(2-hydroxy-1,1-dimethylethyl)ammonium toluene-4-sulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with propan-1-ol, 2-amino-2-methyl-, 4-methylbenzenesulphonate using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system. The dose range was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was 15 to 5000 μg/plate. Propan-1-ol, 2-amino-2-methyl-, 4-methylbenzenesulphonate was considered to be non-mutagenic under the conditions of this test (Envigo, 2016).

 

The second in vitro study was conducted in human lymphocytes to investigated the induction of chromosomal damage due to the treatment with propan-1-ol, 2-amino-2-methyl-, 4-methylbenzenesulphonate (LPT, 2020). The test material was investigated up to cytotoxic concentrations of 2000 µg/mL medium in the absence and in the presence of metabolic activation employing two exposure times (4 & 24 h) without S9 mix and one exposure time (4 h) with S9 mix. All treatment conditions (250 -200 µg/ml) revealed no indications of chromosomal damage in the in vitro micronucleus test.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

uvrB-

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Details on mammalian cell type (if applicable):

Strains Genotype Type of mutations indicated
TA1537 his C 3076; rfa-; uvrB-: frame shift mutations
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-: base-pair substitutions
TA100 his G 46; rfa-; uvrB-;R-factor

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

Strain Genotype Type of mutations indicated
WP2uvrA trp-; uvrA-: base-pair substitution

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

The test item was tested using the following method. The maximum concentration was 5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.

Vehicle / solvent:

Distilled water

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

Positive controls:

yes

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene (2AA)

Details on test system and experimental conditions:

The five strains of bacteria used, and their mutations, are defined above.
All of the Salmonella strains are histidine dependent by virtue of a mutation through the histidine
operon and are derived from S. typhimurium strain LT2 through mutations in the
histidine locus. Additionally due to the "deep rough" (rfa-) mutation they possess a faulty
lipopolysaccharide coat to the bacterial cell surface thus increasing the cell permeability to
larger molecules. A further mutation, through the deletion of the uvrB- bio gene, causes an
Report Envigo Study Number: TJ05YB
Page 13
inactivation of the excision repair system and a dependence on exogenous biotin. In the
strains TA98 and TA100, the R-factor plasmid pKM101 enhances chemical and UV-induced
mutagenesis via an increase in the error-prone repair pathway. The plasmid also confers
ampicillin resistance which acts as a convenient marker (Mortelmans and Zeiger, 2000). In
addition to a mutation in the tryptophan operon, the E. coli tester strain contains a uvrA- DNA
repair deficiency which enhances its sensitivity to some mutagenic compounds. This
deficiency allows the strain to show enhanced mutability as the uvrA repair system would
normally act to remove and repair the damaged section of the DNA molecule (Green and
Muriel, 1976 and Mortelmans and Riccio, 2000).
The bacteria used in the test were obtained from:
• University of California, Berkeley, on culture discs, on 04 August 1995.
• British Industrial Biological Research Association, on a nutrient agar plate, on

All of the strains were stored at approximately -196 °C in a Statebourne liquid nitrogen
freezer, model SXR 34.
In this assay, overnight sub-cultures of the appropriate coded stock cultures were prepared in
nutrient broth (Oxoid Limited; lot number 1758279 10/20) and incubated at 37 °C for
approximately 10 hours. Each culture was monitored spectrophotometrically for turbidity
with titres determined by viable count analysis on nutrient agar plates.

The test item was fully soluble in sterile distilled water at 50 mg/mL in solubility checks
performed in–house. Sterile distilled water was therefore selected as the vehicle.

The test item was tested using the following method. The maximum concentration was
5000 μg/plate (the maximum recommended dose level). Eight concentrations of the test item
(1.5, 5, 15, 50, 150, 500, 1500 and 5000 μg/plate) were assayed in triplicate against each
tester strain, using the direct plate incorporation method.
Without Metabolic Activation
0.1 mL of the appropriate concentration of test item, solvent vehicle or appropriate positive
control was added to 2 mL of molten, trace amino-acid supplemented media containing
0.1 mL of one of the bacterial strain cultures and 0.5 mL of phosphate buffer. These were
then mixed and overlayed onto a Vogel-Bonner agar plate. Negative (untreated) controls
were also performed on the same day as the mutation test. Each concentration of the test
item, appropriate positive, vehicle and negative controls, and each bacterial strain, was
assayed using triplicate plates.
With Metabolic Activation
The procedure was the same as described previously (see 3.3.2.2) except that following the
addition of the test item formulation and bacterial culture, 0.5 mL of S9-mix was added to the
molten, trace amino-acid supplemented media instead of phosphate buffer.
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the
presence of revertant colonies using an automated colony counting system. The plates were
viewed microscopically for evidence of thinning (toxicity).

