2-Butyne-1,4-diol, polymer with 2-(chloromethyl)oxirane, brominated, dehydrochlorinated, methoxylated

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant, guideline study, no restrictions, fully adequate for assessment

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-Butyne-1,4-diol, polymer with 2-(chloromethyl)oxirane, brominated, dehydrochlorinated, methoxylated

EC Number:

614-503-3

Cas Number:

68441-62-3

Molecular formula:

(C3H7O2)xC4H4O2Br2(C4H9O2)y with x + y = 2.5

IUPAC Name:

2-Butyne-1,4-diol, polymer with 2-(chloromethyl)oxirane, brominated, dehydrochlorinated, methoxylated

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): IXOL B350
- Other name: Polyol IXOL B350
- Chemical name: 2-Butyne-1,4-diol, polymer with 2-(chloromethyl)oxirane, brominated, dehydrochlorinated, methoxylated.
- Chemical name (short): Halogenated PolyetherPolyol
- Molecular weight: 441
- CAS Number: 68441-62-3
- Physical state: Viscous liquid, dark brown
- Analytical purity: Treated as 100% pure
- Lot/batch No.: 20091118-761
- Expiration date of the lot/batch: 18 November 2011
- Storage condition of test material: At room temperature in the dark
- Stability under storage conditions: Stable

Method

Target gene:

thymidine kinase (TK) locus on chromosome 11

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

0.37, 0.31, 0.26, 0.18, 0.13, 0.06, 0.03, 0.02, 0.01 and 0 mmol/L in the absence of S9-mix
0.7, 0.6, 0.5, 0.37, 0.26, 0.06, 0.03, 0.02, 0.01 and 0 mmol/L in the presence of S9-mix

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 3-methylcholanthrene and methylmethanesulfonate

Details on test system and experimental conditions:

Prior to the main study two dose range finding studies were performed. In the first dose range finding study single cultures were exposed for 24 hours in the absence of S9-mix to 5 concentrations of IXOL B350 ranging from 0.63 to 10 mmol/l. In the second dose range finding study single cultures were exposed for 24 hours in the absence and 4 hours in the presence of S9-mix to concentrations of IXOL B350 ranging from 0.016 to 0.25 mmol/l or 0.25 to 1.0 mmol/l, respectively.
In the main study single cultures were exposed for 24 hours in the absence and 4 hours in the presence of S9-mix to 15 or 13 concentrations of IXOL B350 ranging from 0.004 to 0.70 mmol/l or 0.0019 to 1.0 mmol/l, respectively. Finally, 9 concentrations were evaluated for mutagenicity in both the absence and presence of S9-mix.

Cell treatment without metabolic activation
In the assay without metabolic activation the cells were exposed to the test substance according to the following procedure; 0.1 ml test substance solution or 0.1 ml positive control and 4.9 ml culture medium without serum were added to ca. 3,000,000 L5178Y cells in 5 ml culture medium (with 10 % horse serum) to a final volume of 10 ml. Two cultures treated with the vehicle (DMSO) were used as negative controls; one single culture treated with MMS was used as positive control substance at a final concentration of 0.1 mmol/l. Single cultures were used for each concentration of the test substance. The cells were exposed for 24 h at ca. 37 oC and ca. 5 % CO2 in a humidified incubator.

The dose levels of the test substance used ranged from 0.004 to 0.70 mmol/l IXOL B350. At the start and end of the treatment, all cell cultures were checked visually and selected cultures were checked for viability by trypan blue exclusion.

Cell treatment with metabolic activation
In the assay with metabolic activation the cells were exposed to the test substance according to the following procedure; 0.1 ml test substance solution or 0.1 ml positive control and 3.9 ml culture medium without serum were added to 1 ml 20% (v/v) S9-mix and 5 ml culture medium (with 10% horse serum) containing ca. 5,000,000 L5178Y cells to a final volume of 10 ml. Two cultures treated with the vehicle (DMSO) were used as negative controls; one single culture treated with MCA was used as positive control substance at a final concentration of 10 µg/ml. Single cultures were used for each concentration of the test substance. The cells were exposed for 4 h at ca. 37 oC and ca. 5 % CO2 in a humidified incubator.

The dose levels of the test substance used ranged from 0.0019 to 1.0 mmol/l IXOL B350. At the start and end of the treatment, all cell cultures were checked visually and selected cultures were checked for viability by trypan blue exclusion.

