Chlorosulphuric acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test:

The test chemical did not induce gene mutation in Salmonella typhimurium and E.coli strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro chromosome aberration study:

The test chemical induced chromosomal aberration in the Chinese hamster ovary K1 (CHO-K1) cell line used in the presence and absence of S9 metabolic activation system and hence it is likely to be clastogenic in vitro.

In vitro mammalian cell gene mutation assay:

The test chemical did not induce mammalian cell gene mutation assay in Fischer L5178Y mouse-lymphoma cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

1. Histidine
2. Streptomycin
3. Histidine

Species / strain / cell type:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Remarks:

1

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

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

Remarks:

2

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

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

Remarks:

3

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9 metabolic activation system

Test concentrations with justification for top dose:

1. 0, 0.001, 0.01, 0.1, 1.0, 5.0 uL/plate
2. 0, 0.002, 0.002, 0.002, 0.003 or 0.005 %
3. trial 1 and 2: 10 - 1000 ug/plate
trial 2a: 10 - 5000 ug/plate
trial 2b: 100 - 3333 ug/plate
(only with TA 98 / TA 100 without S9-mix)

Vehicle / solvent:

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

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

3. - Vehicle(s)/solvent(s) used: ethylene glycol dimethylether (EGDE)
- Justification for choice of solvent/vehicle: Test chemical solubility in solvent

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Deionized water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: NF (TA98, TA1538; -S9), QM (TA1537; -S9), 2 AA (All strains; +S9)

Remarks:

1

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

Remarks:

2

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

ethylene glycol dimethylether (EGDE)

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

other: 4-nitro-o-phenylene-diamine (TA 98, TA 1537; -S9) and 2-aminoanthracene (TA 98, TA 100, TA 1535, TA 1537; +S9)

Remarks:

3

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: 48 hrs
- Expression time (cells in growth medium): 48 hrs
- Selection time (if incubation with a selection agent): 48 hrs
- 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: One

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

2. 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)
Sterptomycin 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

3. METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: No data
- Exposure duration: No data
- Expression time: No data
- 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: Triplicate

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:

1. The result was considered positive if the test material produced a positive dose-related response over three concentrations with the lowest increase equal to three times the spontaneous revertant rate for TA1535, TA1537 or TA1538 strains while for TA98 and TA100 strains a doubling of the rate at the highest increase over three dose levels was considered positive.

2. 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

3. The plates were observed for number of revertants/plate

Statistics:

1. No data
2. Poisson distribution was used to estimate the variance and the statistical significance of the result was evaluated
3. No data

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537, TA 1538

Remarks:

1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

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

Remarks:

2

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:

not specified

Species / strain:

S. typhimurium, other: TA 98, TA 1535, TA 1537

Remarks:

3

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥3333 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Remarks:

3

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥3333 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Remarks:

3

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥3333 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

1. No data

2. TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data on pH values given
- Effects of osmolality: No data
- Evaporation from medium: No data
- Water solubility: No data
- Precipitation: 5000 ug/plate
- Other confounding effects: No data

RANGE-FINDING/SCREENING STUDIES: No data

COMPARISON WITH HISTORICAL CONTROL DATA: No data

ADDITIONAL INFORMATION ON CYTOTOXICITY: No data

3. No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce gene mutation in Salmonella typhimurium and E.coli strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Bacterial gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in deionized water and used at dose level of 0, 0.001, 0.01, 0.1, 1.0, 5.0 uL/plate. The study was performed as per the plate incorporation assay and the number of revertant colonies was scored after incubation at 37°C for 48 hr. The result was considered positive if the test material produced a positive dose-related response over three concentrations with the lowest increase equal to three times the spontaneous revertant rate for TA1535, TA1537 or TA1538 strains while for TA98 and TA100 strains a doubling of the rate at the highest increase over three dose levels was considered positive.The test chemical did not induce gene mutation in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another study, 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.

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical. The study was performed using Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 in the presence and absence of S9 metabolic activation system. The test chemical was soluble inethylene glycol dimethylether (EGDE) and used at dose level of 10 - 1000 ug/plate in trial 1 and 2, 10 - 5000 ug/plate in trial 2a and 100 - 3333 ug/plate in trial 2b with TA 98 / TA 100 without S9-mix. Concurrent solvent and positive control chemicals were also included in the study. Significant and reproducible dose-dependent increase up to the highest investigated concentration only in TA 100 without S9-mix. No effects were observed in the strains TA 98, TA 1535 and TA 1537. Based on the observations made, the test chemical did not induce gene mutation inSalmonella typhimurium TA 98, TA 1535, TA 1537 in the presence and absence of S9 metabolic activation system. It however induced gene mutation in the strain TA100 in the absence of S9 metabolic activation system. Overall the test chemical is not likely to classify as a gene mutant in vitro.

