Chlorosulphuric acid

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data is from peer reviewed publication

Data source

Reference

Reference Type:

publication

Title:

Gene mutation toxicity study of the test chemical

Author:

Demerec et al

Year:

1951

Bibliographic source:

The American Naturalist

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

Gene mutation toxicity study by the streptomycin method was performed to determine the mutagenic nature of the test chemical

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Sulphuric acid

EC Number:

231-639-5

EC Name:

Sulphuric acid

Cas Number:

7664-93-9

Molecular formula:

H2O4S

IUPAC Name:

Acide Sulfurique

Details on test material:

- Name of test material: Sulfuric acid
- IUPAC name: Dihydrogensulfate
- Molecular formula: H2O4S
- Molecular weight: 98.0778 g/mol
- Substance type: Inorganic
- Physical state: No data
- Purity: No data
- Impurities (identity and concentrations): No data

Method

Target gene:

Streptomycin

Species / strain

Species / strain / cell type:

E. coli, other: B/Sd-4/1,3,4,5 and B/Sd-4/3,4

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

not specified

Metabolic activation system:

No data

Test concentrations with justification for top dose:

0, 0.002, 0.002, 0.002, 0.003 or 0.005 %

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: The test chemical was soluble in Distilled water

Controls

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Distilled water

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: 3 hrs
- Expression time:
Streptomycin agar plates: 48 hrs (2 days)
Strptomycin free plates: 6 days
- Selection time (if incubation with a selection agent): No data
- Fixation time (start of exposure up to fixation or harvest of cells): No data

SELECTION AGENT (mutation assays): No data
SPINDLE INHIBITOR (cytogenetic assays): No data
STAIN (for cytogenetic assays): No data

NUMBER OF REPLICATIONS: No data

NUMBER OF CELLS EVALUATED: No data

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: No data

OTHER EXAMINATIONS:
- Determination of polyploidy: No data
- Determination of endoreplication: No data
- Other: No data

OTHER: No data

Rationale for test conditions:

No data

Evaluation criteria:

The streptomycin agar and free plates were scored for the presence of revertant colonies and the frequency of mutants was calculated by dividing the number of colonies by the number of viable bacteria plated

Statistics:

Poisson distribution was used to estimate the variance and the statistical significance of the result was evaluated

Results and discussion

Test results

Species / strain:

E. coli, other: B/Sd-4/1,3,4,5 and B/Sd-4/3,4

Metabolic activation:

not specified

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Any other information on results incl. tables

Table: Results of the gene mutation test with the test chemical

Concentration %	Treatment (hrs)	Survival %	Treated			Control
			No. of plates	Total no. of bacteria	Mutants/10^8bacteria	No. of plates	Total no. of bacteria	Mutants/10^8bacteria
0.003	3	13	10	1.2 X 10^8	11.8	4	8.5 X 10^7	8.3
0.002	3	12	8	8.0 X 10^7	70.0	4	7.0 X 10^7	147
0.002	3	11	8	1.1 X 10^8	28.7	4	8.4 X 10^7	35.4
0.005	3	7	9	4.6 X 10^7	10.9	4	5.4 X 10^7	33.1
0.002	3	1.9	8	2.2 X 10^7	74	4	9.2 X 10^7	98.1

Applicant's summary and conclusion

Conclusions:

The test chemical did not induce gene mutation in E. coli Strains B/Sd-4/1,3,4,5 and B/Sd-4/3,4 and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using E. coli Strains B/Sd-4/1,3,4,5 and B/Sd-4/3,4. The test chemical was dissolved in distilled water and used at dose levels of 0, 0.002, 0.002, 0.002, 0.003 or  0.005 %.

 

For every experiment, bacteria were grown for 24 hours at 37˚C in an aerated broth culture containing 10 micrograms of streptomycin per milliliter. They reached a saturation titer of approximately 2 to 3 x 10⁹cells per milliliter. Each culture was started from an inoculum, usually large, taken from streptomycin-agar slants kept in a refrigerator. Before treatment the bacteria were washed in saline and resuspended in distilled water. A sample of the new suspension was added to the desired solution of chemical in distilled water, and incubated at 37˚C for a certain period of time; no growth occurs under these conditions. Another sample of the same suspension was added to an equal amount of distilled water and incubated for the same period of time, as a control. At the end of the treatment period, both treated and control suspensions were assayed by plating suitable dilutions on streptomycin-agar plates. At the same time they were plated (0.1 ml per Petri dish), either undiluted or diluted not more than 1:10 in plain broth, onto a number of streptomycin-free plates, using a glass spreader and a turntable. The assay plates were incubated for 48 hours, after which it was possible to count the colonies and calculate the titers of the two suspensions at the end of treatment, and the percentage of survivors. The streptomycin-free plates (mutant plates) were incubated for at least six days. After this time the colonies were scored, and the frequency of mutants calculated by dividing the number of colonies by the number of (viable) bacteria plated.

 

The test chemical did not increase the frequency of revertant mutants in the strains of E. coli used. The test chemical did not induce gene mutation in E. coli Strains B/Sd-4/1,3,4,5 and B/Sd-4/3,4 and hence it is not likely to classify as a gene mutant in vitro.