Test for Mutagenicity: Experiment 2 – Pre-Incubation Method
As Experiment 1 was deemed negative, Experiment 2 was performed using the
pre-incubation method in the presence and absence of metabolic activation.
Dose selection
The dose range used for Experiment 2 was determined by the results of Experiment 1 and
was 15 to 5000 μg/plate.

Six test item dose levels per bacterial strain were selected in the second mutation test in order
to achieve both a minimum of four non-toxic dose levels and the potential toxic limit of the
test item following the change in test methodology from plate incorporation to
pre-incubation.
Without Metabolic Activation
0.1 mL of the appropriate bacterial strain culture, 0.5 mL of phosphate buffer and 0.1 mL of
the test item formulation, solvent vehicle or 0.1 mL of appropriate positive control were
incubated at 37 ± 3 °C for 20 minutes (with shaking) prior to addition of 2 mL of molten,
trace amino-acid supplemented media and subsequent plating onto Vogel-Bonner plates.
Negative (untreated) controls were also performed on the same day as the mutation test
employing the plate incorporation method. All testing for this experiment was performed in
triplicate.

With Metabolic Activation
The procedure was the same as described previously (see 3.3.3.2) except that following the
addition of the test item formulation and bacterial strain culture, 0.5 mL of S9-mix was added
to the tube instead of phosphate buffer, prior to incubation at 37 ± 3 °C for 20 minutes (with
shaking) and addition of molten, trace amino-acid supplemented media. All testing for this
experiment was performed in triplicate.
Incubation and Scoring
All of the plates were incubated at 37 ± 3 °C for approximately 48 hours and scored for the
presence of revertant colonies using an automated colony counting system. The plates were
viewed microscopically for evidence of thinning (toxicity).

Acceptibility criteria
The reverse mutation assay may be considered valid if the following criteria are met:
All bacterial strains must have demonstrated the required characteristics as determined by
their respective strain checks according to Ames et al., (1975), Maron and Ames (1983) and
Mortelmans and Zeiger (2000).
All tester strain cultures should exhibit a characteristic number of spontaneous revertants per
plate in the vehicle and untreated controls (negative controls). Acceptable ranges are
presented as follows:
TA1535 7 to 40
TA100 60 to 200
TA1537 2 to 30
TA98 8 to 60
WP2uvrA 10 to 60
Based on combined historical negative and solvent control ranges for 2014 and 2015.

All tester strain cultures should be in the range of 0.9 to 9 x 109 bacteria per mL.
Diagnostic mutagens (positive control chemicals) must be included to demonstrate both the
intrinsic sensitivity of the tester strains to mutagen exposure and the integrity of the S9-mix.
All of the positive control chemicals used in the study should induce marked increases in the
frequency of revertant colonies, both with or without metabolic activation.
There should be a minimum of four non-toxic test item dose levels.
There should be no evidence of excessive contamination.

Evaluation criteria:

There are several criteria for determining a positive result. Any, one, or all of the following
can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and
Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester
strain (especially if accompanied by an out-of-historical range response (Cariello and
Piegorsch, 1996)).
A test item will be considered non-mutagenic (negative) in the test system if the above
criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the
data generated will prohibit making a definite judgment about test item activity. Results of
this type will be reported as equivocal.

Statistics:

Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05)
for those values that indicate statistically significant increases in the frequency of revertant
colonies compared to the concurrent solvent control.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not determined

Vehicle controls validity:

not applicable

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:

not determined

Vehicle controls validity:

not applicable

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:

not determined

Vehicle controls validity:

not applicable

Untreated 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:

not determined

Vehicle controls validity:

not applicable

Untreated 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:

not determined

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Prior to use, the master strains were checked for characteristics, viability and spontaneous
reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and
the S9-mix used in both experiments was shown to be sterile. The test item formulation was
also shown to be sterile.
Results for the negative controls (spontaneous mutation rates) were considered to be acceptable.
These data are for concurrent untreated control plates performed on the same day as the Mutation Test.
The individual plate counts, the mean number of revertant colonies and the standard
deviations, for the test item, positive and vehicle controls, both with and without metabolic
activation were evaluated.