Based on the data available, the test chemical did not induce gene mutation in Salmonella typhimurium and E.coli strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the various test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

No data

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

1

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were grown in Fischer's Medium for mouse leukaemic cells supplemented with 10% horse serum, sodium pyruvate and penicillin-streptomycin. The cloning medium consisted of growth medium with 20% horse serum and 0.37% agar.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes, Cell stocks were determined to be mycoplasma free.
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1 / 2 / 3

Details on mammalian cell type (if applicable):

- Type and identity of media: Ham's F12 medium supplemented with sodium bicarbonate (16.7 mM), kanamycin (60 µg/ml) and 10% fetal calf serum (FCS).
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes, The cells were confirmed to be free of mycoplasma.
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Liver S-9 homogenate prepared from random bred male CD-1 mice

Test concentrations with justification for top dose:

1. Without activation : 0, 0.1, 0.2, 0.4 uL/mL (1.2, 2.4, 4.8 mM)
With activation : 0, 0.2, 0.4, 0.8 uL/mL (2.4, 4.8, 9.6 mM)

2. With S9: 0, 2, 4 , 6 or 8 mM
Without S9: 0, 2, 4 , 6 , 8 or 10 mM

3. With S9: 0, 6, 8, 10 or 12 mM
Without S9: 0, 8, 10, 12, 14 or 16 mM

Vehicle / solvent:

1. - Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: The test chemical was soluble in ethanol

2./3.
- Vehicle(s)/solvent(s) used: No data
- Justification for choice of solvent/vehicle: No data

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

Ethanol

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Remarks:

1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Remarks:

2./3.

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 20 hrs
- Selection time (if incubation with a selection agent): 3 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): No data

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

NUMBER OF REPLICATIONS: One

NUMBER OF CELLS EVALUATED: 50 cells at each dose level

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

2./3. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: With S9: 6 hrs
Without S9: 24 hrs
- Expression time: 18 hrs
- 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: 100 metaphases were evaluated

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:

1. Compounds that induced aberrations in 8% or more of the cells were considered to possess clastogenic (chromosome damaging) potential.
2./3. The cell line was observed for chromosome aberration studies

Statistics:

1. In chromosome aberration studies, a value of 8% or more of the cells with aberrations was considered the lower limit of significance based on a concurrent and historical control frequency.

2./3. No data

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

Chinese hamster Ovary (CHO)

Remarks:

CHO-K1 / 2 / 3

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce chromosome aberrations in the mammalian cell line in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical. The study was performed using Fischer L5178Y mouse-lymphoma cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in ethanol and used at dose level of 0, 0.1-0.4 and 0.2-0.8µg/mL. The cell line was exposed to the test chemical for 4 hrs. BUdR was added to treated cells for 20 hr, or two cell doublings, to permit its incorporation into the DNA. The cells were then prepared for Chromosome aberration staining. Metaphase arrest was accomplished by adding colchicine (0.5µg/ml final concentration) to the growing cultures during the last 3 hr of incubation. The cells were harvested with 0.075 M-hypotonic KCl solution followed by fixing in three changes of Carnoy's fixative (methanol-glacial acetic acid, 3:1). The rinsed slides were then air-dried overnight. 50 metaphase cells were evaluated for chromosome aberration. Concurrent solvent and positive control chemicals were also included in the study. Based on the observations made,The test chemical did not induce chromosome aberrations inFischer L5178Y mouse-lymphoma cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another study, in vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of two test chemicals. The study was performed using Chinese hamster ovary K1 (CHO-K1) cells in the presence and absence of S9 metabolic activation system. The cell line was maintained in Ham's F12 medium supplemented with sodium bicarbonate (16.7 mM), kanamycin (60µg/ml) and 10% fetal calf serum (FCS). The cultures were incubated at 37°C in an atmosphere of 5°70 CO2 in air and subcultures were made every 3-4 days. The cells were confirmed to be free of mycoplasma. One test chemical was tested at dose level of 0, 2, 4, 8 or 10 mM in the absence of S9 and 0, 2, 4, 6 or 8 mM in the presence of S9. Another test chemical was tested at concentration of 0, 6, 8, 10 or 12 mM in the presence of S9 metabolic activation system and at dose level of 0, 8, 10, 12, 14 or 16 mM.The test chemical was added to the F12 medium, and subsequently the pH of the medium was measured. The pH of the medium was measured prior to and after treatment using a pH meter. In the absence of S9 mix,the test chemical was added to the 3-day-old cultures after a medium change for 24 hrs. In metabolic activation with S9 mix, cells were treated with S9 mix and the test chemical for 6 h. Recovery times were 18 h. The cells were then cultured for 24 h. In all experiments, chromosome preparations were made 24 h after the start of treatment. The number of cells with chromosomal aberrations was recorded on 100 metaphases. Chromatid breaks and exchanges were observed. Based on the observations made, the test chemical induced chromosomal aberration in the Chinese hamster ovary K1 (CHO-K1) cell line used in the presence and absence of S9 metabolic activation system and hence it is likely to be clastogenic in vitro. The results of the study indicate that low pH can cause chromosomal effects in this assay; findings seen with the inorganic acids tested are therefore considered to be artefactual.

