Sodium 3-(allyloxy)-2-hydroxypropanesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Three valid tests for genetic toxicity are available. A bacterial reverse gene mutation test shows no adverse effects. A mammalian forward gene mutation test shows no adverse effects. A mammalian chromosome aberration test shows no adverse effects.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26. June 2012 - 17. April 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase locus Tk1

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640-HAT Medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

2180; 1100; 550; 280; 140; 70; 40; 20 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: medium without supplemnets
- Justification for choice of solvent/vehicle: Medium without supplements was chosen as solvent, because the test item was com-pletely soluble, and this solvent doesn’t have any effects on the viability of cells in the test system. The stock solution was used to prepare the geometric series of the concentrations to be tested.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

7.1 Pre-Test for Cytotoxicity
A pre-test was performed in order to determine the concentration range of the mutageni-city experiments. Both pH value and osmolarity were determined at the maximal concen-tration of the test item and in the solvent control without metabolic activation.
Cytotoxicity is characterised by the relative suspension growth or the relative survival of treated cells in comparison with the controls. As recommended in the regulatory guideline the maximum concentration of the test item should be 5 µl/mL, 5 mg/mL or 0.01mol/l, whichever is the lowest.
According to these guidelines the maximum concentration that was used for the test item was 21.8 mg/mL (= 0.01 mol/l).
In the pre-experiment, eight concentrations (concentrations ranging from 21.8 mg/mL to 0.2 mg/mL nominal) of the test item were used and tested with and without S9. The expo-sure time was 4 hours and the exposure date 13. Aug. 2012.
1*107 cells were exposed to each concentration of the test item for 4 hours with and with-out metabolic activation. The serum concentration was 5% during the treatment. Following treatment, the cells were washed twice by centrifugation (1750 rpm, 5 min) and resuspension in saline G.
For determination of the dose range of the test item, two methods were chosen:
7.1.1 Determination of the Relative Suspension Growth (RSG):
After washing, the cells were resuspended in 10 mL complete culture medium RPMI 1640 with 5% HS for a growth period of 48 hours (counting from start of exposition). The cell density was determined immediately after treatment and at each day of the growth period and adjusted to 3*105 cells/mL immediately . The relative suspension growth (RSG) of the treated cell cultures was calculated at the end of the growth period according to the method of Clive and Spector.
7.1.2 Determination of Survival by Cloning Efficiency (CE)
The microwell version of the pre-test is a fluctuation-assay and the key parameter for analysis is the number of negative wells on each 96-well plate.
After counting the cells after the washing steps, two cells were seeded per well of each 96-well plate and incubated for 7 – 10 days. The percentage relative survival (%RS) of each test item culture was determined by comparing the cloning efficiency of test item treated cultures and control cultures.
7.2 Experiment I
Because of a too high mutant frequency in the solvent controls, the first experiment had to be repeated. The data of the invalid experiment are not included in this final report. All data and information given here apply to the valid experiment I. The exposure date of the valid experiment was 04. Dec. 2012.
In the mutation experiment, 107 cells per 10 cm culture dish suspended in 10 ml RPMI medium with 5 % horse serum were exposed to the chosen concentrations of the test item in the presence and in the absence of metabolic activation each. Positive and solvent controls were performed in parallel. The cytotoxicity was again determined by the Relative Suspension Growth (RSG) as described for the pre-experiment (chapter 7.2.1, page 20). However in experiment I, the cell density was determined at each day of the growth period and adjusted to 3*105 cells/mL.
After 4 h, the test item was removed by centrifugation (1750 rpm, 5 min) and the cells were washed twice with the buffer solution saline G. Subsequently the cells were cultured in 30 ml complete culture medium containing 5 % horse serum and incubated for an expres-sion and growth period of in total 48 hours (counting from start of exposition). As the initial cell number was 106 cells/mL in 10 mL medium, the number of the cells after resuspension in 30 mL medium should be approximately 3*105 cells/mL.
The cell density was determined each day and adjusted to 3*105 cells/mL (3*106 cells per 10 mL). The relative suspension growth (RSG) of the treated cell cultures was calculated by the day 1 fold-increase in cell number multiplied by the day 2 fold-increase in cell number according to the method of Clive and Spector (literature see chapter 5, page 11).
After the expression period, the cultures were selected. Cells from each experimental group were seeded into two microtiter plates so that each well contained approxi¬mately 4*103 cells in selection medium with TFT. The viability (cloning efficiency) was determined by seeding about two cells per well into microtiter plates (using complete culture medium with 5 % horse serum). The plates were incubated at 37° 1.5 °C in 5 % CO2 for 10 days (viability) resp. 12 days (selection). Then, the plates were evaluated.
7.3 Experiment II
Because the results of experiment I were negative, a subsequent experiment was per-formed for verification.
7.4 Because of an invalid result of the approach with metabolic activation the sec-ond experiment had to be partially repeated and was therefore performed in two independent experimental parts (the first one without metabolic activation (exposure date: 05. Feb. 2013) and the second one with metabolic activation (exposure date 20. Feb. 2013). Both experimental parts were performed in the same fashion as Experiment I except for the incubation time of the test item without metabolic activation (24 hours) The cytotoxicity was again determined by the Relative Suspension Growth (RSG) as described in the pre-experiment (chapter 7.2.1, page 20). However in experiment II, the cell density was deter-mined immediately after treatment and at each day of the growth period and adjusted to 3*105 cells/mL immediately. Evaluation
7.4.1 Cell Numbers
Cell numbers in suspension were determined using an aliquot of the well homogenised suspension whose cell number was determined in a cell counter.
7.4.2 Colonies
Colonies were counted manually. In accordance with their size, the colonies were classi-fied into two groups. The colony size distribution was determined in the controls and at all evaluated concentrations of the test item. Criteria to determine colony size were the abso-lute size of the colony (more than 1/3 of a well for large colonies) and the optical density of the colonies (the optical density of the small colonies is generally higher than the optical density of the large ones).
All generated data of each experiment (test item, solvent controls and positive control) were recorded in the raw data.

