Potassium dimethyl 5-sulphonatoisophthalate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenicity of the test substance was assessed in the bacterial reverse mutation test using five strains, Salmonella typhimurium TA100, TA1535, TA98, and TA1537 and Escherichia coli WP2 uvrA. The test was conducted up to 5000 µg/plate as the maximum dose using 8 doses with a common ratio of 4. According to the results, the number of revertant colonies treated with the lest substance was less than twice that treated with the negative control (solvent) in any tester strain with or without S9 mix. Moreover, microbial growth inhibition or precipitation of the test substance was not observed at any dose in all tester strains with and without S9 mix.
From these results described, it was concluded that the test substance was not mutagenic under the conditions employed in this study.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot number of test material: sponsor, 70404
- Purity: 96.8 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, shielded from light in airtight container.

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:

Type and composition of metabolic activation system: S9 mix

Test concentrations with justification for top dose:

5000 µg/plate

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-(2-Fury )-3-( 5-nitro-2-fury l) acrylamide (AF-2) and 2-Aminoanthracene (2-AA)

Details on test system and experimental conditions:

According to the test guidelines, the preliminary test was performed at the following doses:
With and without S9 mix: 5000, 1250, 313, 78.1, 19.5, 4.88, 1.22, 0.305 µg/plate.

According to the results of the preliminary test, revertant colonies did not increase in any tester strains with or without S9 mix. Microbial growth inhibition was not observed in all strains. Based on these results, main tests I and II were performed at the following doses:
With and Without S9 mix: 5000, 2500, 1250, 625, 313 µg/plate

Pre-incubation method:
(1) For each treatment, 0.1 mL of the test substance solution, negative (solvent) control or 0.1 mL of positive control solution was added into a sterilized test tube with an aluminum top.
(2) For assays without S9 mix, 0.5 mL of 0.1 mol/L sodium phosphate buffer (pH 7.4) and 0.1 mL ofthe bacterial suspension were added to the test substance solutions.
(3) For assays with S9 mix, 0.5 mL of S9 mix was mixed in place of 0.1 mol/L sodium phosphate buffer.
(4) The mixture was incubated with gentle shaking (shaking frequency: 120 times/minute) at 37°C for 20 minutes (pre-incubation).
(5) After pre-incubation, 2 mL of the molten top agar was added to this mixture and then poured onto the minimal glucose agar plate.
(6) After the overlaid agar had solidified, the plates were incubated for 48 hours at 37°C.

Observation :
Precipitation: Precipitation was judged by observation of the plate surface macroscopically.
Microbial growth inhibition: Background lawn of the bacterial cells that have amino-acid requirement was observed by a stereoscopic microscope (40x; Nikon, SMZ-10), and microbial growth inhibition was judged by the relationship between the test substance treated plates and the non-treated plate.

Measurement of colony number.
Revertant colonies were measured using an automatic colony counter (Toyo Sokki Co. Ltd., CA-7) or manual counting. Revertant colonies in the positive control treatment groups and those in TAIOO ofthe test substance treatment groups were measured using an automatic colony counter and the others were measured by manual counting. Correction ofthe measuring area was employed using instrumental analysis.

Number of plate:
Preliminary test: 1 plate/dose; (negative and positive control treatment groups: 2 plates/dose)
Maintest I: 3 plates/dose
Maintest II: 3 plates/dose

Evaluation criteria:

The main tests were accepted as valid if all the following criteria were satisfied. The preliminary test was accepted to obtain the data by which doses were set in the main test.
(1) The negative (solvent) control values (mean) and the positive control values (mean) are within the proper ranges calculated based on the historical data at the testing facility.
(2) The positive control values (mean) show clear positive responses in the respective tester strains, as evidenced by the number of revertant colonies being greater than 2-fold of the respective negative (solvent) control value.
(3) There are more than 4 doses showing no microbial growth inhibition and more than 5 doses applicable to the evaluation in the main test.
(4) The result of the sterility test indicates that there is no bacterial or fungous contamination.
(5) There are no plates that become invalid for measurement due to contamination or other unexpected situations.

Statistics:

The mean and standard deviation of the revertant colonies were calculated for the negative (solvent) control, the positive control, and the test substance treatment groups. The mean and standard deviation were expressed by rounding to the first decimal place.

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

Positive controls validity:

valid

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

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

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

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Conclusions:

In the preliminary and two main tests, the number of revertant colonies induced by the test substance was less than twice the corresponding negative control value for all tester strains with and without S9 mix. Furthermore, all tests indicated that they were performed accurately as the tests satisfied acceptable the specified criteria.
From the results described above, it was concluded that the test substance was not mutagenic under the conditions employed in this study.

Executive summary:

Mutagenicity of the test substance was assessed in the bacterial reverse mutation test using five strains, Salmonella typhimurium TA100, TA1535, TA98, and TA1537 and Escherichia coli WP2 uvrA. The test was conducted by the pre-incubation method with and without S9 mix.
A preliminary test was performed at 5000 µg/plate as the maximum dose using 8 doses with a common ratio of 4. According to the results, the number of revertant colonies treated with the lest substance was less than twice that treated with the negative control (solvent) in any tester strain with or without S9 mix. Moreover, microbial growth inhibition or precipitation of the test substance was not observed at any dose in all tester strains with and without S9 mix.
Main test I was performed at 5000 µg/plate as the maximum dose using 5 doses with a common ratio of 2 based on the results of the preliminary test. According to the results, the number of revertant colonies treated with the test substance was less than twice that treated with the negative control in any tester strain with or without S9 mix. Moreover, microbial growth inhibition or precipitation of the test substance was not observed at any dose in all tester strains with and without S9 mix.
Main test II was performed using the same doses as main test I. According to the results, the number of revertant colonies treated with the test substance was less than twice that treated with the negative control in any tester strain with or without S9 mix. Moreover, microbial growth inhibition or precipitation of the test substance was not observed at any dose in all tester strains with and without S9 mix.

From the results described above, it was concluded that the test substance was not mutagenic under the conditions employed in this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)