Based on the observations made, the test chemical induced chromosomal aberration in the Chinese hamster ovary K1 (CHO-K1) cell line used in the presence and absence of S9 metabolic activation system and hence it is likely to be clastogenic in vitro.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Remarks:

Experimental data from various test chemicals

Justification for type of information:

Data for the target chemical is summarized based on the variuos test chemicals

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

other: Refer below principle

Principles of method if other than guideline:

WoE derived based on the experimental data from various test chemicals

GLP compliance:

not specified

Type of assay:

other: In vitro mammalian chromosome aberration test

Target gene:

Tk locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

Fischer cells / 1

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were grown in Fischer's Medium for mouse leukaemic cells supplemented with 10% horse serum, sodium pyruvate and penicillin-streptomycin. The cloning medium consisted of growth medium with 20% horse serum and 0.37% agar.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes, Cell stocks were determined to be mycoplasma free.
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

mouse lymphoma L5178Y cells

Remarks:

2

Details on mammalian cell type (if applicable):

- Type and identity of media: Stock cultures were maintained in Fischer's medium for leukemic cells of mice at approx. 37 °C on gyratory tables. Fischer's medium (F0) was supplemented with 2 mM glutamine (HEM), 110 µg/ml sodium pyruvate 0.05% Pluronic F68 and and 10% by volume of heat-inactivated donor horse serum.
- Properly maintained: No data
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

Liver S-9 homogenate prepared from random bred male CD-1 mice

Test concentrations with justification for top dose:

1. Without activation: 0.4, 0.5, 0.6, 0.7, 0.8 µL/mL (4.8-9.6 mM)
With activation: 0.8, 1.0, 1.2, 1.4, 1.6 µL/mL (9.6-23.0 mM)

2. With metabolic activation: pH 6.3, 6.6, 6.9, 7.2, 7.5
Without metabolic activation: pH 6.0, 6.2, 6.5, 6.7, 6.9, 7.0

Vehicle / solvent:

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

2. No data

Untreated negative controls:

yes

Remarks:

Medium

Negative solvent / vehicle controls:

yes

Remarks:

Water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: With activation: Dimethylnitrosoamine 0.3 µL/mL

Remarks:

1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): 10 days
- 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: One

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

2. METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: No data
- Exposure duration: 4 hrs
- Expression time: 2 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: Duplicate

NUMBER OF CELLS EVALUATED: 100 metaphases were evaluated

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:

1. A dose-response relationship over three concentrations of the four dose levels employed is observed, or an increase in the mutation frequencies at the highest dose is at least 2.5 times greater than the solvent control value.