The maximum dose level of the test item in the first experiment was selected as the maximum
recommended dose level of 5000 μg/plate. There was no visible reduction in the growth of
the bacterial background lawn at any dose level, either in the presence or absence of
metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and
consequently the same maximum dose level was used in the second mutation test. Similarly
there was no visible reduction in the growth of the bacterial background lawn at any dose
level, either in the presence or absence of metabolic activation (S9-mix), in the second
mutation test (pre-incubation method). No test item precipitate was observed on the plates at
any of the doses tested in either the presence or absence of S9-mix.
There were no significant increases in the frequency of revertant colonies recorded for any of
the bacterial strains, with any dose of the test item, either with or without metabolic
activation (S9-mix) in Experiment 1 (plate incorporation method). Similarly, no significant
increases in the frequency of revertant colonies were recorded for any of the bacterial strains,
with any dose of the test item, either with or without metabolic activation (S9-mix) in
Experiment 2 (pre-incubation method).
The vehicle (sterile distilled water) control plates gave counts of revertant colonies within the
normal range. All of the positive control chemicals used in the test induced marked increases
in the frequency of revertant colonies, both with or without metabolic activation. Thus, the
sensitivity of the assay and the efficacy of the S9-mix were validated

Conclusions:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with propan-1-ol, 2-amino-2-methyl-, 4-methylbenzenesulphonate using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system. The dose range was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was 15 to 5000 μg/plate. Propan-1-ol, 2-amino-2-methyl-, 4-methylbenzenesulphonate was considered to be non-mutagenic under the conditions of this test.

Executive summary:

Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 and Escherichia coli strain WP2uvrA were treated with propan-1-ol, 2-amino-2-methyl-, 4-methylbenzenesulphonate using both the Ames plate incorporation and pre-incubation methods at up to eight dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolizing system. The dose range was 1.5 to 5000 μg/plate. The experiment was repeated on a separate day (pre-incubation method) using fresh cultures of the bacterial strains and fresh test item formulations. The dose range was 15 to 5000 μg/plate. Propan-1-ol, 2-amino-2-methyl-, 4-methylbenzenesulphonate was considered to be non-mutagenic under the conditions of this test.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-02-26 to 2020-04-30

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

2016-07-29

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EC method B.49: In vitro Mammalian Cell Micronucleus Test

Version / remarks:

2017-04-28

Deviations:

no

Principles of method if other than guideline:

The test for 24 h exposure without S9 mix has been extende to a 20 h recovery periode after the culture medium was removed.

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Batch: NC19B11650

Species / strain / cell type:

lymphocytes:

Details on mammalian cell type (if applicable):

human donors (young, healthy, non smocker)
treated with Phytohemagglutinin

Cytokinesis block (if used):

5 µg/ml Cytochalasin B
lot no. (BO11910; AppliChem GmbH 64291 Darmstadt (Germany)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from Aroclor 1254 induced male Sprague-Dawley rat livers rat liver

Test concentrations with justification for top dose:

0; 3.16; 10; 31.6, 100; 316; 1000 and 2000 µg/ml (+/- S9) (no cytotoxicity was observed at the highest concentration)

Vehicle / solvent:

DMSO dimethyl sulfoxide 1% ( v/v)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

1% DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

with and without metabolic activation

Positive control substance:

colchicine

cyclophosphamide

mitomycin C

Remarks:

positive with metabolic activation: cyclophosphamide

Details on test system and experimental conditions:

SYSTEM OF TESTING
- Species/cell type: human peripheral lymphocytes
- Metabolic activation system: S9 mix based on S9 fraction from Aroclor  1254 induced male Sprague-Dawley rat livers
ADMINISTRATION: 
- Dosing:   0; 3.16; 10; 31.6, 100; 316; 1000 and 2000 µg/ml (+/- S9)
- Number of technical replicates: 2
- Application: 1 % (v/v) Vehicle dimethyl sulfoxide (DMSO). 50 µl in 5 ml treatment medium;
 incubated for 4 h, thereafter  20 h recovery time with 5 µg/ml Cytocalasin B
or incubation for 24 h, thereafter 20 h recovery periode with 5 µg/ml Cytocalasin B
all at 37 °C; after 1.5 cell cycles the cells have been harvested and centrifuged for 10 min at 800 rpm and resuspended in KCl (0.56%).
After 17 min at 37°C the cell suspensions were centrifuged for 10 minutes at 800 rpm.
The supernatant was discarded and 5 mL of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid v/v) added.
The cells were fixed for 30 min followed by centrifugation at 800 rpm. The supernatant was discarded, and the cell pellet was
resuspended in 0.5 mL of fresh fixative and 30% glacial acetic acid Two drops of this cell suspension were dropped onto a
prewarmed, pre-cleaned microscope slide. The slides were then stained using 10% Giemsa and left to air-dry at room temperature.
- Positive and negative control groups and treatment:    
vehicle (solvent) control: 1 % (v/v) dimethyl sulfoxide (DMSO)   
positive -S9:  Mitomycin C, 0.2 and 0.1 µg/ml
Colchine, 0.02 and 0.01 µg/ml
positive +S9:  cyclophosphamide, 20 and 10 µg/ml
DETERMINATION OF CYTOTOXICITY
- Method: induction of micronuclei
OTHER EXAMINATIONS:
- ph, osmolarity, mononucleated, binucleate and multinucleated cells

Rationale for test conditions:

reactive substance

Evaluation criteria:

Acceptance of a test is based on the following criteria:
1) The concurrent negative control is considered acceptable for addition to the laboratory historical negative control database (Poisson-based 95% control limits). Where concurrent negative control data fall outside the 95% control limits, they may be acceptable for inclusion in the historical control data as long these data are not extreme outliers.
2) Concurrent positive controls induce responses that are compatible with those generated in the laboratory's historical positive control data base and produce a statistically significant increase compared with the concurrent negative control.
3) Adequate number of cells, cell proliferation criteria and concentrations are analysable

Statistics:

not named

Key result

Species / strain:

lymphocytes: human periperhal lymphoctes

Remarks:

Phytothemagglutin treated

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Remarks:

0.2 µg/ml Mitomycin C -> 23.5 micronucleaed cells/1000 cells (4h -S9) 20µg/ml Cyclophosphamide-> 22.0 micronucleaed cells/1000 cells (4h +S9)

Additional information on results:

GENOTOXIC EFFECTS:
- With metabolic activation: None
- Without metabolic activation:  None
PRECIPITATION CONCENTRATION:
 no  precipitation up to (2000 µg/ml)   The test substance had no relevant effect on osmolarity or pH of the  
medium at concentrations up to 2000 µg/ml.
CYTOTOXIC CONCENTRATION in pre-tests: 
- With and without metabolic activation: None

Table:  pH values and osmolality

Concentrationof Propan… [µg/mL medium]	pH value	Osmolality [mOsmol/kg]
Medium	8.23	290
0, vehicle control	8.26	430
3.16	8.27	440
10	8.29	430
31.6	8.28	430
100	8.27	430
316	8.27	430
1000	8.25	430
2000	8.22	430

Conclusions:

On the basis of the result of this study it is concluded that the test substance has no mutagenic activity in vitro in the micronucleus assay neither in the absence nor in the presence of metabolic activation under the experimental conditions of this study.

Executive summary:

The substance was tested for its ability to induce micronucleus generation in in human peripheral lymphoctes in vitro. Three experiments were carried out with and without the addition of Aroclor-induced rat liver S9 mix. On the basis of the results, the test substance did not cause any increase in the micronuclei without and with S9 mix performed with two technicla replicates each. Thus, under the experimental conditions of this study, the test substance has no mutagenic activity in vitro in the micronucleus assay neither in the absence nor in the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

The test substance was investigated for its mutagenicity in three different in vitro tests (Ames, Micronucleus test and HPRT test). In all three tests the test substance showed no mutagenicity and has not to be classified according to the criteria of EC Regulation 1272/2008.