Bis(hydroxylammonium) sulphate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test procedure acc. to standardized protocol under the auspices of reputated institutions (NCI, NIEHS/USA); however, the test material was only tested up to a concentration of 333 µg per plate

Data source

Reference

Reference Type:

publication

Title:

Reproducibility of Microbial Mutagenicity Assays: I. Tests with Salmonella typhimurium and Escherichia coli Using a Standardized Protocol

Author:

Dunkel VC, Zeiger E, Brusick D, McCoy E, McGregor D, Mortelmans K, Rosenkranz HS, Simmon VF

Year:

1984

Bibliographic source:

Environ. Mutagen. 6 (Suppl. 2): 1-254

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

; the highest dose tested was 333.3 µg/plate

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Hydroxylammonium chloride

EC Number:

226-798-2

EC Name:

Hydroxylammonium chloride

Cas Number:

5470-11-1

Molecular formula:

ClH.H3NO

IUPAC Name:

hydroxyammonium chloride

Details on test material:

- Name of test material (as cited in study report): Hydroxylamine HCl (Tridom Fluka)
- Analytical purity: > 95 %

Method

Target gene:

Determination of the rate of induced back mutations of several bacteria mutants from histidine auxotrophy (his-) to histidine prototrophy (his+).

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Species / strain 3

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

0.3, 1.0, 3.3, 10.0, 33.3, 100.0 and 333.3 µg/plate

Vehicle / solvent:

Depending on the testing laboratory the test material was solved in distilled water or on DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: see "Details on test system and conditions"

Details on test system and experimental conditions:

The results reported for the test material belonged to an interlaboratory study with four institutes involved (Inveresk Research International, IRI; Litton Bionetics Inc., LBI; New York Medical College, NYM; SRI International, SRI): Compilation of the results of 59 chemicals. The assay was carried out with Arochlor-induced and non-induced liver microsomes from rat, mouse and hamster.

Preparation of S-9 and S-9 Mix:
Male Fisher 344 rats and B6C3F1 [C57B16 x C3H(He)] mice were supplied to all laboratories by the NCI Carcinogenesis Testing Program. Male Syrian hamsters were obtained by each laboratory from different suppliers: Wrights of Essex, Latchingdon, England (IRI); Mammalian Genetics and Animal Production Section, Division of Cancer Treatment, NCI, Bethesda, MD (LBI and NYM); and Simonsen Laboratories, Inc., Gilroy, California (SRI). Livers were used from animals either without treatment or following administration of Aroclor 1254. Treated animals were given 500 mg/kg bw Aroclor 1254 in corn oil by intraperitoneal injection 5 days before killing. Food was removed on the fourth day following injection, and on the fifth day the animals were killed by cervical dislocation or decapitation. The livers were removed aseptically and washed in cold 0.15 M KCI. All steps were carried out at 0-4 °C with cold, sterile solutions and glassware. The livers were minced with sterile surgical scissors in three volumes of 0.15 M KCI (3 ml/g wet liver), and homogenized with a Potter-Elvehjem apparatus with a Teflon pestle (NYM initially used a Waring Blender and then changed to the Potter-Elvehjem apparatus). The homogenate was centrifuged for 10 min at 9000 g. The supernatant (S-9) was removed, dispensed into screw-cap vials in convenient amounts, and stored at -80°C or lower. The S-9 activation mix was prepared daily as needed. The final concentrations of cofactors per ml liver was NADP, 4 μmol, glucose-6-phosphate 5 μmol, MgCl2 8 μmol, KCI 33 μmol and sodium phosphate buffer, pH 7.4, 100 μmol. Initially the S-9 in the mix was either 10 % or 20% . This was later standardized at 10 % .

Chemicals:
All test materials were tested under code and were supplied to each laboratory by the National Cancer Institute Chemical Repository (ITTRI, Chicago, IL). Chemicals were stored at -20°C or +4°C as recommended by the Repository. They were weighed on the day of assay, and stock solutions were prepared immediately prior to use. Information on the solubility of each chemical was supplied by the Repository. The laboratories made their independent decisions regarding the choice of solvent for each chemical.

All laboratories reported difficulty in obtaining reproducible positive responses in E coli with the selected positive controls sodium azide and 2-aminoanthracene. They were therefore instructed to use other positive controls (N-methyl-N'-nitro-N-nitrosoguanidine, AF-2, and 2-nitrotoluene).
In order to provide a basis for the comparison of data for each coded chemical, seven preset doses were tested : 0.3, 1 .0, 3.3, 10 .0, 33.3, 100.0 and 333.3 μg/plate.

Mutagenesis Assays:
The test material was tested in all strains either without S9-mix (NA), or with S9mix, induced with Aroclor 1254 from the liver of mouse (MLI), rat (RLI) or hamster (HLI), or with S9-mix from the liver of mouse (MLN), rat (RLN) or hamster (HLN) which was not induced.

Plate test:
The bacterial strain, test chemical, and S-9 mix, when required, were added to 2 ml ot molten top agar at 45 °C in the order and volumes given in the protocol. Two laboratories added buffer in lieu of S-9 mix in experiments without activation. The contents were then mixed and poured onto minimal agar plates. All plates were prepared in triplicate, and concurrent positive and negative controls were run at all times. The composition of top agar and bottom agar used in each laboratory was defined. Plates were incubated at 37°C for 48 hr prior to counting.

