Benzene-1,4-diammonium sulphate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Gene mutation in vitro:

Ames test:

The test chemical benzene-1,4-diammonium sulphate (CAS no 16245 -77 -5) did not induce gene mutation in Salmonella typhimurium strains in the presence and absence of S9 metabolci activation system and hence it is not likely to classify as a gene mutant in vitro.

Chromosome aberration test:

The test chemical benzene-1,4-diammonium sulphate (CAS no 16245 -77 -5) did not induce gene mutation in the cell line used at the HGPRT and Tk locus 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 of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across 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 structurally and functionally similar read across chemicals

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium, other: TA98, TA100, TA1535, TA1537, TA102

Remarks:

1

Details on mammalian cell type (if applicable):

Not applicable

Additional strain / cell type characteristics:

not specified

Species / strain / cell type:

S. typhimurium, other: TA98 and TA100

Remarks:

2/3

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:

Aroclor 1254- induced rat liver metabolic activation system (S-9)

Test concentrations with justification for top dose:

1. Range-finder Experiment and Mutation Experiment 1 Final concentration (μg/plate): 1.6, 8, 40, 200, 1000, 5000Mutation Experiment 2 Final concentration (μg/plate): 156.3, 312.5, 625, 1250, 2500, 50002. 0, 10, 100, 1000 or 10000 µg/plate

Vehicle / solvent:

1. - Vehicle(s)/solvent(s) used: purified water- Justification for choice of solvent/vehicle: test substance is a solid; dilution needed and very soluble in vehicle2. - Vehicle(s)/solvent(s) used: DMSO- Justification for choice of solvent/vehicle: The chemical was soluble in DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-nitrofluorene (TA98 -S9), sodium azide (TA100 and TA1535 -S9), 9-aminoacridine (TA1537 -S9), Mitomycin C (TA102 -S9), benzo[a]pyrene (TA98 +S9), 2- aminoanthracene (TA100, TA1535, TA1537, and TA102 +S9)

Remarks:

1

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-Aminoanthracene (All strains; +S9); NOP (All strains; -S9)

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; For all assays, bacteria were cultured for 10 hours at 37±1°C in nutrient broth (containing ampicillin for strains TA98 and TA100 and ampicillin and tetracycline for strain TA102).DURATION- Preincubation period: 2 hours- Exposure duration: 1 hour at 37±1°C- Expression time (cells in growth medium): Not reported- Selection time (if incubation with a selection agent): Not reported - Fixation time (start of exposure up to fixation or harvest of cells): Not reported SELECTION AGENT (mutation assays): The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation was prepared from male Sprague Dawley rats induced with Aroclor 1254.NUMBER OF REPLICATIONS: triplicate plates without and with S-9; negative (solvent) controls were included in each assay, in quintuplicate without and with S-9. In each experiment, bacterial strains were treated with diagnostic mutagens in triplicate in the absence of S-9.NUMBER OF CELLS EVALUATED: Colonies were counted electronically using a Seescan Colony Counter (Seescan plc) or manually where confounding factors such as split agar affected the accuracy of the automated counter.DETERMINATION OF CYTOTOXICITY - Method: relative total growth2. METHOD OF APPLICATION: preincubation assayDURATION- Preincubation period: No data available- Exposure duration: No data available- Expression time (cells in growth medium): No data available- Selection time (if incubation with a selection agent): No data available- Fixation time (start of exposure up to fixation or harvest of cells): No data availableSELECTION AGENT (mutation assays): No data availableSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: In triplicate for each strainNUMBER OF CELLS EVALUATED: No data availableDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, bacterial growth was observedOTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No data availableOTHER: The presence of a bacterial lawn indicated the absence or presence of gross toxicity.3. METHOD OF APPLICATION: in agar (plate incorporation)DURATION- Preincubation period: No data available- Exposure duration: No data available- Expression time (cells in growth medium): No data available- Selection time (if incubation with a selection agent): No data available- Fixation time (start of exposure up to fixation or harvest of cells): No data availableSELECTION AGENT (mutation assays): No data availableSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: In triplicate for each strainNUMBER OF CELLS EVALUATED: No data availableDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, bacterial growth was observedOTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No data availableOTHER: The presence of a bacterial lawn indicated the absence or presence of gross toxicity.

