study: ASCplus®
Lot Number: 1812004
CAS No.: 78521-39-8
EC No.: 278-934-5
Composition: 6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid 74.76 %; water 25 %
Molecular formula: C13H19NO4S
Purity: > 99.5 % (6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid 74.76 %; water 25 %)
Appearance: White powder

Method

Target gene:
TA100: his G46
TA98: his D3052
TA102: his G428 (pAQ1)
TA1535: his G46
TA 1537
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1537
Additional strain / cell type characteristics:
other: Strain is histidine-dependent
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: Strain is histidine-dependent
Species / strain / cell type:
S. typhimurium TA 98
Additional strain / cell type characteristics:
other: Strain is histidine-dependent
Species / strain / cell type:
S. typhimurium TA 1535
Additional strain / cell type characteristics:
other: Strain is histidine-dependent
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: Strain is histidine-dependent
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: Trinova Biochem GmbH, Gießen.
- method of preparation of S9 mix : produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intra-peritoneally.
Test concentrations with justification for top dose:
The test was performed in four experiments in the presence and absence of metabolic activation:
- Experiment 1:was invalid as a contamination of the bacterial cultures was assumed.

- 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.

- Experiment 2: Based on the results of the experiment 1b, the test item was tested with the following concentrations 5000; 2500; 1250; 625; 313, 156 μg/plate in TA98 (+/-S9), TA100 (+/-S9), TA102 (+/-S 9), TA1535 (+/-S9), TA1537 (+/-S9) using the pre-incubation method.

Experiment 2b: Based on the results of experiment 2, the experiment 2b was performed with the following concentrations 5000; 2500; 1250; 625; 313, 156 μg/plate in TA102 (-S9)
Vehicle / solvent:
- Vehicle/solvent used:

Dimethylsulfoxide (DMSO), CAS No. 67-68-5, batch: 2018270919, for the positive controls 4-Nitro-1,2-phenylene diamine, benzo-a-pyrene and 2-amino-anthracene and the test item.

Demineralized water, prepared by LAUS GmbH, from an ion-exchanger, batch: T20200303, for the positive control sodium azide
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
other: Benzo-a-Pyrene; 4-Nitro-1,2-phenylene Diamine; 2-Amino-Anthracene
Details on test system and experimental conditions:
Experiment 1b
METHOD OF APPLICATION: in agar (plate incorporation).
The following materials were gently vortexed in a test tube and poured onto the selective agar plates:
100 μL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control)
500 μL S9-mix or phosphate buffer (for test without metabolic activation).
100 μL bacteria suspension
2000 μL overlay agar (top agar)
The plates were closed and left to solidify for a few minutes, then inverted and placed in the dark
incubator at 37 ± 1 °C.
Exposure duration: 48hr
NUMBER OF REPLICATIONS: 3

Experiment 2
METHOD OF APPLICATION: pre-incubation method.
The following materials were gently vortexed in a test tube and incubated at 37 ± 1 °C for 20 minutes:
100 μL test solution at each dose level, solvent (negative control) or reference muta-gen solution (positive control)
500 μL S9-mix (see chapter 6.4.13, page 17 for test with metabolic activation) or phosphate buffer (for test without metabolic activation).
100 μL bacteria suspension (see chapter 6.2.2, page 12, test system, culture of the strains)
After the pre-incubation for 20 minutes, 2000 μL top agar was added and the tube was gently slewed. The mixture was poured onto the selective agar plate.
The plates were closed and left to solidify for a few minutes, then inverted and placed in the incubator at 37 ± 1 °C.
Exposure duration: 48hr
NUMBER OF REPLICATIONS: 3

Experiment 2b
METHOD OF APPLICATION: pre-incubation method, as above.
Exposure duration: 48hr
NUMBER OF REPLICATIONS: 3


Evaluation criteria:
A substance is considered to be mutagenic, if a reproducible increase with or without metabolic activation of revertant colonies per plate exceeding an increase factor of 2 for the bacteria strains TA98, TA100, TA102, TA1535 and TA1537 compared to negative controls 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.
A substance is not mutagenic if it does not meet these criteria.
If the criteria listed above are not clearly met, the results will be assessed as equivocal and will be discussed.
Statistics:
The colonies will be counted visually and the numbers will be recorded. A validated spreadsheet software (Microsoft Excel®) will be used to calculate mean values and standard deviations of each treatment, negative control and positive control.
The mean values and standard deviations of each threefold determination are calculated as well as the increase factor 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 (mean revertants minus mean spontaneous revertants) is given.

Results and discussion

Test resultsopen allclose all
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.

Any other information on results incl. tables

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.

Applicant's summary and conclusion

Conclusions:
Based on the results of this study it is concluded that the 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 test substance was determined in accordance with the OECD Guideline for Testing of Chemicals 471. The 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.