2. The cell line was observed for gene mutation at tk locus

Statistics:

No data

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

Fischer cells / 1

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

With metabolic activation: 0.8 µL/mL (100% growth inhibition) and Without metabolic activation: Ca 1.0 µL/mL (12.5% growth inhibition at 0.8 µL/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

mouse lymphoma L5178Y cells

Remarks:

2

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce mammalian cell gene mutation assay in Fischer L5178Y mouse-lymphoma cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Executive summary:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using Fischer L5178Y mouse-lymphoma cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in water and used at dose level of 0.4, 0.5, 0.6, 0.7, 0.8 uL/mL (4.8-9.6 mM) in the presence of S9 and 0.8, 1.0, 1.2, 1.4, 1.6 uL/mL (9.6-23.0 mM) in the absence of S9. The cell line was exposed to the test chemical for 4 hrs in the presence and absence of S9 metabolic activation system. The culture was allowed to express the TK- mutation in growth medium for 3 days prior to mutant determination. Cells were counted daily and diluted to a concentration of 3 x 10⁵cells/ml, considered optimal for growth. After 3 days, an aliquot of 3 x 10⁶cells at each dose was seeded into the selection medium and cultures were incubated for 10 days. Mutant colonies (resistant to BUdR) were stained and counted in an Artek electronic colony counter, set to discriminate colonies 200 pm or larger in size. A portion of this first aliquot culture was diluted to give 300 cells/plate prior to the addition of BUdR and seeded into non-selective medium to measure cell survival. Mutant cell counts from the selection plates were corrected for cell survival and expressed as a mutant frequency/clonable cell. Based on the observations made, thetest chemical did not induce mammalian cell gene mutation assay in Fischer L5178Y mouse-lymphoma cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical. The study was performed using mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system. The test chemical was used at pH 6.3, 6.6, 6.9, 7.2, 7.5 in the presence of S9 metabolic activation system and at pH 6.0, 6.2, 6.5, 6.7, 6.9, 7.0 in the absence of S9 metabolic activation system.A series of sterile, 15-ml conical centrifuge tubes were inoculated with 3 x 10⁶ cells per tube. The cells were sedimented by centrifugation at approx. 150 x g for 10 min. The cells were then resuspended in medium that had been adjusted to the appropriate pH. RPMI 1640 medium containing 10% heat-inactivated horse serum was used for assays in which the pH was controlled with organic buffers. All other experiments were suspended in F10 medium. The total volume for treatment was 10 ml; for activation assays, the 10-ml volume included the activation mix.The dosed tubes were placed in a shaker incubator at approximately 80 rpm at 37 ° C. After 4 h, the cells were washed twice and resuspended in 10 ml of F10 and returned to the shaker incubator. A standard expression time of 2 days was used. On day 1, the viable cell count was determined for each culture and the cell densities were adjusted to 3 × 10⁵cells/ml. On day 2, each tube was adjusted to contain a 10-ml cell suspension at 3 × 10⁶cells/ml. A sample containing 3 × 106 cells was added to 100 ml of Fl0 medium containing trifluorothymidine (TFT) and approx. 0.3% Noble agar (Difco, Detroit, MI). Each sample was then poured into a set of three 100-nm culture dishes (Falcon). A sample of 300 cells from each culture was also seeded into a set of three 100-mm culture dishes in F10 medium containing 0.3% agar without any selective agent in order to determine cloning efficiencies. The dishes were then incubated for 10-12 days in a 37°C incubator with 5% CO2/humidified atmosphere for colony development. At the end of the incubation period, colonies were scored on an Artek 880 electronic colony counter. Based on the observations made,the test chemical induced gene mutation at tk locus inmouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system and hence it is likely to be mutagenic in vitro. The results of the study indicate that low pH can cause gene mutation in this assay; findings seen with the inorganic acids tested are therefore considered to be artefactual.

Based on the observations made, the test chemical did not induce mammalian cell gene mutation assay in Fischer L5178Y mouse-lymphoma cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Gene mutation in vitro:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

Ames test:

Bacterial gene mutation assay was performed to determine the mutagenic nature of the test chemical 1. The study was performed using Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in deionized water and used at dose level of 0, 0.001, 0.01, 0.1, 1.0, 5.0 uL/plate. The study was performed as per the plate incorporation assay and the number of revertant colonies was scored after incubation at 37°C for 48 hr. The result was considered positive if the test material produced a positive dose-related response over three concentrations with the lowest increase equal to three times the spontaneous revertant rate for TA1535, TA1537 or TA1538 strains while for TA98 and TA100 strains a doubling of the rate at the highest increase over three dose levels was considered positive.The test chemical did not induce gene mutation in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, TA 1538 in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another study, gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical 2. 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.

