Polymer with 2-Butyne-1,4-Diol and (Chloromethyl-)Oxirane, Brominated, Dehydrochlorinated, Methoxylated

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

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 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes (incl. QA statement)

Remarks:

TNO Quality of life

Type of assay:

bacterial reverse 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:

his

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

S9 liver homogenate

Test concentrations with justification for top dose:

62, 185, 556, 1667 and 5000 µg/plate

Vehicle / solvent:

DMSO

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: benzo(a)pyrene and 2-aminoanthracene

Details on test system and experimental conditions:

One bacterial reverse mutation test was performed. The test substance was dissolved in DMSO at a concentration of 50 mg/ml based on a purity of 99%. A clear, slight-brown solution was obtained. Serial dilutions in DMSO were made. Five concentrations were tested, ranging from 62 to 5000 µg/plate. Negative controls (DMSO) and positive controls were run simultaneously with the test substance.

Fresh bacterial cultures were prepared by inoculation of nutrient broth with a thawed aliquot of the stock culture and subsequent incubation for approximately 10-16 h at 37°C while shaking. Briefly, the mutagenicity assay was carried out as follows. To 2 ml molten top agar (containing 0.6 % agar, 0.5 % NaCl and 0.05 mM L-histidine.HCl/0.05 mM biotin or 0.05 mM tryptophane for the S. typhimurium strains, and E. coli WP2 uvrA strain, respectively), maintained at ca. 46 oC, were added subsequently: 0.1 ml of a fully grown culture of the appropriate strain, 0.1 ml of the test substance or of the negative control or of the positive control substance solution, and 0.5 ml S9-mix for the experiments with metabolic activation or 0.5 ml sodium phosphate 100 mM (pH 7.4) for the experiments without metabolic activation. The ingredients were thoroughly mixed and the mix was immediately poured onto minimal glucose agar plates (1.5 % agar in Vogel and Bonner medium E with 2 % glucose). All determinations were made in triplicate. The plates were incubated at ca. 37 oC for approximately 48-72 hours. Subsequently, the his+ and trp+ revertants were counted. Toxicity is defined as a reduction (by at least 50%) in the number of revertant colonies and/or a clearing of the background lawn of bacterial growth as compared to the negative (vehicle) control and/or the occurrence of pinpoint colonies.

Evaluation criteria:

A test substance is considered to be positive in the bacterial gene mutation test if the mean number of revertant colonies on the test plates shows a
concentration-related increase or if a reproducible two-fold or more increase is observed compared the negative controls.
A test substance is considered to be negative in the bacterial gene mutation test if it produces neither a dose-related increase in the mean number of revertant colonies nor a reproducible positive response at any of the test points.

Statistics:

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

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

from 556 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at 5000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

from 185 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 5

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with

Genotoxicity:

positive

Remarks:

at 5000 µg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 6

Species / strain:

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

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Test results 7

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the presence of S9-mix, in strains TA 1535, TA 98, TA 100 and WP2 uvrA, IXOL B350 did induce a dose related increase in the mean number of revertants compared to the background spontaneous reversion rate observed with the negative control. A minimal 2-fold increase was observed in strain TA 1535, at and above 185 µg/plate; in strain TA 98, at 5000 µg/plate; in strain TA 100, at and above 556 µg/plate and in strain WP2 uvrA, at 5000 µg/plate. The maximal increase observed was 27-fold in strain TA 1535 at 5000 µg/plate.
In the absence of S9-mix in all strains, IXOL B350 did not induce a minimal 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control.

Any other information on results incl. tables

Table: Number of revertants counted in bacterial reverse mutation test with IXOL B350