Evaluation criteria:

A mutation assay is considered acceptable if it meets the following criteria (the current recommendations of the International Workshop on Genotoxicity Testing (IWGT)) (Moore et al., 2007):
1. All plates, from either the viability or the mutagenicity portion of the experiment are analysable.
2. The total suspension growth (TSG) of the solvent control corresponds to an increase factor of at least 4
3. The range of the solvent control mutant frequency is in the range of 50 – 170 per 106 cells.
4. The positive controls (MMS and CPA) should yield an absolute increase in total mu-tant frequency that is an increase above spontaneous background mutant frequency of at least 300 per 106 cells. At least 40% of the induced mutation frequency (IMF) should be reflected in the small colony MF. Alternatively, the positive controls should induce at least 150 small colonies.
5. The upper limit of cytotoxicity observed in the positive control culture should be the same as for the experimental cultures (i.e. the relative total growth – RTG – should be greater than 10% of the concurrent selective control group).
6. The highest concentration of the test item should be 0.01 M or 5 mg/mL (resp. 5 µL/mL), unless limited by toxicity or solubility of the test item. If toxicity occurs, the highest evaluated concentration should result in approximately 10 - 20% relative total growth, if possible. If precipitation is noted, the highest analysed concentration should be the lowest concentration where precipitation is observed by the naked eye.

Statistics:

A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies. With the assessment of this regression, it can be evalu-ated whether mutations increase with increasing dose of the test item. A p-value of 0.05 or lower (significance level 95%) is considered as critical.
The positive controls were tested at one concentration only, therefore, no dose depend-ency could be evaluated, although the positive controls showed considerable increases in mutants. In the following table, the statistical significance values are presented. The chi-square test was used.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not examined, pH was about 7.4 to 7.6 in all experiments
- Effects of osmolality: not examined, osmolality was about 277 - 303 mOs mol/kg
- Evaporation from medium: no data
- Water solubility: the substance is soluble in water
- Precipitation: not observed
- Other confounding effects: no

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test item did not induce mutations at the thymidine kinase locus TK1 in the mouse lymphoma assay using the cell line L5178Y TK+/- in the absence and presence of metabolic activation under the experimental conditions reported. Sodium 3-(allyloxy)-2-hydroxypropanesulphonate (HAPS) is considered to be “non-mutagenic under the conditions of the mouse lymphoma assay”.

Executive summary:

This study was performed to investigate the potential of sodium 3-(allyloxy)-2-hydroxypropanesulphonate (HAPS) to induce mutations at the thymidine kinase locus (TK1) on chromosome 11 and/or structural chromosomal aberrations using the mouse lymphoma L5178Y TK^+/-cells.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without metabolic activation (liver enzyme S9 fraction) and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence of metabolic activation and 4 hours in the presence of metabolic activation.

The highest concentration (2180 µg/mL) applied was chosen with regard to the solubility of the test item in organic solvents and aqueous media.

The tested concentrations, the evaluated experimental points, and the results are summarised in tables 3-a and 3-b.

No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. No relevant shift of the ratio of small versus large colonies was observed up to the maximal concentration of the test item.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No study available and not needed because information from in vitro studies is conclusive.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Additional information from genetic toxicity in vitro:

None of the available in-vitro tests show genetic toxicity.

Justification for selection of genetic toxicity endpoint
The Endpoint has been selected as it is the most recent study performed.