Bis(2-chloroethoxy)methane

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

Study period:

06 December 2005 - 27 january 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is performed according to internationally accepted guidelines and under GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-chloroethoxy)methane

EC Number:

203-920-2

EC Name:

Bis(2-chloroethoxy)methane

Cas Number:

111-91-1

Molecular formula:

C5H10Cl2O2

IUPAC Name:

1-chloro-2-[(2-chloroethoxy)methoxy]ethane

Details on test material:

Name: Diformal
Appearance: clear colorless liquidt
chemical name: Bis-(2-chlorethyl)-formal
CAS: 111-91-1
EINECS: 2039202
Purity: 88.7%
Homogenicity: homogenous
Stability: see expiry data
Date of receipt: 29. Nov. 2005
Expiry date: 28. Mai. 2006
Storage: room temperature

Method

Target gene:

histidine operon

Species / strain

Species / strain / cell type:

other: Salmonella typhimurium LT2: strains TA1535, TA97a, TA98, TA100 and TA102

Additional strain / cell type characteristics:

other: TA97a: hisD6610, TA98: hisD3052 TA100 and TA1535 hisG46, TA102: hisG428, TA97a, TA98 und TA100 uvrB; TA97a, TA98, TA100 and TA102 Plasmid pKM 101 and are lipopolysaccharidesidechain deficient (rfa)

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

First experiment: 5019, 1506, 502, 151 or 50 µg/plate
Second experiment: 4987, 2494, 1247 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: the test substance is completely soluble in DMSO and it is known that DMSO does not have any effects on the tester strains.

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: - S9-mix: NPD 80 µg/Plate: TA97a, 98 und 102. Na-azid 6 µg/Plate: TA100 and 1535. +S9-mix: BaP 40 µg/Plate: TA98. 2-AA 3 µg/Plate, TA97a, 100, 102 and 1535.

Details on test system and experimental conditions:

METHOD OF APPLICATION: First experiment: in agar (plate incorporation). Second experiment: preincubation.

DURATION
- Preincubation period: second experiment: 20 minutes
- Exposure duration: 48 hours
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): histidine
SPINDLE INHIBITOR (cytogenetic assays): not applicable
STAIN (for cytogenetic assays): not applicable

NUMBER OF REPLICATIONS: 4 plates per strains per dose

NUMBER OF CELLS EVALUATED: all colonies were counted manually

DETERMINATION OF CYTOTOXICITY
- Method: the background lawn and number of spontanous reventant colonies was evaluated.


OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:


OTHER:

Evaluation criteria:

A positive response is defined as a twofold increase of the number of revertant colonies as compared to the negative control.

Statistics:

None

Results and discussion

Test results

Species / strain:

other: Salmonella typhimurium LT2: strains TA1535, TA97a, TA98, TA100 and TA102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

on the second experiment slight toxicity was observed in the highest concentration tested (5000 µg/plate) in strains TA97a and TA98. Further toxicity was not observed.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: not observed
- Other confounding effects: no data
RANGE-FINDING/SCREENING STUDIES: no data
COMPARISON WITH HISTORICAL CONTROL DATA: the values for the solvent and positive controls are with the hitorical control values of the laboratory
ADDITIONAL INFORMATION ON CYTOTOXICITY: no data

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

First experiment:

strain		TA97a		TA98		TA100		TA102		TA1535
Metabolic activation		-	+	-	+	-	+	-	+	-	+
Water	Average	115	119	7	9	148	120	150	168	6	9
	SD	18.3	23.2	2.9	3.6	21	40	48.5	37.9	1.9	3.0
DMSO	Average	131	136	7	12	143	156	150	186	11	15
	SD	31.0	11.3	2.2	2.0	26	35	48.0	11.3	3.9	5.1
Positive control	Average	791	673	3.81	831	664	713	446	1101	1001	79
	SD	244	302	105	341	201	294	19	200	0	18
	F(I)	6.04	4.95	54.4	69.3	4.49	4.57	2.97	5.92	167	5.27
5019 µg/plate	Average	120	114	13	5	117	141	133	97	22	32
	SD	33	34	2	3	43	7	44	16	7	6
	F(I)	0.92	0.84	1.86	0.42	0.82	0.90	0.89	0.52	2.00	2.13
1506 µg/plate	Average	106	95	8	7	89	104	88	113	13	16
	SD	31	27	8	2	14	9	38	10	4	4
	F(I)	0.81	0.70	1.14	0.58	0.62	0.67	0.59	0.61	1.18	1.07
502 µg/plate	Average	87	149	10	7	69	134	152	104	9	10
	SD	7	25	3	4	5	32	8	19	5	3
	F(I)	0.66	1.10	1.43	0.58	0.48	0.86	1.01	0.56	0.82	0.67
151 µg/plate	Average	129	133	12	5	79	167	123	114	7	9
	SD	30	42	2	3	44	23	40	33	3	3
	F(I)	0.98	0.98	1.71	0.42	0.55	1.07	0.82	0.61	0.64	0.60
50 µg/plate	Average	122	92	8	13	168	123	106	83	13	9
	SD	13	30	3	6	8	37	6	6	3	3
	F(I)	0.93	0.68	1.14	1.08	1.17	0.79	0.71	0.45	1.18	0.60

