Dodecyl(2-hydroxy-3-sulphonatopropyl)dimethylammonium

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A battery of in vitro tests have been conducted. Data are available from an Ames study, an in vitro chromosome aberration study and an in vitro mammalain mutation study. These show the test material is not mutagenic or clastogenic.

Link to relevant study records

Reference

Reference 1

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

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

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

Target gene:

hprt

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

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.

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.

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

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From August 20, 2014 to August 29, 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Test method is according to OECD guideline 471; non-GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

The number of replicate cultures is two/dose, not three/dose.

GLP compliance:

no

Remarks:

The original study is reported in Japanese: the English translation of the study is available but is not considered to be to GLP standard

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

-Lot No. : 1720
-Purity: 30.7%

Species / strain / cell type:

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

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:

-Dose finding test:
1.2, 4.9, 20, 78, 313, 1250, 5000 µg/plate.

-Main test:
without S9: 9.8, 20, 39, 78, 156, 313 µg/plate.
with S9 : 39, 78, 156, 313, 625, 1250 µg/plate.

Vehicle / solvent:

Distilled water was selected as the vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

furylfuramide

other: 2-Anthramine, 6-Chloro-9-[3-(2-chloroethylamino)propylamino]-2-methoxyacridine dihydrochloride

Details on test system and experimental conditions:

-Test conditions
Strains used: TA100, TA1535, TA98, and TA1537 (Salmonella typhimurium) and WP2 uvrA (Escherichia coli)
Test for Induction of: S9 (±), pre-incubation method (37°C, 20 minutes)
Culture period: 48 hours
Solvent used: Distilled water
Dose: Dose finding test: Total of 7 doses as determined from the highest dose of 5000 μg/plate* with a common ratio of 4
Main test: S9 (-), total of 6 doses as determined from the highest dose of 313 μg/plate* with a common ratio of 2
S9 (+), total of 6 doses as determined from the highest dose of 1250 μg/plate* with a common ratio of 2
No. of Replicate Cultures: Two/dose
* Purity corrected value

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Because the test article had a purity of less than 95%, the doses were corrected for purity. The doses are hereinafter described as those corrected for purity.

The potential of the test article to induce mutations was examined using Salmonella typhimurium strains including TA100, TA1535, TA98, and TA1537 and an Escherichia coli strain of WP2 uvrA. A dose finding test for each strain was performed using a total of 7 doses as determined from the highest dose of 5000 μg/plate with a common ratio of 4. Based on the effect to inhibit the growth of each strain, the main test was performed using a total of 6 doses as determined from the highest dose with a common ratio of 2, where the growth inhibition dose was used as the highest dose.

The results of the dose finding and main tests showed that the test article did not induce a two-fold or more increase in the number of reverse mutation colonies as compared to the negative control for any of the strains, irrespective of whether the metabolic activation was present or not. The results also showed no dose-dependent increase in the number.

The positive control induced a two-fold or more increase in the number of reverse mutation colonies as compared to the negative control for all the strains, demonstrating the validity of the test. It was observed that the test article inhibited the growth of each strain at the doses listed in the following table. At 1250 μg/plate or higher with S9 (+), the test article was precipitated (white).

Doses causing growth inhibition
Strain S9 (-) S9 (+)
TA100 156 - 5000 μg/plate 625 - 5000 μg/plate
TA1535 156 - 5000 μg/plate 1250 - 5000 μg/plate
WP2 uvrA 313 - 5000 μg/plate 1250 - 5000 μg/plate
TA98 156 - 5000 μg/plate 1250 - 5000 μg/plate
TA1537 156 - 5000 μg/plate 1250 - 5000 μg/plate

Conclusions:

It was determined that the test article had no potential to induce mutations (negative) under the present test conditions.

Executive summary:

The potential of the test article to induce mutations was examined using Salmonella typhimurium strains including TA100, TA1535, TA98, and TA1537 and an Escherichia coli strain of WP2 uvrA. A dose finding test for each strain was performed using a total of 7 doses as determined from the highest dose of 5000 μg/plate with a common ratio of 4. Based on the effect to inhibit the growth of each strain, the main test was performed using a total of 6 doses as determined from the highest dose with a common ratio of 2, where the growth inhibition dose was used as the highest dose. Because the test article had a purity of less than 95%, the doses were corrected for purity. The doses are hereinafter described as those corrected for purity.

The results of the dose finding and main tests showed that the test article did not induce a two-fold or more increase in the number of reverse mutation colonies as compared to the negative control for any of the strains, irrespective of whether the metabolic activation was present or not. The results also showed no dose-dependent increase in the number. Therefore it was determined that the test article had no potential to induce mutations (negative) under the present test conditions.