Preincubation test:
The bacterial strain, test chemical, and S-9 mix, when required, were added to tubes in the volumes given in the protocol. The contents were mixed and incubated at 37 °C for 30-30 min (IRI, 30 min ; LBI, NYM, and SRI 20 min). At the end of the incubation period 2 ml of top agar was added to each tube, and the contents were mixed and poured onto the minimal agar plates. Plates were incubated at 37°C for 48 hr. Since the prime objective of this study was to evaluate interlaboratory reproducibility, the laboratories were only required to do a single assay with each coded chemical.

Evaluation criteria:

In order to compare test data from the four laboratories, rules were devised for designationg positive (+), negative (-) or equivocal or borderline (?) results. A positive response was defined as a dose-related increase with at least two doses being greater than or equal to twofold background, unless the background was Iess than ten, in which case a threefold increase was required. A question mark (?) was defined as a test in which only a single dose was equal to or greater than twofold (or threefold) background. Low-Ievel non-dose-related increases were scored as negative, as were single doses whose increase was the result of widely divergent replicate plate counts.
An experiment was designated "no test" (NT) if the majority of plates were contaminated or showed a toxic response, contols were not run, or, in the case of TA100, if the mean background count plus the standard error of the mean was equal to or greater than 300. It must be emphasized that these rules were selected in order to facilitate comparison of data and are generally not used in any of the authors' laboratories. There were numerous instances in which the laboratory's or the authors' judgment disagreed with the results obtained from using these fixed criteria. However, these rules were necessary for comparing the results; they are not recommended for use beyond this comparison study.

Results and discussion

Test results

Species / strain:

other: S. typhimurium TA 1535, 1537, 1538, 98, 100 and E.coli WP-2 uvrA

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

other: A slight decrease in the number of back-revertants was sometimes seen in the highest concentration tested.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The substance was tested in all strains, with or without the addition of induced or not-induced S9-mix (from rat, mouse or hamster liver). There was no dose-related increase found with at least two doses being greater or equal to twofold background. However, the maximum revertant factor was in some single cases increased > 2-fold. This result was obtained in all testing laboratories involved. As the test material was tested up to 333.3 µg/per plate only, no prediction in terms of genotoxic effects of the test material at higher doses (up to 5000 µg/plate) is possible.
A slight decrease in the number of back-revertants was sometimes seen in the highest concentration tested.

Remarks on result:

other: other: S. typhimurium TA 1535, 1537, 1538, 98, 100 and E.coli WP-2 uvrA

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Summary of the maximum revertant factors of all data obtained from the 4 test labs involved

Plate test (0.3 - 333.3 µg/plate)
Strain	Metabolic activation system	maximum revertant factor	dose-related increase with at least 2 doses ≥ 2-fold	Assessment
TA 98	no	1.93	no	negative
	rat liver S9 not induced	1.9	no	negative
	rat liver S9 induced	1.9	no	negative
	mouse liver S9 not induced	3.81	no	?
	mouse liver S9 induced	2.13	no	?
	hamster liver S9 not induced	1.4	no	negative
	hamster liver S9 induced	1.78	no	negative
TA100	no	1.15	no	negative
	rat liver S9 not induced	1.3	no	negative
	rat liver S9 induced	1.47	no	negative
	mouse liver S9 not induced	1.29	no	negative
	mouse liver S9 induced	1.3	no	negative
	hamster liver S9 not induced	1.01	no	negative
	hamster liver S9 induced	1.05	no	negative
TA 1535	no	1.31	no	negative
	rat liver S9 not induced	1.92	no	negative
	rat liver S9 induced	2.32	no	negative
	mouse liver S9 not induced	1.35	no	negative
	mouse liver S9 induced	2.57	no	?
	hamster liver S9 not induced	2.16	no	?
	hamster liver S9 induced	1.9	no	negative
TA 1537	no	1.13	no	negative
	rat liver S9 not induced	2.17	no	?
	rat liver S9 induced	1.46	no	negative
	mouse liver S9 not induced	2.83	no	?
	mouse liver S9 induced	1.66	no	negative
	hamster liver S9 not induced	2.88	no	?
	hamster liver S9 induced	1.88	no	negative
TA 1538	no	1.73	no	negative
	rat liver S9 not induced	5.58	no	?
	rat liver S9 induced	1.47	no	negative
	mouse liver S9 not induced	5.73	no	?
	mouse liver S9 induced	1.46	no	negative
	hamster liver S9 not induced	2.25	no	?
	hamster liver S9 induced	1.44	no	negative
WP2 uvr A	no	2.0	no	?
	rat liver S9 not induced	1.28	no	negative
	rat liver S9 induced	1.73	no	negative
	mouse liver S9 not induced	1.26	no	negative
	mouse liver S9 induced	2.0	no	?
	hamster liver S9 not induced	1.52	no	negative
	hamster liver S9 induced	1.93	no	negative

maximum revertant factor = max. no. of revertants per plate / no. of revertants in resprective vehicle control (both values tested in the same lab)

? = ambiguous