Gene mutation toxicity study was performed to determine the mutagenic nature of the test chemical 3. The study was performed using Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537 in the presence and absence of S9 metabolic activation system. The test chemical was soluble inethylene glycol dimethylether (EGDE) and used at dose level of 10 - 1000 ug/plate in trial 1 and 2, 10 - 5000 ug/plate in trial 2a and 100 - 3333 ug/plate in trial 2b with TA 98 / TA 100 without S9-mix. Concurrent solvent and positive control chemicals were also included in the study. Significant and reproducible dose-dependent increase up to the highest investigated concentration only in TA 100 without S9-mix. No effects were observed in the strains TA 98, TA 1535 and TA 1537. Based on the observations made, the test chemical did not induce gene mutation inSalmonella typhimurium TA 98, TA 1535, TA 1537 in the presence and absence of S9 metabolic activation system. It however induced gene mutation in the strain TA100 in the absence of S9 metabolic activation system. Overall the test chemical is not likely to classify as a gene mutant in vitro.

Based on the data available, the test chemical did not induce gene mutation in Salmonella typhimurium and E.coli strains in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro chromosome aberration study:

Data available for the various test chemicals was reviewed to determine the mutagenic nature of the test chemical. The studies are as mentioned below:

In vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of the test chemical 1. The study was performed using Fischer L5178Y mouse-lymphoma cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in ethanol and used at dose level of 0, 0.1-0.4 and 0.2-0.8µg/mL. The cell line was exposed to the test chemical for 4 hrs. BUdR was added to treated cells for 20 hr, or two cell doublings, to permit its incorporation into the DNA. The cells were then prepared for Chromosome aberration staining. Metaphase arrest was accomplished by adding colchicine (0.5µg/ml final concentration) to the growing cultures during the last 3 hr of incubation. The cells were harvested with 0.075 M-hypotonic KCl solution followed by fixing in three changes of Carnoy's fixative (methanol-glacial acetic acid, 3:1). The rinsed slides were then air-dried overnight. 50 metaphase cells were evaluated for chromosome aberration. Concurrent solvent and positive control chemicals were also included in the study. Based on the observations made,The test chemical did not induce chromosome aberrations inFischer L5178Y mouse-lymphoma cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In another study, in vitro mammalian chromosome aberration study was performed to determine the mutagenic nature of two test chemicals 1 and 2. The study was performed using Chinese hamster ovary K1 (CHO-K1) cells in the presence and absence of S9 metabolic activation system. The cell line was maintained in Ham's F12 medium supplemented with sodium bicarbonate (16.7 mM), kanamycin (60µg/ml) and 10% fetal calf serum (FCS). The cultures were incubated at 37°C in an atmosphere of 5°70 CO2 in air and subcultures were made every 3-4 days. The cells were confirmed to be free of mycoplasma. One test chemical was tested at dose level of 0, 2, 4, 8 or 10 mM in the absence of S9 and 0, 2, 4, 6 or 8 mM in the presence of S9. Another test chemical was tested at concentration of 0, 6, 8, 10 or 12 mM in the presence of S9 metabolic activation system and at dose level of 0, 8, 10, 12, 14 or 16 mM.The test chemical was added to the F12 medium, and subsequently the pH of the medium was measured. The pH of the medium was measured prior to and after treatment using a pH meter. In the absence of S9 mix,the test chemical was added to the 3-day-old cultures after a medium change for 24 hrs. In metabolic activation with S9 mix, cells were treated with S9 mix and the test chemical for 6 h. Recovery times were 18 h. The cells were then cultured for 24 h. In all experiments, chromosome preparations were made 24 h after the start of treatment. The number of cells with chromosomal aberrations was recorded on 100 metaphases. Chromatid breaks and exchanges were observed. Based on the observations made, the test chemical induced chromosomal aberration in the Chinese hamster ovary K1 (CHO-K1) cell line used in the presence and absence of S9 metabolic activation system and hence it is likely to be clastogenic in vitro. The results of the study indicate that low pH can cause chromosomal effects in this assay; findings seen with the inorganic acids tested are therefore considered to be artefactual.

Based on the observations made, the test chemical induced chromosomal aberration in the Chinese hamster ovary K1 (CHO-K1) cell line used in the presence and absence of S9 metabolic activation system and hence it is likely to be clastogenic in vitro.

