Dodecyl(2-hydroxy-3-sulphonatopropyl)dimethylammonium

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 August 2017 to 21 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

other: in vitro mammalian mutation study

Test material

Test material information

Constituent 1

Reference substance name:

Dodecyl(2-hydroxy-3-sulphonatopropyl)dimethylammonium

EC Number:

236-164-7

EC Name:

Dodecyl(2-hydroxy-3-sulphonatopropyl)dimethylammonium

Cas Number:

13197-76-7

Molecular formula:

C17H37NO4S

IUPAC Name:

dodecyl(2-hydroxy-3-sulphonatopropyl)dimethylammonium

Test material form:

solid

Specific details on test material used for the study:

Test material: Betadet S-20-S
Batch: 16021 (also referred to as ESTS168/17)
Appearance: White powder
Molecular weight: 351.545
Storage: 15-25°C; protected from light
Purity: 98.8%
Expiry date: 30 May 2018

Method

Target gene:

hprt

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

2000 µg/mL, the maximum concentration recommended for this assay.

Vehicle / solvent:

Purified water

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

At least 10^7 cells in a volume of 16 mL of RPMI (containing 5% serum) were placed in a series of sterile disposable 50 mL centrifuge tubes. For all treatments 2 mL vehicle, test article or positive control solution (comprising 0.2 mL positive control and 1.8 mL purified water) was added. S-9 mix or 150 mM KCl was added as described. Each treatment, in the absence or presence of S-9, was in duplicate (single cultures only used for positive control treatments) and the final treatment volume was 20 mL.
After 3 hours’ incubation at 37±1°C with gentle agitation, cultures were centrifuged (200 g) for 5 minutes, washed with the appropriate tissue culture medium, centrifuged again (200 g) for 5 minutes and finally resuspended in 20 mL RPMI 10 medium. Cell densities were determined using a Coulter counter and, where sufficient cells survived, the concentrations adjusted to 2 x 10^5 cells/mL. Cells were transferred to flasks for growth throughout the expression period or were diluted to be plated for survival.

Following adjustment of the cultures to 2 x 10^5 cells/mL after treatment, samples from these were diluted to 8 cells/mL to assess survival. Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells, averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (seven days). Wells containing viable clones were identified by eye using background illumination and counted.Cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed. Sub-culturing was performed as required with the aim of retaining an appropriate concentration of cells/flask. From observations on recovery and growth of the cultures during the expression period, cultures treated with 0, 25, 50, 100, 125, 150, 175, 200 and 225 µg/mL were selected to be plated for viability and 6TG resistance. At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 10^5 cells/mL in readiness for plating for 6TG resistance. Samples from these were diluted to 8 cells/mL to assess viability.
Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (12 days). Wells containing viable clones were identified by eye using background illumination and counted.
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 10^5 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 µg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 10^4 cells/well). Plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (13 days) and wells containing clones were identified as above and counted.

Rationale for test conditions:

The test concentrations were selected based on the results of a range-finder experiment where excessive toxicity was observed at concentrations of 500 µg/mL and above.

Evaluation criteria:

For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF 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. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical negative control database (mean MF +/ 2 standard deviations.
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 article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

Following 3 hour treatment in the absence of S-9 sporadic increases in mutant frequency that were statistically significant (P≤0.05) compared to the vehicle control, were observed at concentrations of 125 and 200 µg/mL. These increases were outside the range generated by the last twenty studies performed in this laboratory (1.36 to 8.46 mutants per 10^6 viable cells) for both concentrations. However, the increases were not concentration related and there was no significant linear trend. These increases were therefore considered of questionable biological relevance.

Following 3 hour treatment in the presence of S-9, no statistically significant increases in mutant frequency, compared to the vehicle control values, were observed at any concentration analysed and there were no statistically significant linear trends. The mutant frequency observed at the lowest concentration analysed (25 µg/mL) was outside the range generated by the last twenty studies (1.74 to 7.70 mutants per 10^6 viable cells), however it was not statistically significant and no linear trend was observed. This observation was therefore considered not biologically relevant.

