6-[(p-tosyl)amino]hexanoic acid, compound with 2,2',2''-nitrilotriethanol (1:1)

Genetic toxicity: in vitro

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

Test material information

Constituent 1

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 / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1537

Additional strain / cell type characteristics:

other: Strain is histidine-dependent

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 100

Additional strain / cell type characteristics:

other: Strain is histidine-dependent

Species / strain 3

Species / strain / cell type:

S. typhimurium TA 98

Additional strain / cell type characteristics:

other: Strain is histidine-dependent

Species / strain 4

Species / strain / cell type:

S. typhimurium TA 1535

Additional strain / cell type characteristics:

other: Strain is histidine-dependent

Species / strain 5

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: S9 was obtained by Trinova Biochem GmbH, Gießen. Batch nos. 4183, 4053, 4063, 4120, 4126
Produced from the livers of male Sprague-Dawley rats which were treated with 500 mg Aroclor 1254/kg body weight intraperitoneally.
- method of preparation of S9 mix
Phosphate buffer 22.5 mL
0.1M NADP-solution 1.0 mL
1M G6P-solution 0.125 mL
Salt solution 0.5 mL
Rat liver S9
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the duration of the test.

Test concentrations with justification for top dose:

Top dose concentration of 5000 µg/plate used inline with OECD 471 for soluble substances

Experiment 1b
Nominal concentrations Real concentrations
5000 µg/plate 5007 µg/plate
1500 µg/plate 1502 µg/plate
500 µg/plate 501 µg/plate
150 µg/plate 150 µg/plate
50 µg/plate 50 µg/plate


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

Nominal concentrations Real concentrations
5000 µg/plate 5008 µg/plate
2500 µg/plate 2504 µg/plate
1250 µg/plate 1252 µg/plate
625 µg/plate 626 µg/plate
313 µg/plate 313 µg/plate
156 µg/plate 157 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: In a non-GLP pre-test, the solubility of the test item was determined in demineralized water (demin. water) and dimethyl sulfoxide (DMSO). The test item is soluble in a concentration of 50 g/L in DMSO.
Based on these results of the non-GLP pre-test, DMSO was used as solvent in the experiments.
Therefore, a test item solution containing 50 ± 5 g/L in DMSO was prepared for all experiments.

Controls

Untreated negative controls:

yes

Remarks:

Demineralized water

Negative solvent / vehicle controls:

yes

Remarks:

Dimethylsulfoxide (DMSO), CAS No. 67-68-5, batch: 2018270919

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:

Specification: Species Salmonella typhimurium LT2
Strains: TA98, TA100, TA102, TA1535 and TA1537
All strains met the criterion of at least 109 bacteria/mL (correlating to 100 colonies/plate after dilution)

NUMBER OF REPLICATIONS:
- Number of cultures per concentration (single, duplicate, triplicate): Per bacteria strain and concentration, three plates with (+S9) and three plates without metabolic activation (-S9) were used.
- Number of independent experiments: Four experiments (1, 1b, 2 and 2b) were performed. Experiment 1 was invalid for all bacterial strains due to probable contamination and experiment 2 was invalid for TA102 (-S9).
The invalid experiments are not reported.

METHOD OF TREATMENT/ EXPOSURE:
Experiment 1b:
- Method: plate incorporation method
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 mutagen 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)
 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.

Experiment 2b:
- Method: 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 mutagen 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
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.

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48h
- Harvest time after the end of treatment (sampling/recovery times): not specified


METHODS FOR MEASUREMENT OF CYTOTOXICITY
- The toxicity of the following concentration was tested: 5000 µg/plate. Per strain, 2 plates with and without metabolic activation were incubated with the corresponding dose of the test item on maximal soft agar.
Criterion: The determination of titre should give a number of at least 109 cells/mL, correlating to at least 100 colonies/plate after dilution. Exact colony counts could not be determined due to the strong growth of the bacteria, but by visual inspection it was obvious that the values exceed 100 colonies/ plate.
In the cytotoxicity test, the test item shows no cytotoxicity towards all tested bacterial strains in the highest test item concentration (5000 µg/plate).

Rationale for test conditions:

The study procedures described were based on the most recent Guideline OECD 471 and EU Method B.13/14.
The test item ASCplus® (6-[[(4-methylphenyl)sulphonyl]amino]hexanoic acid) was tested in the Salmonella typhimurium reverse mutation assay
with five strains of Salmonella typhimurium (TA98, TA100, TA102, TA1535 and TA1537).
The test was performed in four experiments in the presence and absence of metabolic
activation, with +S9 standing for the presence of a metabolic activation, and -S9 standing
for absence of metabolic activation.

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 results

Test results 1

Key result

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

Test results 2

Key result

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

Test results 3

Key result

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

Test results 4

Key result

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

Test results 5

Key result

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

Additional information on results:

Ames test:
- Signs of toxicity:
Experiment 1b: 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.

Experiment 2b:
The test item showed no precipitates on the plates and the bacterial background lawn and
the number of colonies was not affected at any of the test item concentrations

Remarks on result:

other: non-mutagenic

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.