In vitro mammalian cell gene mutation study:

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical 1. The study was performed using Fischer L5178Y mouse-lymphoma cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in water and used at dose level of 0.4, 0.5, 0.6, 0.7, 0.8 uL/mL (4.8-9.6 mM) in the presence of S9 and 0.8, 1.0, 1.2, 1.4, 1.6 uL/mL (9.6-23.0 mM) in the absence of S9. The cell line was exposed to the test chemical for 4 hrs in the presence and absence of S9 metabolic activation system. The culture was allowed to express the TK- mutation in growth medium for 3 days prior to mutant determination. Cells were counted daily and diluted to a concentration of 3 x 10⁵cells/ml, considered optimal for growth. After 3 days, an aliquot of 3 x 10⁶cells at each dose was seeded into the selection medium and cultures were incubated for 10 days. Mutant colonies (resistant to BUdR) were stained and counted in an Artek electronic colony counter, set to discriminate colonies 200 pm or larger in size. A portion of this first aliquot culture was diluted to give 300 cells/plate prior to the addition of BUdR and seeded into non-selective medium to measure cell survival. Mutant cell counts from the selection plates were corrected for cell survival and expressed as a mutant frequency/clonable cell. Based on the observations made, thetest chemical did not induce mammalian cell gene mutation assay in Fischer L5178Y mouse-lymphoma cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

In vitro mammalian cell gene mutation assay was performed to determine the mutagenic nature of the test chemical 2. The study was performed using mouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system. The test chemical was used at pH 6.3, 6.6, 6.9, 7.2, 7.5 in the presence of S9 metabolic activation system and at pH 6.0, 6.2, 6.5, 6.7, 6.9, 7.0 in the absence of S9 metabolic activation system.A series of sterile, 15-ml conical centrifuge tubes were inoculated with 3 x 10⁶cells per tube. The cells were sedimented by centrifugation at approx. 150 x g for 10 min. The cells were then resuspended in medium that had been adjusted to the appropriate pH. RPMI 1640 medium containing 10% heat-inactivated horse serum was used for assays in which the pH was controlled with organic buffers. All other experiments were suspended in F10 medium. The total volume for treatment was 10 ml; for activation assays, the 10-ml volume included the activation mix.The dosed tubes were placed in a shaker incubator at approximately 80 rpm at 37 ° C. After 4 h, the cells were washed twice and resuspended in 10 ml of F10 and returned to the shaker incubator. A standard expression time of 2 days was used. On day 1, the viable cell count was determined for each culture and the cell densities were adjusted to 3 × 10⁵cells/ml. On day 2, each tube was adjusted to contain a 10-ml cell suspension at 3 × 10⁶cells/ml. A sample containing 3 × 106 cells was added to 100 ml of Fl0 medium containing trifluorothymidine (TFT) and approx. 0.3% Noble agar (Difco, Detroit, MI). Each sample was then poured into a set of three 100-nm culture dishes (Falcon). A sample of 300 cells from each culture was also seeded into a set of three 100-mm culture dishes in F10 medium containing 0.3% agar without any selective agent in order to determine cloning efficiencies. The dishes were then incubated for 10-12 days in a 37°C incubator with 5% CO2/humidified atmosphere for colony development. At the end of the incubation period, colonies were scored on an Artek 880 electronic colony counter. Based on the observations made,the test chemical induced gene mutation at tk locus inmouse lymphoma L5178Y cells in the presence and absence of S9 metabolic activation system and hence it is likely to be mutagenic in vitro. The results of the study indicate that low pH can cause gene mutation in this assay; findings seen with the inorganic acids tested are therefore considered to be artefactual.

Based on the observations made, the test chemical did not induce mammalian cell gene mutation assay in Fischer L5178Y mouse-lymphoma cells in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

On the basis of observations made and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. This chemical undergoes immediate hydrolysis in water of body fluids after contact with any tissue. The hydrolysis products are test chemical 1 and 2. Some mutagenic effects though observed but results of the studies performed with inorganic acids test chemical 1 and 2 indicate that low pH can cause false positive effects in these assay. Hence further testing is not scientifically justified. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.

Justification for classification or non-classification

On the basis of observations made and applying the weight of evidence approach, the test chemical does not exhibit gene mutation in vitro. This chemical undergoes immediate hydrolysis in water of body fluids after contact with any tissue. The hydrolysis products are test chemical 1 and 2. Some mutagenic effects though observed but results of the studies performed with inorganic acids test chemical 1 and 2 indicate that low pH can cause false positive effects in these assay. Hence further testing is not scientifically justified. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.