The positive control induced a two-fold or more increase in the number of reverse mutation colonies as compared to the negative control for all the strains, demonstrating the validity of the test.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

From 3rd of October to 18th of December, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

-Lot No. : 1720
-Purity: 30.7%

Species / strain / cell type:

other: CHL/IU (Chinese hamster lung fibroblast cell line)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

-Short-term treatment process (with and without metabolic activation): 0.125, 0.177, 0.250, 0.354 and 0.500 mg/mL
Since the cytotoxic effect estimated in terms of cell growth inhibition of greater than 50% was observed at a dose of 0.500 mg/mL in both treatment groups in the cell growth inhibition test, the highest dose was established as 0.500 mg/mL.

-Continuous treatment process (24 and 48-hour treatment): 0.0442, 0.0625, 0.0884 and 0.125 mg/mL
Since the cytotoxic effect estimated in terms of cell growth inhibition of greater than 50% was observed at a dose of 0.125 mg/mL in both treatment groups in the cell growth inhibition test, the highest dose was established as 0.125 mg/mL.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Saline
- Justification for choice of solvent/vehicle: The test substance was soluble in saline at 35.2 mg/mL

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

mitomycin C

Details on test system and experimental conditions:

-Short-term treatment method
(1) Cell growth inhibition test (Preliminary test for dose finding)
The test was conducted with or without the metabolic activation system using one plate per dosage. Two×104 cells per plate were seeded, incubated for 3 days and used. The test article solutions, 0.3 mL each, were added to test tubes containing the culture solution, which were prepared in accordance with the dosage levels, and mixed. When using the metabolic activation system, 0.5 mL of S9 mix was further added and mixed. Immediately after adding S9 mix, the culture solution in the plate was replaced with the prepared solution, and the plate was incubated in a CO2 incubator for 6 hours. A total volume of fluid during treatment with the test article was 3 mL per plate. After completion of treatment, precipitation of the test article was confirmed, cells were washed with PBS (removal of the test article), and 5 mL of fresh culture solution was added. Then, cells were incubated for more 18 hours. After completion of incubation, cells were observed under inverted phase-contrast microscopy, and the cell growth index was determined. Cell growth index was determined as a relative increase in cell counts (RICC, %). Cytotoxicity was determined as 100−RICC. Where the lower limit of RICC calculated was to be 0 and expressed as such.

(2) Chromosomal aberration test
The test was conducted using two plates per dosage. Preculture and treatment with the test article were performed in the similar manner to that in the cell growth inhibition test, and chromosome specimens were prepared. The highest dose inhibiting cell growth by 55±5% (45±5% as RICC) as cytotoxicity was established.

-Continuous treatment method
(1) Cell growth inhibition test (Preliminary test for dose finding)
The test was conducted by 24-hour treatment and 48-hour treatment using one plate per dosage. Two×104 cells per plate were seeded, incubated for 3 days and used. The test article solution, 0.5 mL each, was added to test tubes containing the culture solution, which were prepared in accordance with the dosage levels, and mixed. Immediately after adding the test article solution, the culture solution in the plate was replaced with the prepared solution, and the plate was incubated in a CO2 incubator for 24 hours or 48 hours. A total volume of fluid during treatment with the test article was 5 mL per plate. After completion of incubation, the presence or absence of precipitation of the test article was confirmed and cells were observed. Then, the cell growth index was determined. Cell growth index was determined as a relative increase in cell counts (RICC, %) similarly to that in the short-term treatment method. Cytotoxicity was determined as 100−RICC. Where, the lower limit of RICC calculated was to be 0 and expressed as such.

(2) Chromosomal aberration test
The test was conducted using two plates per dosage. Preculture and treatment with the test article were performed in the similar manner to that in the cell growth inhibition test, and chromosome specimens were prepared. The highest dose inhibiting cell growth by 55±5% (45±5% as RICC) as cytotoxicity was established similarly to that in the short-term treatment method.

-Preparation of chromosome specimens
Colcemide solution (GIBCO) was added to the plate at a concentration of 0.2 μg/mL 2 hours before completion of incubation in order to obtain metaphase cells. After completion of incubation, the culture solution was transferred into a centrifuge tube and centrifuged together with cells that were detached from the plate with 0.25% trypsin solution. After removal of the supernatant, 4 mL of 0.075 M potassium chloride solution was added and subjected to 15-minute hypotonic treatment in a constant temperature water tank at 37°C. Cells were semi-fixed by adding a few drops of the fixative (a mixed solution of alcohol and acetic acid) and centrifuged. Then, the fixative was replaced with a fresh fixative. The replacement of the fixative was repeated several times, and cells were sufficiently fixed. Cell suspension at an adequate concentration was prepared, and a drop of it was placed on a slide glass. Then the slide glass was air-dried and stained with 2% Giemsa solution.

