7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

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:

27 May - 25 June 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Landesamt für Umwelt, Wasserwirtschaft und Gewerbeaufsicht, Mainz, Germany

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

EC Number:

276-957-5

EC Name:

7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

Cas Number:

72869-86-4

Molecular formula:

C23H38N2O8

IUPAC Name:

2-[({2,2,4-trimethyl-6-[({2-[(2-methylprop-2-enoyl)oxy]ethoxy}carbonyl)amino]hexyl}carbamoyl)oxy]ethyl 2-methylprop-2-enoate; 2-[({2,4,4-trimethyl-6-[({2-[(2-methylprop-2-enoyl)oxy]ethoxy}carbonyl)amino]hexyl}carbamoyl)oxy]ethyl 2-methylprop-2-enoate

Method

Target gene:

his operon (for S. typhimurium strains)

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

co-factor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

First experiment: 50, 150, 500, 1501 and 5004 µg/plate with and without metabolic activation
Second experiment: 312, 624, 1247, 2493 and 4986 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: DMSO was chosen because the test substance was completely soluble, and this solvent does not have any effects on the viability of the bacteria or the number of spontaneous revertants.

Controls

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

benzo(a)pyrene

other: 4-Nitro-1,2-phenylene diamine (4-NPD); 2-Amino-Anthracene (2-AA)

Remarks:

+S9: 2-AA (1 µg/plate, TA100, TA102, TA1535, TA97a); B[a]P (20 µg/plate, TA98); -S9: sodium azide (1 µg/plate, TA100 and TA1535); 4-NPD (20 µg/plate, TA102, TA97a, TA98)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) (experiment 1); preincubation (experiment 2)

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: quadruplicates each in 2 independent experiment

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants or a clearing of the bacterial background lawn

Evaluation criteria:

A test substance is considered to have a mutagenic potential if a significant, reproducible increase of revertant colonies per plate (increase factor ≥ 2) in at least one strain can be observed. A concentration-related increase over the range tested can also be taken as a sign of mutagenic activity.

Statistics:

Mean values, standard deviations and the increase factor of revertant induction were calculated.

Results and discussion

Test results

Species / strain:

S. typhimurium, other: TA97a, TA98, TA100, TA102 and TA1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

but tested up to limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA: The determined values for the spontaneous revertants of the negative controls and all positive control values were in the range of historical data.

Any other information on results incl. tables

Table 1. Test results of Experiment 1 (plate incorporation)

With or without S9 mix	Test substance concentration [μg/plate]	Mean number of revertant colonies per plate (average of 4 plates ± standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA102	TA1535	TA97a	TA98
-	0 (H2O)	110 ± 12.0	264 ± 47.0	6 ± 1.9	105 ± 4.2	7 ± 1.4
-	0 (DMSO)	97 ± 6.8	296 ± 21.6	6 ± 2.1	96 ± 13.4	10 ± 1.0
-	50	97 ± 14	307 ± 7	6 ± 1	104 ± 21	4 ± 1
-	150	96 ± 17	310 ± 25	6 ± 1	105 ± 2	7 ± 3
-	500	102 ± 9	315 ± 14	7 ± 4	89 ± 18	7 ± 1
-	1501	108 ± 1	268 ± 36	8 ± 3	107 ± 5	7 ± 4
-	5004	99 ± 10	275 ± 49	6 ± 2	92 ± 6	7 ± 3
Positive controls, -S9 mix	Name	NaN3	4-NPD	NaN3	4-NPD	4-NPD
	Concentrations [μg/plate]	1	20	1	20	20
	Mean No. of colonies/plate (average of 4 ± SD)	630 ± 44	1285 ± 185	374 ± 18	445 ± 35	278 ± 56
+	0 (H2O)	101 ± 15.8	345 ± 20.0	7 ± 1.5	97 ± 16.7	5 ± 2.8
+	0 (DMSO)	108 ± 0.8	305 ± 51.7	9 ± 4.2	90 ± 13.3	7 ± 3.1
+	50	107 ± 2	304 ± 21	6 ± 3	109 ± 3	6 ± 1
+	150	109 ± 3	276 ± 33	7 ± 3	109 ± 7	6 ± 1
+	500	100 ± 6	309 ± 17	8 ± 3	111 ± 10	8 ± 2
+	1501	94 ± 15	315 ± 59	4 ± 2	99 ± 15	6 ± 2
+	5004	118 ± 21	330 ± 40	13 ± 4	96 ± 15	7 ± 2
Positive controls, +S9 mix	Name	2-AA	2-AA	2-AA	2-AA	B[a]P
	Concentrations [μg/plate]	1	1	1	1	20
	Mean No. of colonies/plate (average of 4 ± SD)	524 ± 102	1093 ± 53	105 ± 12	319 ± 22	99 ± 9

NaN3: Sodium azide

4-NPD: 4-Nitro-1,2-phenylene diamine

2-AA: 2-aminoanthracene

B[a]P: Benzo-a-pyrene

 

Table 2. Test results of Experiment 2 (preincubation)

With or without S9 mix	Test substance concentration [μg/plate]	Mean number of revertant colonies per plate (average of 4 plates ± standard deviation)
		Base-pair substitution type			Frameshift type
		TA 100	TA102	TA1535	TA97a	TA98
-	0 (H2O)	121 ± 10.0	299 ± 52.4	12 ± 3.2	103 ± 4.1	10 ± 0.0
-	0 (DMSO)	93 ± 7.1	274 ± 45.0	12 ± 1.9	110 ± 9.0	10 ± 3.7
-	312	106 ± 12	282 ± 22	8 ± 5	92 ± 14	11 ± 6
-	624	91 ± 9	232 ± 16	7 ± 1	110 ± 19	11 ± 2
-	1247	110 ± 30	255 ± 46	4 ± 4	109 ± 10	10 ± 3
-	2493	124 ± 12	192 ± 9	8 ± 3	114 ± 11	13 ± 4
-	4986	100 ± 7	188 ± 18	9 ± 6	98 ± 9	10 ± 2
Positive controls, -S9 mix	Name	NaN3	4-NPD	NaN3	4-NPD	4-NPD
	Concentrations [μg/plate]	1	20	1	20	20
	Mean No. of colonies/plate (average of 4 ± SD)	386 ± 37	1127 ± 178	316 ± 17	903 ± 73	978 ± 72
+	0 (H2O)	97 ± 10.9	217 ± 12.9	10 ± 3.0	98 ± 7.4	14 ± 3.4
+	0 (DMSO)	109 ± 18.1	290 ± 30.2	10 ± 3.4	111 ± 2.1	11 ± 2.6
+	312	109 ± 8	279 ± 34	8 ± 2	94 ± 10	11 ± 2
+	624	106 ± 7	220 ± 15	5 ± 3	104 ± 7	11 ± 2
+	1247	101 ± 10	218 ± 21	12 ± 2	104 ± 9	13 ± 3
+	2493	95 ± 3	250 ± 51	10 ± 6	102 ± 3	11 ± 3
+	4986	92 ± 6	235 ± 29	8 ± 2	103 ± 4	7 ± 2
Positive controls, +S9 mix	Name	2-AA	2-AA	2-AA	2-AA	B[a]P
	Concentrations [μg/plate]	1	1	1	1	20
	Mean No. of colonies/plate (average of 4 ± SD)	499 ± 34	1345 ± 115	124 ± 12	826 ± 63	50 ± 5

NaN3: Sodium azide

4-NPD: 4-Nitro-1,2-phenylene diamine

2-AA: 2-aminoanthracene

B[a]P: Benzo-a-pyrene