Hydroxylamine-O-sulphonic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Equivocal effects, in vitro bacterial mutagenicity testings of the test substance hydroxylamine-O-sulphonic acid (CAS 2950-43-8) led to contradictory results. No data available for mutagenicity testing in mammalian cells.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Details on mammalian cell type (if applicable):

- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-mix prepared from Wistar rat livers

Test concentrations with justification for top dose:

Exp 1: 0; 33; 100; 333; 1000; 2500; 5000 µg/plate (all strains/standard plate test)
Exp 2: 0; 10; 33; 100; 333; 1000; 2500 µg/plate (all strains/preincubation test)
Exp 3: 0; 3.3; 10; 33; 100; 333; 1000 µg/plate (TA 98/preincubation test)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: due to the good solubility of the test substance

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

other: 2-aminoanthracene; N-methyl-N`-nitro-N-nitrosoguanidine; 4-nitro-o-phenylenediamine

Details on test system and experimental conditions:

Standard plate test
Salmonella typhimurium
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM histidine + 0.5 mM biotin) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were be poured onto Minimal glucose agar plates within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager. Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Escherichia coli
Test tubes containing 2-mL portions of soft agar (overlay agar), which consists of 100 mL agar (0.8% [w/v] agar + 0.6% [w/v] NaCl) and 10 mL amino acid solution (minimal amino acid solution for the determination of mutants: 0.5 mM tryptophan) were kept in a water bath at about 42 - 45°C, and the remaining components were added in the following order:
0.1 mL test solution or vehicle (negative control)
0.1 mL fresh bacterial culture
0.5 mL S9 mix (with metabolic activation)
or
0.5 mL phosphate buffer (without metabolic activation)
After mixing, the samples were be poured onto Minimal glucose agar plates within approx. 30 seconds. After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies (his+ revertants) were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager. Colonies were counted manually, if precipitation of the test substance hinders the counting using the Image Analysis System.

Preincubation Test
0.1 mL test solution or vehicle, 0.1 mL bacterial suspension and 0.5 mL S9 mix (with metabolic activation) or phosphate buffer (without metabolic activation) were incubated at 37°C for the duration of about 20 minutes using a shaker. Subsequently, 2 mL of soft agar was added and, after mixing, the samples were poured onto the agar plates within approx. 30 seconds.
After incubation at 37°C for 48 – 72 hours in the dark, the bacterial colonies were counted. The colonies were counted using the Sorcerer Image Analysis System with the software program Ames Study Manager (Perseptive Instruments Ltd., Haverhill, UK). Colonies were counted manually, if precipitation of the test substance hindered the counting using the Image Analysis System.

In each experiment 3 test plates per dose or per control were used.

The following positive controls were used to check the mutability of the bacteria and the activity of the S9 mix:
With S9 mix
• 2-aminoanthracene (2-AA) (TA 1535, TA 100, TA 1537, TA 98, Escherichia coli WP2 uvrA)

Without S9 mix
• N-methyl-N'-nitro-N-nitrosoguanidine (TA 1535, TA 100)
• 4-nitro-o-phenylenediamine( TA 98)
• 9-aminoacridine (AAC) (TA 1537)
• 4-nitroquinoline-N-oxide (4-NQO(E. coli WP2 uvrA)

Evaluation criteria:

The test substance was considered positive in this assay if the following criteria were met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. at least doubling (bacteria strains with high spontaneous mutation rate, like TA 98, TA 100 and E.coli WP2 uvrA) or tripling (bacteria strains with low spontaneous mutation rate, like TA 1535 and TA 1537) of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TOXICITY: A bacteriotoxic effect was observed depending on the strain and test conditions from about 100 μg/plate onward.
SOLUBILITY: No precipitation of the test substance was found with and without S9 mix.
A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Thus, under the experimental conditions of this study, the test substance Hydroxylamine-O-sulphonic acid is not mutagenic in the Salmonella typhimurium/Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:

Bacterial in vitro assay:

 

Only limited data are available for the test substance to evaluate the mutagenic potential.

An Ames test was performed with Hydroxylamine-O-sulphonic acid for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains, as Salmonella typhimurium and Escherichia coli, in a reverse mutation assay (BASF, 2015).

The test substance was exposed at concentrations of 33 µg - 5000 µg/plate (Standard plate test) and 3.3 µg - 2500 µg/plate (preincubation test) to the test strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA with and without S9 mix. A relevant increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system A bacteriotoxic effect was observed depending on the strain and test conditions from about 100 μg/plate onward. Thus, under the experimental conditions of this study, the test substance Hydroxylamine-O-sulphonic acid is not mutagenic in the Salmonella typhimurium/ Escherichia coli reverse mutation assay in the absence and the presence of metabolic activation.

 

In an older Ames test performed with hydroxylamine-O-sulphonic acid (BASF, 2003), several Salmonella typhimurium strains were exposed to the test substance as follows: 20, 100, 500, 2500 and 5000 µg/plate (TA 98, TA 100 first experiment), 125, 250, 500, 750 and 1000 µg/plate (TA 98, TA 100 second experiment) and 125, 250, 500, 1000 and 2000 µg/plate (TA 102). No increase in the number of his+ revertants was observed in the standard plate test, with or without the addition of S9 mix in all S. typhimurium strains tested (TA98, TA100, TA102). A bacteriotoxic effect (reduced his- background growth) was observed in the standard plate test with and without S9-mix at concentrations > 500-750 µg per plate. The results of the negative and positive controls were as expected and confirmed the validity and sensitivity of the test system used.

In another bacterial reverse mutation test, Hydroxylamine-O-sulfonic acid was only mutagenic in the excision-repair proficient strain his G 46, but failed to induce mutations in the excision repair deficient strain TA 100. At a concentration of 5 mg/plate hydroxylamine-O-sulfonic acid inhibited growth of his G 46 and TA 100 (Kalus, 1987).

 

Furthermore, in another bacterial in vitro test / in situ investigation on DNA it was demonstrated that hydroxylamine-O-sulphonic acid evoked changes in DNA molecules, documented by spectroscopic measurements, and inhibited the pol A-strain preferentially in the (pol A+/pol A- assay) (Rosenkranz, 1973). The positive result in the non-standard bacterial Rec-assay was also observed in further experiments published by the same author. The test substance hydroxylamine-O-sulfonic acid displayed a positive result in the non-relevant E.coli-based Pol A+/Pol A- assay. Nevertheless, negative results were obtained in the Ames test with Salmonella typhimurium TA1535 and TA1538 (Rosenkranz, 1976).

 

 

Other findings:

 

A publication of Kawazoe (1972) investigated the reaction of guanosine, a target moiety of DNA presumably involved in certain chemical carcinogenesis, with hydroxylamine-O-sulfonic acid. Results may suggest a possibility that amination with hydroxylamine-0-sulfonic acid might be a model for the DNA damage involved in carcinogenesis by N-arylhydroxylamines.

However, no further data are available to DNA-binding of the test substance in mammalian cells and no data on in vitro tests of mammalian cells.

 

 

Conclusion:

Based on the limited data set with equivocal results from different tests in bacterial systems, no conclusion on classification and labeling can be drawn. Data of mutagenicity testing in mammalian cells is lacking.

Justification for selection of genetic toxicity endpoint
Estimated Klimisch rating: 1

Justification for classification or non-classification

Equivocal effects were observed in in vitro mutagenicity testings with hydroxylamine-O-sulphonic acid (CAS 2950-43-8). Due to contradictory results and lack of data no conclusion on classification is possible according to Classification, Labelling, and Packaging Regulation (EC) No 1272/2008.