Rationale for test conditions:

No data

Evaluation criteria:

1. The test article was considered to be mutagenic if: 1) the assay was valid, 2) Dunnett's test gave a significant response (p ≤ 0.01) and the data set(s) showed a significant dose correlation, 3) the positive responses described above were reproducible.2. The plates were observed for a dose dependent increase in the number of revertants/plate

Statistics:

1. The m-statistic was calculated to check that the data were Poisson distributed, and Dunnett's test was used to compare the counts of each dose with the control. The presence or otherwise of a dose response was checked by linear regression analysis.2. A one-way analysis of variance and a pairwise comparison were conducted on the data.

Species / strain:

S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA102

Remarks:

1

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA100, TA1535, TA1537 and TA102

Remarks:

1

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Remarks:

1

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Species / strain:

S. typhimurium, other: TA98 and TA100

Remarks:

2/3

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

Additional information on results:

1. No data2/3. TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data available- Effects of osmolality: No data available- Evaporation from medium: No data available- Water solubility: No data available- Precipitation: No data available- Other confounding effects: No data availableRANGE-FINDING/SCREENING STUDIES: No data availableCOMPARISON WITH HISTORICAL CONTROL DATA: No data availableADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxicity was observed at a dose level of 10000 µg/plate in the absence of S9 metabolic activation system

Remarks on result:

other: No mutagenic potential

Conclusions:

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

Executive summary:

Data available for the test chemical was reviewed to determine the mutagenic nature of benzene-1,4-diammonium sulphate (CAS no 16245 -77 -5). The studies are as mentioned below:

Benzene-1,4-diamine was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver postmitochondrial fraction (S-9), in two separate experiments. Following the test substance treatments of all the tester strains, increases in revertant numbers were observed in strain TA98 in the presence of S-9 which were statistically significant when the data were analysed at the 1% level using Dunnett's test. These increases were dose-related and reproducible over two independent experiments, and were therefore considered to be indicative of the test substance’s mutagenic activity in this strain following metabolic activation. No comparable increases were seen in strain TA98 in the absence of S-9, and no other increases sufficient to be considered as indicative of mutagenic activity were observed in any of the other tester strains. The test substance induced mutation in Salmonella typhimurium strain TA98 in the presence of a rat liver metabolic activation system (S-9) when tested under the conditions employed in this study.

Gene mutation assay was performed to evaluate the mutagenic nature of the test chemical. Plate incorporation and preincubation assay was performed using S. typhimurium TA98 and TA100 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO at dose levels of 0, 10, 100, 1000 or 10000 µg/plate. Concurrent solvent and positive controls were run concurrently for each strain in each test. The plates were observed for a dose dependent increase in the number of revertants/plate. The test chemical did not induce gene mutation in theS. typhimuriumTA98 and TA100 in the presence and absence of S9 metabolic activation system and hence the chemical is negative for mutation in vitro.

Based on the data available for the test chemicals, benzene-1,4-diammonium sulphate (CAS no 16245 -77 -5) did not induce gene mutation in Salmonella typhimurium strains in the presence and absence of S9 metabolci activation system and hence it is not likely to classify as a gene mutant in vitro.

Reference 2

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 of read across substances

Justification for type of information:

Data for the target chemical is summarized based on the structurally similar read across chemicals

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 structurally and functionally similar read across chemicals

GLP compliance:

not specified

Type of assay:

other: Mammalian cell gene mutation assay

Target gene:

1. HGPRT locus2. Tk locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Remarks:

1

Details on mammalian cell type (if applicable):

- Type and identity of media: F12 medium (hypoxanthine-free F12 supplementedwith 5% dialyzed, heat-inactivated fetal bovine serum, 0.11% NaHC03 and 25 pg ml-' gentamicin sulfate).- Properly maintained: Yes- Periodically checked for Mycoplasma contamination: Yes- 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):