SD = standard deviation

F(I) = induction factor

 

 

Second experiment:

strain		TA97a		TA98		TA100		TA102		TA1535
Metabolic activation		-	+	-	+	-	+	-	+	-	+
Water	Average	67	138	10	9	141	137	188	180	10	10
	SD	37.8	36.4	5.0	1.8	10	7	12.7	23.0	4.1	3.1
DMSO	Average	177	116	14	8	134	140	116	144	12	11
	SD	69.6	96.3	5.7	2.2	14	30	9.7	4.8	2.4	0.0
Positive control	Average	1001	359	995	39	729	504	1019	631	1001	106
	SD	0	124	13	49	184	111	36	74	0	59
	F(I)	5.66	3.09	71.1	4.88	5.17	3.60	8.78	4.38	100.	9.64
4987 µg/plate	Average	35	38	6	2	74	123	44	74	13	16
	SD	40	31	3	1	16	53	11	23	6	7
	F(I)	0.20	0.33	0.43	0.25	0.55	0.88	0.38	0.51	1.08	1.45
2494 µg/plate	Average	118	96	8	6	131	142	156	167	18	15
	SD	62	34	2	2	11	14	7	29	2	2
	F(I)	0.67	0.83	0.57	0.75	0.98	1.01	1.34	1.16	1.50	1.36
1247 µg/plate	Average	227	162	10	6	122	130	159	158	14	18
	SD	31	39	4	2	15	18	19	26	4	7
	F(I)	1.28	1.40	0.71	0.75	0.91	0.93	1.37	1.10	1.17	1.64

SD = standard deviation

F(I) = induction factor

 

Comparison with historical control

strain		TA97a		TA98		TA100		TA102		TA1535
Metabolic activation		-	+	-	+	-	+	-	+	-	+
Water	Range	72-153	57-162	6-16	6-26	93-179	92-178	91-187	95-239	6-19	5-25
	Exp 1	115	119	7	9	148	120	150	168	6	9
	Exp 2	67	138	10	9	141	137	188	180	10	10
DMSO	Range	22-176	58-174	4-19	3-24	90-162	73-172	79-199	70-237	5-19	6-17
	Exp 1	131	136	7	12	143	156	150	186	11	15
	Exp 2	177	116	14	8	134	140	116	144	12	11
Pos. Control	Range	304-1017	254-1001	223-1001	31-305	389-1170	160-1001	414-1144	118-1001	139-1001	63-359
	Exp 1	791	673	3.81	831	664	713	446	1101	1001	79
	Exp 2	1001	359	995	39	729	504	1019	631	1001	106

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

A significant or dose-related increase in the number of revertant colonies was not observed in any of the tested strains with or without metabolic activation. It is concluded that under the conditions of this test the test substance is not mutagenic in the Ames test.

Executive summary:

An ames test was performed accroding to OECD 471 and EC B.13/14 and under GLP. Two independent experiments were performed. The vehicle that was used is DMSO. The strains used were TA97a, TA98, TA100, TA102 and TA1535. The strains were exposed for 48 hours.

In the first experiment 5 concentrations were tested ranging from 50 -5000 µg/plate with and without metabolic activation. No toxicity to the test strains was observed. At the highest concentration a weak increase of revertant colonies was observed for TA1535. Since this was the only effect observed it was not seen as a sign of mutagneicity.

In the second experiment 3 concentartions were tested between 5000 -1250 µg/plate. No signs of mutagenicity were observed and slight toxicity was observed in strain TA97a and TA98 at the highest concentration tested.

In both studies the number of spontaneous revertants in the control group was within the historical control range and controls for confirmation of the genotype of the tester strains and the sterility check were in order. The number of revertant colonies in the positive controls showed a significant mutagenic response with and without S9 -mix.

Under the conditions of this test the test substance is not mutagenic.