Any other information on results incl. tables

3 hour treatment in the absence of S9

Concentration (µg/mL)	Relative survival (%)	Mutant frequency (6TG resistant mutants/10^6viable cells)
0 (Vehicle control)	100	4.08
25	70	5.78
50	57	3.64
100	44	5.37
125	48	9.01*
150	29	6.98
175	29	3.31
200	22	10.62*
225	10	2.82
Positive control – NQO 0.15 µg/mL	52	33.57
Positive control – NQO 0.2 µg/mL	35	64.51

NQO: 4-nitroquinoline 1-oxide
*: Significant at the 5% level with Dunnett’s test

Test for linear trend: Not significant (negative trend)
Historical control MF range for last 20 studies performed at laboratory: 1.36 to 8.46 mutants per 10⁶viable cells

3 hour treatment in the presence of S9

Concentration (µg/mL)	Relative survival (%)	Mutant frequency (6TG resistant mutants/10^6viable cells
0 (Vehicle control)	100	5.02
25	103	9.69
50	86	4.21
100	69	6.62
125	58	5.68
150	39	5.67
175	22	7.31
200	27	7.04
225	16	5.02
Positive control – B[a]P 2 µg/mL	80	31.25
Positive control – B[a]P 3 µg/mL	105	32.02

B[a]P: Benzo[a]pyrene

Test for linear trend: Not significant
Historical control MF range for last 20 studies performed at laboratory: .1.74 to 7.70 mutants per 10⁶viable cells

Applicant's summary and conclusion

Conclusions:

It is concluded that Betadet S-20-S did not induce biologically relevant increases in mutant frequency at the hprt locus in mouse lymphoma L5178Y cells when tested up to the limit of cytotoxicity for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9) under the experimental conditions described.

Executive summary:

In an in vitro mammalian cell mutation assay, performed according to the OECD No.476 and in compliance with the GLP, Betadet S-20-S diluted in purified water was tested in the L5178Yhprt+mouse lymphoma cell line in the presence and the absence of mammalian metabolic activation (S9 mix).

Prior to the mutagenicity experiments, a preliminary assay was conducted in order to assess the cytotoxicity of the test substance. The substance was tested for its toxicity at dose levels between 0 and 2000 µg/mL as recommended in the OECD guideline.

In the main experiment, L5178Y hprt⁺mouse lymphoma cells (single copy of the hypoxanthine guanine phosphoribosyl transferase gene on the X chromosome) were exposed to the test item at twelve dose levels, in duplicate, together with vehicle control (water) for 3 hours in the absence and in the presence of metabolic activation. Positive control cultures (single cultures) were also included comprising treatments with 4-nitroquinoline 1-oxide (NQO) or Benzo[a]pyrene (B[a]P) without and with metabolic activation respectively.

The concentration range of test item was selected based on the toxicity observed in the Range-Finder Experiment. Due to toxicity, the highest analysable concentration was 225 µg/mL in the absence and in the presence of S9, which gave relative survivals of 10% and 16%, respectively.

The vehicle controls had acceptable mutant frequency values and the positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

Statistically significant increases in mutant frequency were observed in the absence of metabolic activation at 125 and 200 µg/mL which exceeded the historical vehicle control range. Since the increases were not concentration related and there was no significant linear trend, this was considered to be of questionable biological relevance.

An increase in mutant frequency which was outside the historical control range was observed in the presence of metabolic activation at 25 µg/mL. Since this was the lowest concentration analysed, was not statistically significant and there was no linear trend, this was considered not be biologically relevant.

Under the experimental conditions of this study, the test item, Betadet S-20-S, did not induce biologically relevant increases in mutant frequency at the hprt locus in mouse lymphoma L5178Y cells when tested up to the limit of cytotoxicity for 3-hours in the absence and in the presence of a rat liver metabolising system.