1,1-Bis(acryloyloxymethyl)ethyl isocyanate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 - 25 July 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

2 plates per concentration were used, no standard deviations were calculated; According to the guideline at least 5 analysable concentrations should be used. Due to the cytotoxicity only 4 concentrations were valid for several strains in experiment 1 and 2.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2005

Report date:

2005

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted 21 July 1997

Deviations:

yes

Remarks:

(2 plates per concentration were used, no standard deviations were calculated)

Test guideline 2

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Deviations:

not specified

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

2-isocyanato-2-methyl-3-(prop-2-enoyloxy)propyl prop-2-enoate

Cas Number:

886577-76-0

Molecular formula:

C11H13NO5

IUPAC Name:

2-isocyanato-2-methyl-3-(prop-2-enoyloxy)propyl prop-2-enoate

Test material form:

liquid

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL- Storage condition of test material: Refrigerated, shading- Solubility and stability of the test substance in the solvent/vehicle: No exothermic reaction nor generation of gas in DMSO, solubility in DMSO was 50 mg/mL and more.

Method

Target gene:

his operon (for S. typhimurium strains)trp operon (for E. coli strain)

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:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with phenobarbital/5,6-benzoflavone

Test concentrations with justification for top dose:

Pre-experiment: 1.2, 4 .9, 20, 78, 313, 1250 and 5000 μg/plate with and without metabolic activationMain Experiment:Experiment I: The pre-experiment served as Experiment I.Experiment II:10, 20, 39, 78, 156 and 313 μg/plate without metabolic activation10, 20, 39, 78, 156, 313, 625 and 1250 μg/plate with metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO - Justification for choice of solvent/vehicle: The solvent was chosen because of its solubility properties.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

furylfuramide

other: -S9: ICR-191 (1.0 µg/plate) for TA1537; +S9: 2-Aminoanthracene (2-AA; 2.0 and 10.0 µg/plate) for TA1535 and WP2 uvrA, respectively

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubationDURATION- Preincubation period: 20 min- Exposure duration: 48 hNUMBER OF REPLICATIONS: 2 replications each in 2 independent experimentsDETERMINATION OF CYTOTOXICITY- Method: growth inhibition

Evaluation criteria:

In the dose-finding test and the main test, if the number of revertant colonies on the test plates increased significantly in comparison with that on the control plates (based on twice as many as that of the negative control), and dose-response and reproducibility were also observed, the test substance was to be judged positive.

Statistics:

Mean values were calculated.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: ≥ 313 µg/plate with and without metabolic activation; Experiment 2: ≥156 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: ≥ 313 µg/plate with and without metabolic activation; Experiment 2: ≥156 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: ≥ 313 µg/plate with and without metabolic activation; Experiment 2: ≥156 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: ≥ 313 µg/plate with and without metabolic activation; Experiment 2: ≥156 µg/plate with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Experiment 1: ≥ 313 µg/plate without metabolic activation; ≥ 1250 µg/plate with metabolic activation; Experiment 2: ≥ 313 µg/plate without metabolic activation; ≥ 625 µg/plate wit metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS- Precipitation: Precipitation of the test substance on the plates was not observed either with or without metabolic activation.RANGE-FINDING/SCREENING STUDIES:The pre-experiment served as Experiment I (all strains were tested in the pre-experiment).

Any other information on results incl. tables

Table 1: Results of pre-experiment/Experiment I

With or without S9-Mix	Test substance concentration (μg/plate)	Average number of colonies of 2 plates
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
–	Solvent control (DMSO)	126	20	31	18	12
–	1.2	102	15	26	16	17
–	4.9	117	18	26	19	13
–	20	124	23	28	16	16
–	78	160	13	26	25	11
–	313	73*	8*	23*	9*	0*
–	1250	0*	0*	19*	0*	0*
–	5000	0*	0*	0*	0*	0*
Positive controls, –S9	Name	AF-2	SA	AF-2	AF-2	ICR-191
	Concentrations (μg/plate)	0.01	0.5	0.01	0.1	1.0
	Average number of colonies of 2 plates	625	426	177	597	2361
+	Solvent control (DMSO)	130	14	36	31	18
+	1.2	83	11	30	21	12
+	4.9	112	11	17	29	24
+	20	112	13	32	36	23
+	78	119	12	29	32	29
+	313	83*	11*	32	15*	5*
+	1250	71*	11*	27*	13*	6*
+	5000	0*	0*	0*	0*	0*
Positive controls, +S9	Name	B(a)P	2AA	2AA	B(a)P	B(a)P
	Concentrations (μg/plate)	5.0	2.0	10.0	5.0	5.0
	Average number of colonies of 2 plates	730	285	325	181	92

AF-2 = Furylfuramide

SA = Sodium azide

2AA = 2-Aminoanthracene

B(a)P = Benzo(a)pyrene

* = Growth inhibition of the tested bacteria strain by the tested substance was observed

Table 2: Results of Experiment II

With or without S9-Mix	Test substance concentration (μg/plate)	Average number of colonies of 2 plates
		Base-pair substitution type			Frameshift type
		TA 100	TA1535	WP2uvrA	TA98	TA1537
–	Solvent control (DMSO)	101	18	28	27	11
–	10	111	15	26	20	12
–	20	91	19	27	26	12
–	39	113	21	20	30	9
–	78	121	22	20	21	10
–	156	100*	12*	21	17*	9*
–	313	73*	11*	21*	14*	3*
Positive controls, –S9	Name	AF-2	SA	AF-2	AF-2	ICR-191
	Concentrations (μg/plate)	0.01	0.5	0.01	0.1	1.0
	Average number of colonies of 2 plates	552	451	162	594	2196
+	Solvent control (DMSO)	94	14	28	34	16
+	10	104	15	NT	36	20
+	20	89	14	NT	32	19
+	39	97	16	20	32	20
+	78	102	12	24	31	18
+	156	109*	15*	21	30*	12*
+	313	85*	9*	21	24*	6*
+	625	NT	NT	26*	NT	NT
+	1250	NT	NT	26*	NT	NT
Positive controls, +S9	Name	B(a)P	2AA	2AA	B(a)P	B(a)P
	Concentrations (μg/plate)	5.0	2.0	10.0	5.0	5.0
	Average number of colonies of 2 plates	785	330	359	230	93

AF-2 = Furylfuramide

SA = Sodium azide

2AA = 2-Aminoanthracene

B(a)P = Benzo(a)pyrene

* = Growth inhibition of the tested bacteria strain by the tested substance was observed

NT = Not tested

Table 3: Historical control data (mean values)

Strain		Mean
		-S9 mix	+S9 mix
TA100	Solvent control	120	123
	Positive control	626	713
TA1535	Solvent control	19	13
	Positive control	442	312
WP2uvrA	Solvent control	28	29
	Positive control	153	383
TA98	Solvent control	22	30
	Positive control	581	205
TA1537	Solvent control	12	20
	Positive control	2334	78