-Identification methods
Each plate had an identification number or each test tube had a label with an identification number so that each plate or test tube could be identified when there were some plates or tubes for each treatment method, test article, test dose or the same dosage.

-Methods of cell counting
A part of the suspension of cells detached from the plate with 0.25% trypsin solution was collected and stained with trypan blue. Then, cells were counted using a cell counter (Bio-Rad, TC10).

-Observation of chromosome specimens
Observations were conducted in a blinded manner, and in principle, 200 metaphase cells that were well-confluent were observed under microscope at a magnification of 600 to 1000× for each dosage.

(1)Structural aberrations
Since the chromosome mode of CHL/IU cell line was 25, cells with 25±2 chromosomes were subjected to observations. Structural aberrations were evaluated in accordance with the following classification, and a cell with at least one aberration were recorded as one aberrant cell. In parallel, the type of aberration was also recorded. For example, when there were 2 break types and 3 exchange types in one cell, the number of aberrant cells was recorded as one, and the type of aberration was recorded as one break type and one exchange type.


Chromatid break (abbreviated as ctb)
Chromatid exchange (abbreviated as cte)
Chromosome break (abbreviated as csb)
Chromosome exchange (abbreviated as cse)
Others (fragmentation etc.)

(2) Numerical aberrations
When polyploid cells (three (triploid) or more set of chromosomes, abbreviated as poly) or cells with endoreduplication (abbreviated as end) were observed, the number of these cells was recorded.

(3) Gap
Chromatid gap or chromosome gap (abbreviated as g) was recorded distinctively from other aberrations and was not contained in structural aberrations. A gap was considered to be an unstained region of a lesser width than the width of a chromatid on the same chromatid axis, and the unstained region of a greater width than the width of a chromatid was considered to be a break.

Evaluation criteria:

-Judgment by dosage
The incidence (%) of structural aberrations and numerical aberrations according to the criteria described by Ishidate et al. was evaluated using the following criteria.

<5% : Negative (−)
≥5% and <10% : Equivocal (±)
≥10% : Positive (+)

-Final Judgment
When the value in the treatment group markedly increased as compared to the negative control value and a dose dependency was observed, the result was considered to be positive in the final judgment. In addition, when a positive result or false positive result was obtained at only one dosage, a confirmatory study was conducted, and when reproducibility was confirmed, the overall result was considered to be positive.

Statistics:

No statistical methods were used for data analyses.

Key result

Species / strain:

other: CHL/IU (Chinese hamster lung fibroblast cell line)

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:

-Short-term treatment method
The test was conducted at the doses described above with or without the metabolic activation system. No precipitation of the test article was observed at any doses in both treatment groups at the start and the end of treatment. Cell growth inhibition of greater than 50% was observed at a dose of 0.354 mg/mL in both treatment groups. The incidence of structural and numerical chromosome aberrations was less than 5.0% at any doses in both treatment groups.

-Continuous treatment method
The test was conducted at the doses described above by 24-hour and 48-hour treatment method. No precipitation of the test article was observed at any doses in both treatment groups at the start and the end of treatment. Cell growth inhibition of greater than 50% was observed at a dose of 0.125 mg/mL in both treatment groups. The incidence of structural and numerical chromosome aberrations was less than 5.0% at any doses in both treatment groups.

Conclusions:

It is considered that the test article is negative for the potential to induce chromosome aberration. In addition, the negative and positive controls provided expected results under all the treatment conditions, demonstrating the validity of the test.

Executive summary:

 A chromosome aberration test of a lauryl hydroxysulfobetaine solution (aqueous solution) was performed using CHL/IU cells. Chromosome aberrations were observed in less than 5.0% of the cells for both structural and numerical aberrations at all the doses tested, irrespective of whether the metabolic activation system of the short-term treatment method was present or not. Similarly, they were observed in less than 5.0% at all the doses tested when the test was performed using the 24 or 48-hour continuous treatment method.

Therefore, it is generally considered that the test article is negative for the potential to induce chromosome aberrations. In addition, the negative and positive controls provided expected results under all the treatment conditions, demonstrating the validity of the test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No data are available.

There is no requirement to conduct an in vivo study on the basis of the in vitro assays conducted.

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The in vitro battery of genetic toxicity studies are negative, there is no requirement to classify this product under CLP regulation 1272/2008.