No data

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

No data

Metabolic activation:

with and without

Metabolic activation system:

S9 metabolic activation system

Test concentrations with justification for top dose:

1. Without S9: 0, 5, 10, 20, 30 µg/mLWith S9: 0, 100, 250, 500, 600, 700 µg/mL2. 1.0 - 15.0 μg/ml (without S9-mix)10.0 - 100.0 μg/ml (with S9-mix)

Vehicle / solvent:

1. - Vehicle(s)/solvent(s) used: DMSO/water- Justification for choice of solvent/vehicle: Chemical solubility2. - Vehicle(s)/solvent(s) used: NH4OH - Justification for choice of solvent/vehicle: The test chemical was soluble in NH4OH

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO/water

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: Without S9: N-methyl-N-nitro-N-nitrosoguanidine (MNNG), 4-Nitro-phenylenediamine (NPD) With S9: 3-Methylcholanthrene

Remarks:

1

Untreated negative controls:

yes

Remarks:

as per the guideline

Negative solvent / vehicle controls:

not specified

True negative controls:

not specified

Positive controls:

yes

Remarks:

as per the guideline

Positive control substance:

not specified

Remarks:

2

Details on test system and experimental conditions:

1. METHOD OF APPLICATION: in medium No. of cells: 1000000 cellsDURATION- Preincubation period: No data available- Exposure duration: 20-24 hrs without S9, 4 hrs with S9- Expression time (cells in growth medium): 6-7 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 availableSELECTION AGENT (mutation assays): No data availableSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: Triplicate in 2 independent experimentsNUMBER OF CELLS EVALUATED: Colonies of ca. 50 cells or more were scored.DETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, Toxicity wasmeasured in the main experiments as percentage relative total growth of the treated cultures relative to the total growth of the solvent control cultures.OTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No data availableOTHER: The exposed cells were subcultured in complete F12 at 1 X 106 cells per 100-mm dish for the mutant expression cultures and at 200 cells per 60-mm dish for the Day 1 parallel cytotoxicity test (Day 0 being treatment day). In cases where lower cell numbers were obtained because of toxicity, the total number of available cells was subcultured for mutant expression. After one day of incubation in complete F12, the medium of the mutant expression dishes was changed to low serum F12 (0.1% dialyzed, heat-inactivatedfetal bovine serum). The mutant expression cultures were incubated in low serum medium for 5-6 days with one medium change. After this period, the cultures were refed with complete F12. One day after changing to complete F12, the cells were subcultured into the mutant selection medium (complete F12 as defined previously plus 10 pM 6-thioguanine) at 0.2 x 106 cells per 100-mm dish (five dishes per culture) for mutant selection, and complete F12 at 200 cells per 60-mm dish (three dishes per culture) to determine the Day 9 survival. After 7 days of incubation, both mutation and survival dishes were fixed and stained.2. METHOD OF APPLICATION: in medium DURATION- Preincubation period: No data available- Exposure duration: 4 h- Expression time (cells in growth medium): 72 h- Selection time (if incubation with a selection agent): 11-13 days- Fixation time (start of exposure up to fixation or harvest of cells): No data availableSELECTION AGENT (mutation assays): No data availableSPINDLE INHIBITOR (cytogenetic assays): No data availableSTAIN (for cytogenetic assays): No data availableNUMBER OF REPLICATIONS: Triplicate in 2 independent experimentsNUMBER OF CELLS EVALUATED: No data availableDETERMINATION OF CYTOTOXICITY- Method: mitotic index; cloning efficiency; relative total growth; other: Yes, Toxicity wasmeasured in the main experiments as percentage relative total growth of the treated cultures relative to the total growth of the solvent control cultures.OTHER EXAMINATIONS:- Determination of polyploidy: No data available- Determination of endoreplication: No data available- Other: No data availableOTHER: No data available

Rationale for test conditions:

No data

Evaluation criteria:

1. The HGPRT mutant frequency was calculated on the basis of mutant colonies per 106 clonable cells. Clonable cells were estimated from the Day 9 parallel cytotoxicity tests. The mutation frequency for each of the treated groups was compared with the concurrent solvent control at the P ≤0 1 level of significance using the conditional binomial test' as recommended by Brusick. A chemical was considered to be positive in the CHO/HGPRT mutation assay if the mutation frequencies for two successive test chemical concentrations were at least 15 per 106 clonable cells and significantly higher than the solvent control value. A statistically significant increase at two successive concentrations was used as a criterion in lieu of a dose-response increase, which is often difficult to achieve with weak mutagens. The requirement for at least 15 mutant colonies per 106 clonable cells was based on our historical solvent control responses and data from a negative compound, anthracene, which displayed sporadic variations. Brusick has also reported a background mutant frequency of 1 to 10 x 10-6 and occasionally higher than 15 x 10-6 with the CHO/HGPRT mutation assay2. The treated cell line was observed to fix the DNA damage into a stable tk mutation

Statistics:

No data

Species / strain:

Chinese hamster Ovary (CHO)

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:

mouse lymphoma L5178Y cells

Remarks:

2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

The required toxicity was reached (10-20% survival compared to the concurrent negative controls) without S9-mix. In the presence of S9-mix the required level of toxicity was not achieved.

Vehicle controls validity:

not specified

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

1. TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data- Effects of osmolality: No data- Evaporation from medium: No data- Water solubility: No data- Precipitation: No data- Other confounding effects: No dataRANGE-FINDING/SCREENING STUDIES: Approximately 1 x 106 cells per 100-mm dish were seeded 2&24 h before exposure to test chemicals. A range of chemical concentrations was screened for toxicity without and with metabolic activation. The cells in complete F12 medium were exposed to test chemicals for 20-24 h without activation. The exposed cells were trypsinized, counted using a hemacytometer, and the total cell number per 100-mm dish was calculated. These cells were cloned at 200 cells per 60- mm dish (three dishes per culture) for the cytotoxicity determination. With the S9 activation system, the cellsin serum-free F12 medium (complete F12 without serum) containing S9 mix was exposed to test chemicalsfor ca. 4 h. Following the 4 h exposure with activation, the medium was changed to complete F12 medium and the cultures were incubated for an additional 16-20 h. The cells were counted and cloned as above. The cloning dishes from the treated cultures without or with S9 were incubated for 7 days without a medium change, then fixed and stained. Colonies of ca. 50 cells or more were scored. The number of viablecells was calculated by multiplying the total cell number per 100-mm dish by the cloning efficiency. Based on these results, four or five concentration levels (attempting to obtain 25-35% survival at the highconcentration) were chosen for the mutation assay unless toxicity was limited by insolubility.COMPARISON WITH HISTORICAL CONTROL DATA: No dataADDITIONAL INFORMATION ON CYTOTOXICITY: No data2. TEST-SPECIFIC CONFOUNDING FACTORS- Effects of pH: No data- Effects of osmolality: No data- Evaporation from medium: No data- Water solubility: No data- Precipitation: No data - Other confounding effects: No dataRANGE-FINDING/SCREENING STUDIES:Test concentrations were based on the results of a pre-test on toxicity measuring relative suspension growth. COMPARISON WITH HISTORICAL CONTROL DATA: No dataADDITIONAL INFORMATION ON CYTOTOXICITY: No data

Remarks on result:

other: No mutagenic potential

Conclusions:

The test chemical did not induce gene mutation in the cell line used at the HGPRT and Tk locus 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 test chemical was reviewed to determine the mutagenic nature of Benzene-1,4-diammonium sulphate (CAS no 16245 -77 -5). The studies are as mentioned below:

