Reaction mass of 2-(methylamino)ethanol, compound with sulphur dioxide and bis[(2-hydroxyethyl)ammonium] sulphite

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Disodium disulphite

EC Number:

231-673-0

EC Name:

Disodium disulphite

Cas Number:

7681-57-4

IUPAC Name:

disodium disulphite

Details on test material:

- Name of test material: Sodium metabisulfite (Disodium disulfite)
- Molecular formula: Na2S2O5
- Molecular weight: 190.11 g/mol
- Physical state: solid, white powder
- Storage condition of test material: 15-25 °C, in the dark
- Analytical purity: >96 % (assumed 100 % for assay)
- Purity test date: 16 February 2010
- Lot/batch no.: GFI501601
- Expiration date of the lot/batch: 15 January 2012

Method

Target gene:

HPRT

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 media containing 2.5 µg/mL Amphotericin B, 100 µg/mL Streptomycin, 100 units/mL Penicillin, 0.5 µg/mL Pluronic (except for RPMI 20) and 0, 10 or 20 % (v/v) heat inactivatet horse serum for RPMI A, RPMI 10 and RPMI 20, respectively.

The master stock of L5178Y tk+/- mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes; each batch of frozen cells was confirmed to be mycoplasma free.
- Periodically checked for karyotype stability: yes; each batch of frozen cells was purged of mutants.
- Periodically "cleansed" against high spontaneous background: yes
For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Range-Finder:
- with and without S9-mix: 59.44, 118.9, 237.8, 475.5, 951, 1902 µg/mL
Concentrations selected for the mutation experiments were based on the results of this cytotoxicity range-finder experiment.

Experiment I:
- with and without S9-mix: 200, 300, 400, 600, 800, 1200, 1600, 1902 µg/mL
Experiment II:
- with and without S9-mix: 100, 300, 600, 900, 1200, 1500, 1902 µg/mL
Experiment III:
- with S9-mix: 200, 400, 800, 1000, 1200, 1400, 1600, 1700, 1800, 1902 µg/mL

Cultures selected for mutation assessment:
Experiment I:
- with and without S9-mix: 200, 300, 400, 600, 800, 1200, 1600, 1902 µg/mL
Experiment II:
- with and without S9-mix: 300, 600, 900, 1200, 1500, 1902 µg/mL
Experiment III:
- with S9-mix: 200, 800, 1000, 1400, 1600, 1700, 1800, 1902 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: purified water
- Justification for choice of solvent/vehicle: preliminary solubility data indicated that the test substance was soluble in water for irrigation (purified water) at concentrations up to at least 20.25 mg/mL.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

treatment with the vehicle (purified water) diluted 10 fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitroquinoline-1-oxide; 0.1 and 0.15 µg/mL (dissolved in DMSO)

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

treatment with the vehicle (purified water) diluted 10 fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Remarks:

2 and 3 µg/mL (dissolved in DMSO)

Positive control substance:

other: Benzo(a)pyrene

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION
in medium

DURATION
- Exposure duration: 3 hours at 37° C
- Expression time (cells in growth medium): 7 days during which the HPRT mutation would be expressed: after the incubation period, cultures were centrifuged (200 g), washed and resuspended in RPMI 10. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival (scoreable after 7 days).
- Selection time (if incubation with a selection agent): 12 to 14 days: at the end of the expression period, aliquots of cell suspension were placed into each well of 4 x 96 well microtitre plates (384 wells at 2 x 10E4 cells/well). Plates were incubated at 37 ºC in a humidified incubator gassed with 5 % v/v CO2 in air and wells containing clones were identified and counted.

SELECTION AGENT (mutation assays)
6-Thioguanine (6TG)

NUMBER OF REPLICATIONS
2 replications

EVALUATION
Wells containing clones were identified and counted

DETERMINATION OF CYTOTOXICITY
- Method (relative survival):
Treatment and post treatment dilution of cell cultures for the cytotoxicity range finder experiment was as described for the mutation experiments. However, single cultures only were used and positive controls were not included. Following treatment, cells were centrifuged (200 g) washed with tissue culture medium and resuspended in 20 mL RPMI 10. Cells were plated into each well of a 96 well microtitre plate for determination of relative survival. The plates were incubated at 37 ºC in a humidified incubator gassed with 5 % v/v CO2 in air for 7 days. Wells containing viable clones were identified by eye using background illumination and counted.

OTHER:
- Probable number of clones/well (P) = -ln (empty wells (without clones) /total of TW),
- Plating efficiency (PE) = P/No of cells plated per well,
- Percentage relative survival (% RS) = % RS = [PE (test)/PE (control)] x 100,
- Mutant frequency (MF) = [PE (mutant)/PE (viable)] x 10E6.

Evaluation criteria:

Evaluation criteria
For valid data, the test substance was considered to induce forward mutation at the HPRT locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p<0.05).
2. There was a significant concentration relationship as indicated by the linear trend analysis (p<0.05).
3. The effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test substance treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

ambiguous

Remarks:

Statistically significant increases in mutant frequency were observed at the highest two concentrations (1600 and 1902 µg/mL). However, there was no significant linear trend.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

No significant increases in mutant frequency were observed following treatment with the test substance at any concentration tested and there were no significant linear trends.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

No significant increases in mutant frequency were observed following treatment with the test substance at any concentration tested and there were no significant linear trends.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

No statistically significant increases in mutant frequency were observed following treatment with the test substance at any concentration tested and there were no significant linear trends.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Osmolality and pH measurements on post-treatment media were taken in the cytotoxicity range-finder experiment.
- Effects of pH and osmolality: no marked changes in osmolality or pH were observed in the range-finder experiment at the highest concentrations analysed as compared to the concurrent vehicle controls (individual data not reported).

RANGE-FINDING/SCREENING STUDIES
Six concentrations were tested in the absence and presence of S9 ranging from 59.44 to 1902 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration analysed gave 37 % and 50 % RS in the absence and presence of S9 mix, respectively.

COMPARISON WITH HISTORICAL CONTROL DATA
Yes; comparison of controls with historical means of the positive control substances.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In Experiment I, concentrations, ranging from 200 to 1902 µg/mL, were tested in the absence and presence of S9 mix. Seven days after treatment, the highest concentration analysed was 1902 µg/mL in the absence and presence of S9 mix, which gave 43 % and 65 % RS, respectively.
In Experiment II, concentrations, ranging from 100 to 1902 µg/mL, were tested in the absence and presence of S9 mix. Seven days after treatment, the highest concentration analysed was 1902 µg/mL in the absence and presence of S9 mix, which gave 30 % and 75 % RS, respectively.
In Experiment III, concentrations, ranging from 200 to 1902 µg/mL, were tested in the presence of S9 mix. Seven days after treatment, the highest concentration analysed was 1902 µg/mL, which gave 43 % RS.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'. Remarks: Experiment I