Assessment of cytotoxicity
The cytotoxicity of the test substance was determined by measuring the relative initial cell yield, the relative suspension growth (RSG) and the relative total growth (RTG).

Evaluation criteria:

A response was considered to be positive if the induced mutant frequency (mutant frequency of the test substance minus that of the vehicle negative control) was more than 126 mutants per 1,000,000 clonable cells. A response was considered to be equivocal if the induced mutant frequency was more than 88 mutants (but smaller than 126 mutants) per 1,000,000 clonable cells. Any apparent increase in mutant frequency at concentrations of the test substance causing more than 90% cytotoxicity was considered to be an artefact and not indicative of genotoxicity.

The test substance was considered to be mutagenic in the gene mutation test at the TK-locus if a concentration-related increase in mutant frequency was observed, or if a reproducible positive response for at least one of the test substance concentrations was observed.

The test substance was considered not to be mutagenic in the gene mutation test at the TK-locus if it produced neither a dose-related increase in the mutant frequency nor a reproducible positive response at any of the test substance concentrations.

Statistics:

No statistical analysis was performed. Both numerical significance and biological relevance were considered together in the evaluation.

Results and discussion

Test results

Test results 1

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: the maximum concentration was limited by cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at and above 0.5 mmol/L

Cytotoxicity / choice of top concentrations:

other: the maximum concentration was limited by cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The test substance IXOL B350 was tested and evaluated for mutagenicity in both the absence and presence of metabolic activation (S9-mix). Beside treatment for 4 hours in the presence of a metabolic activation system, an extended treatment for 24 hours in the absence of S9-mix was used. In the presence of S9-mix a dose related increase in mutant frequency was observed. Although the increase was observed at limited data points, since in the Bacterial reverse mutation test with IXOL B350 (8405/18) also a mutagenic response was observed in the presence of S9-mix, it was decided not to perform a second assay to obtain more data points. Since relatively more small then large colonies were observed at concentrations causing a positive response, it can not be excluded that IXOL B350 is clastogenic.
It is concluded that under the conditions used in this study, the test substance IXOL B350 is mutagenic at the TK-locus of mouse lymphoma L5178Y cells.

Any other information on results incl. tables

Table:  Summary of the results:

Dose (mmol/l)	absence of S9-mix (24h)		Dose (mmol/l)	presence of S9-mix (4h)
	MF	RTG		MF	RTG
0.37	83	12	0.7	407	8
0.31	90	18	0.6	298	19
0.26	67	47	0.5	129	48
0.18	70	51	0.37	123	58
0.13	74	56	0.26	77	82
0.06	66	103	0.06	59	103
0.03	64	93	0.03	70	98
0.02	39	94	0.02	58	105
0.01	60	92	0.01	60	82
0	44*	100*	0	49*	100*

* Mean of duplicate cultures RTG: relative total growth is a measure for the cytotoxicity of the test substance.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, Polyol IXOL B350 is mutagenic (clastogenic) at the TK-locus of mouse lymphoma L5178Y cells.

Executive summary:

Polyol IXOL B350 was examined for its potential to induce gene mutations at the TK-locus of cultured mouse lymphoma L5178Y cells, in both the absence and the presence S9-mix (GLP study according to OECD guideline 478). One assay was conducted in which single cultures were treated for 24 hours and 4 hours in the absence and presence of S9-mix, respectively (TNO, 2010d).

The highest concentrations of the substance evaluated for mutagenicity were 0.37 and 0.7 mmol/L in the absence and presence of S9-mix, respectively. The maximum concentrations were limited by cytotoxicity. In the absence of S9-mix no increase in mutant frequency was observed at any test substance concentration evaluated. In the presence of S9-mix a dose related increase in mutant frequency was observed at and above 0.5 mmol/L. In presence of S9-mix at the concentrations causing an increase in mutant frequency, relatively more small than large colonies were formed. The mean percentage of small colonies formed was 64%. Based on these results it cannot be excluded that Polyol IXOL B350 is clastogenic.The negative controls were within historical background ranges and treatment with the positive control yielded the expected significant increase in mutant frequency compared to the negative controls.

It is concluded that under the conditions used in this study, Polyol IXOL B350 is mutagenic (clastogenic) at the TK-locus of mouse lymphoma L5178Y cells.