Mammalian cell gene mutation assay was conducted for the test chemical. The study was performed using the CHO cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO or water and used at dose level of0, 5, 10, 20, 30µg/mLwithout S9 and 0, 100, 250, 500, 600, 700µg/mLwith S9-mix. Approximately 1 x 10⁶ cells per 100-mm dish were seeded 2&24 h before exposure to test chemicals. A range of chemical concentrations was screened for toxicity without and with metabolic activation. The cells in complete F12 medium were exposed to test chemicals for 20-24 h without activation.The exposed cells were subcultured in complete F12 at 1 X 10⁶cells per 100-mm dish for the mutant expression cultures and at 200 cells per 60-mm dish for the Day 1 parallel cytotoxicity test (Day 0 being treatment day). In cases where lower cell numbers were obtained because of toxicity, the total number of available cells was subcultured for mutant expression. After one day of incubation in complete F12, the medium of the mutant expression dishes was changed to low serum F12 (0.1% dialyzed, heat-inactivatedfetal bovine serum). The mutant expression cultures were incubated in low serum medium for 5-6 days with one medium change. After this period, the cultures were refed with complete F12. One day after changing to complete F12, the cells were subcultured into the mutant selection medium (complete F12 as defined previously plus 10 pM 6-thioguanine) at 0.2 x 10⁶cells per 100-mm dish (five dishes per culture) for mutant selection, and complete F12 at 200 cells per 60-mm dish (three dishes per culture) to determine the Day 9 survival. After 7 days of incubation, both mutation and survival dishes were fixed and stained.A chemical was considered to be positive in the CHO/HGPRT mutation assay if the mutation frequencies for two successive test chemical concentrations were at least 15 per 10⁶clonable cells and significantly higher than the solvent control value. The test chemical did not induce gene mutation in the CHO cells at the HGPRT locus 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, Mammalian cell gene mutation assay was conducted for the test chemical as per the OECD 476. The study was performed using the L5178Y Mouse lymphoma cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in ammonium hydroxide and used at dose level of 1.0 - 15.0 μg/ml without S9 and 10.0 - 100.0 μg/ml with S9-mix. The doses for main study were based on the preliminary toxicity study. The cells were treated for 4 hrs with the test chemical an allowed to express for 72 hrs. Selection period was 11-13 days. Concurrent negative and positive control chemicals were also included in the study. In the pre-experiment on toxicity distinct toxic effects could be observed with concentrations higher than 3.0 μg/ml. In both experiments the required toxicity was reached (10-20% survival compared to the concurrent negative controls) without S9-mix. In the presence of S9-mix the required level of toxicity was not achieved. Occasionally an increase in mutant frequency was observed in both experiments with and without S9-mix. However, these results appeared not reproducible and are, therefore, considered as not biologically relevant. Under the experimental conditions used, the test chemical did not induce gene mutations in this gene mutation test in mammalian cells.

Based on the observations made, the test chemical did not induce gene mutation in the cell line used at the HGPRT and Tk locus 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 test chemical was reviewed to determine the mutagenic nature of benzene-1,4-diammonium sulphate (CAS no 16245 -77 -5). The studies are as mentioned below:

Ames test:

Benzene-1,4-diamine was assayed for mutation in five histidine-requiring strains (TA98, TA100, TA1535, TA1537 and TA102) of Salmonella typhimurium, both in the absence and in the presence of metabolic activation by an Aroclor 1254-induced rat liver postmitochondrial fraction (S-9), in two separate experiments. Following the test substance treatments of all the tester strains, increases in revertant numbers were observed in strain TA98 in the presence of S-9 which were statistically significant when the data were analysed at the 1% level using Dunnett's test. These increases were dose-related and reproducible over two independent experiments, and were therefore considered to be indicative of the test substance’s mutagenic activity in this strain following metabolic activation. No comparable increases were seen in strain TA98 in the absence of S-9, and no other increases sufficient to be considered as indicative of mutagenic activity were observed in any of the other tester strains. The test substance induced mutation in Salmonella typhimurium strain TA98 in the presence of a rat liver metabolic activation system (S-9) when tested under the conditions employed in this study.

In another study, gene mutation assay was performed to evaluate the mutagenic nature of the test chemical. Plate incorporation and preincubation assay was performed using S. typhimurium TA98 and TA100 in the presence and absence of S9 metabolic activation system. The chemical was dissolved in DMSO at dose levels of 0, 10, 100, 1000 or 10000 µg/plate. Concurrent solvent and positive controls were run concurrently for each strain in each test. The plates were observed for a dose dependent increase in the number of revertants/plate. The test chemical did not induce gene mutation in theS. typhimuriumTA98 and TA100 in the presence and absence of S9 metabolic activation system and hence the chemical is negative for mutation in vitro.