				TA 1535			TA 1537			TA 98		TA 100			E. Coli
				-S9	+S9		-S9	+S9		-S9	+S9	-S9	+S9		-S9	+S9
0 µg/plate				34	20		14	17		37	43	139	128		36	23
				34	12		11	24		35	50	118	133		30	38
				26	26		11	24		28	59	127	131		20	26
	Mean			31	19		12	22		33	51	128	131		29	29
	StDev			5	7		2	4		5	8	11	3		8	8
62 µg/plate				35	34		18	19		40	49	133	174		25	28
				32	22		20	22		42	53	153	159		22	38
				26	25		14	26		28	56	145	186		24	36
	Mean			31	27		17	22		37	53	144	173		24	34
	StDev			5	6		3	4		8	4	10	14		2	5
185 µg/plate				31	66		8	14		25	59	143	241		35	29
				28	56		16	18		36	67	136	233		28	37
				26	50		20	18		37	53	143	277		20	40
	Mean			28	57		15	17		33	60	141	250		28	35
	StDev			3	8		6	2		7	7	4	23		8	6
556 µg/plate				34	88		16	16		36	61	146	429		46	37
				29	80		12	22		35	77	130	519		35	36
				41	107		13	22		38	61	135	458		42	25
	Mean			35	92		14	20		36	66	137	469		41	33
	StDev			6	14		2	3		2	9	8	46		6	7
1667 µg/plate				26	244		18	19		23	75	117	813		32	48
				38	268		12	35		31	78	119	930		36	55
				29	342		14	34		44	86	143	978		23	46
	Mean			31	285		15	29		33	80	126	907		30	50
	StDev			6	51		3	9		11	6	14	85		7	5
5000 µg/plate				40	512		7	29		40	123	139	776		42	89
				38	470		19	25		36	123	159	1575		35	84
				35	608		10	37		37	129	147	1781		35	86
				P	P		P	P		P	P	P	P		P	P
	Mean			38	530		12	30		38	125	148	1377		37	86
	StDev			3	71		6	6		2	3	10	531		4	3
Pos. Control				609	743		2301	319		822	1984	719	2525		231	1463
				578	767		2319	389		840	2198	822	2647		191	1727
				589	805		3485	384		645	2232	791	2382		223	1681
	Mean			592	772		2702	364		769	2138	777	2518		215	1624
	StDev			16	31		678	39		108	134	53	133		21	141

Mean                    Average number of revertants per plate                      

StDev                    Standard deviation                                                                                                                          

S9                        Liver homogenate from rats treated with aroclor

Pos. Control          Positive control; see text for actual concentrations of reference mutagens

P              Slight precipitation of the test substance on the agar plates

Applicant's summary and conclusion

Conclusions:

In a GLP compliant bacterial reverse mutation test (OECD 471) it is determined that the test substance is mutagenic in the presence of S9-mix and not mutagenic in the absence of S9-mix.

Executive summary:

Polyol IXOL B350 was examined for mutagenic activity in the bacterial reverse mutation test (GLP compliant and according to OECD guideline 471) using the histidine-requiringSalmonella typhimuriumstrains TA1535, TA1537, TA98, TA100 and the tryptophan-requiringEscherichia colistrain WP2uvrA, in the absence and presence of S9 -mix. All strains were used, with five concentrations of the test substance, ranging from 62 to 5000 µg/plate. Negative controls (DMSO) and positive controls were run simultaneously with the test substance. The mean number of his+and trp+revertant colonies of the negative controls were within the acceptable range and the positive controls gave the expected increase in the mean number of revertant colonies. The test substance was not toxic to any strain, in both the absence and presence of S9-mix, as neither a decrease in the mean number of revertants or a clearing of the background lawn of bacterial growth compared to the negative controls was observed. In the presence of S9-mix, in strains TA1535, TA98, TA100 and WP2uvrA, Polyol IXOL B350 did induce a dose related increase in the mean number of revertants compared to the background spontaneous reversion rate observed with the negative control. A minimal 2-fold increase was observed in strain TA1535, at and above 185 µg/plate; in strain TA98, at 5000 µg/plate; in strain TA100, at and above 556 µg/plate and in strain WP2uvrA, at 5000 µg/plate. The maximal increase observed was 27-fold in strain TA1535 at 5000 µg/plate. In the absence of S9-mix in all strains, Polyol IXOL B350 did not induce a minimal 2-fold and/or dose related increase in the mean number of revertant colonies compared to the background spontaneous reversion rate observed with the negative control. It is concluded that the results obtained with the test substance inSalmonella typhimuriumstrains TA1535, TA98 and TA100 and in theEscherichia colistrain WP2uvrA, indicate that Polyol IXOL B350 is mutagenic in the presence of the S9-mix and all strains used indicate that PolyolIXOL B350 is not mutagenic in the absence of S9-mix under the conditions employed in this study.