Based on the data available for the test chemicals, benzene-1,4-diammonium sulphate (CAS no 16245 -77 -5) did not induce gene mutation in Salmonella typhimurium strains in the presence and absence of S9 metabolci activation system and hence it is not likely to classify as a gene mutant in vitro.

Chromosome aberration study:

Mammalian cell gene mutation assay was conducted for the test chemical. The study was performed using the CHO cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in DMSO or water and used at dose level of0, 5, 10, 20, 30µg/mLwithout S9 and 0, 100, 250, 500, 600, 700µg/mLwith S9-mix. Approximately 1 x 10⁶cells per 100-mm dish were seeded 2&24 h before exposure to test chemicals. A range of chemical concentrations was screened for toxicity without and with metabolic activation. The cells in complete F12 medium were exposed to test chemicals for 20-24 h without activation.The exposed cells were subcultured in complete F12 at 1 X 10⁶cells per 100-mm dish for the mutant expression cultures and at 200 cells per 60-mm dish for the Day 1 parallel cytotoxicity test (Day 0 being treatment day). In cases where lower cell numbers were obtained because of toxicity, the total number of available cells was subcultured for mutant expression. After one day of incubation in complete F12, the medium of the mutant expression dishes was changed to low serum F12 (0.1% dialyzed, heat-inactivatedfetal bovine serum). The mutant expression cultures were incubated in low serum medium for 5-6 days with one medium change. After this period, the cultures were refed with complete F12. One day after changing to complete F12, the cells were subcultured into the mutant selection medium (complete F12 as defined previously plus 10 pM 6-thioguanine) at 0.2 x 10⁶cells per 100-mm dish (five dishes per culture) for mutant selection, and complete F12 at 200 cells per 60-mm dish (three dishes per culture) to determine the Day 9 survival. After 7 days of incubation, both mutation and survival dishes were fixed and stained.A chemical was considered to be positive in the CHO/HGPRT mutation assay if the mutation frequencies for two successive test chemical concentrations were at least 15 per 10⁶clonable cells and significantly higher than the solvent control value. The test chemical did not induce gene mutation in the CHO cells at the HGPRT locus 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, Mammalian cell gene mutation assay was conducted for the test chemical as per the OECD 476. The study was performed using the L5178Y Mouse lymphoma cells in the presence and absence of S9 metabolic activation system. The test chemical was dissolved in ammonium hydroxide and used at dose level of 1.0 - 15.0 μg/ml without S9 and 10.0 - 100.0 μg/ml with S9-mix. The doses for main study were based on the preliminary toxicity study. The cells were treated for 4 hrs with the test chemical an allowed to express for 72 hrs. Selection period was 11-13 days. Concurrent negative and positive control chemicals were also included in the study. In the pre-experiment on toxicity distinct toxic effects could be observed with concentrations higher than 3.0 μg/ml. In both experiments the required toxicity was reached (10-20% survival compared to the concurrent negative controls) without S9-mix. In the presence of S9-mix the required level of toxicity was not achieved. Occasionally an increase in mutant frequency was observed in both experiments with and without S9-mix. However, these results appeared not reproducible and are, therefore, considered as not biologically relevant. Under the experimental conditions used, the test chemical did not induce gene mutations in this gene mutation test in mammalian cells.

Based on the observations made, the test chemical did not induce gene mutation in the cell line used at the HGPRT and Tk locus in the presence and absence of S9 metabolic activation system and hence it is not likely to classify as a gene mutant in vitro.

Based on the data available for the test chemical and applying weight of evidence approach, Benzene-1,4-diammonium sulphate (CAS no 16245 -77 -5) does not exhibit gene mutation in vitro. 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

Based on the data available for the test chemical and applying weight of evidence approach, Benzene-1,4-diammonium sulphate (CAS no 16245 -77 -5) does not exhibit gene mutation in vitro. Hence the test chemical is not likely to classify as a gene mutant as per the criteria mentioned in CLP regulation.