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

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test (OECD 471): negative with S. typhimurium strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 with and without metabolic activation

In vitro micronucleus test: positive in V79 cells without metabolic activation at cytotoxic concentrations

HPRT test: negative in V79 cells without metabolic activation

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

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

Target gene:

his operon (for S. typhimurium strains)

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.

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.

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.

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

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Acceptable, well documented publication/study report which meets basic scientific principles: no experiment with metabolic activation; only long-term treatment (24 h)

Principles of method if other than guideline:

The mutagenicity of urethane dimethacrylate (UDMA) was investigated in an in-vitro gene mutation assay (HPRT test). V79 cells were exposed to UDMA for 24 h in the absence of metabolic activation.

GLP compliance:

not specified

Type of assay:

other: mammalian cell gene mutation assay

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 cells were cultivated in minimal essential medium (MEM) supplemented with 10% fetal calf serum, penicillin (100 U/mL) and streptomycin (100 µg/mL) at 37 °C and 5% CO2.

Metabolic activation:

without

Test concentrations with justification for top dose:

25, 50 and 75 µM (= 11.75, 23.5, 35.25 µg/mL)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Ethylmethanesulphonate: 200 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 h
- Expression time (cells in growth medium): day 4 (after seeding) - day 8
- Selection time: day 8 - 10
- Fixation time: day 10

SELECTION AGENT: 7 µg/mL 6-thioguanine

NUMBER OF REPLICATIONS: The experiments were conducted with one plate per concentration for HPRT deficient mutant isolation and the experiments were at least repeated once.

DETERMINATION OF CYTOTOXICITY
- Method: Initial cytotoxicity of the test substance on day 1 after exposure is indicated by cell numbers plating efficiency (PE1).

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Remarks:

No mutagenic activity of UDMA was detected when V79 cells were exposed for 24 h without metabolic activation.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 23.5 µg/mL UDMA

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: A dose-dependent decrease of cell numbers caused by increasing concentrations was observed.

 Table 1

UDMA [µg/mL]	Cell number [%]	PE1 [%]	PE2 [%]	Numbers of mutants/6E6 cells	Mutant frequencies/ 1E6 survivors
0	100	100	100	22	8
11.75	69	116	100	29	10
23.5	36	123	111	20	6
35.25	15	29	111	30	10
EMS 200 µg/mL	-	95	-	1995	635

 PE=plating efficiency

Conclusions:

Interpretation of results:
negative without metabolic activation
other: no experiment with metabolic activation

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Acceptable, well documented publication which meets basic scientific principles: no experiment with metabolic activation, no cytokinesis blocker (it was not shown that the cell population analysed has undergone mitosis), only long-term treatment (24 h); only 1000 cells per slide were evaluated

Principles of method if other than guideline:

The induction of micronuclei by urethane dimethacrylate (UDMA) was investigated in V79 cells. The cells were exposed to UDMA for 24 h in the absence of metabolic activation.

GLP compliance:

not specified

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

V79 cells were cultivated in minimal essential medium (MEM) supplemented with 10% fetal calf serum (FCS), penicillin (100 U/mL), and streptomycin (100 µg/mL) at 37 °C and 5% CO2.

Metabolic activation:

without

Test concentrations with justification for top dose:

25, 50 and 75 µMol/L (= 11.75, 23.5, 35.25 µg/mL)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

ethylmethanesulphonate: 5 mmol/L (=620 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

STAIN (for cytogenetic assays): Schiff reagent

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

NUMBER OF CELLS EVALUATED: 1000 cells/slide

DETERMINATION OF CYTOTOXICITY
- Method: Concentrations leading to a 50% reduction of cell viability (TC50 value; n=3) were calculated from fitted dose-response curves.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

at cytotoxic concentration levels (>= 24 µg/mL) the numbers of micronuclei was slightly increased

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TC50=51.3±5.4 µmol/L (about 24 µg/mL)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY: Concentrations leading to a 50% reduction of cell viability (TC50 value; n=3) were calculated from fitted dose-response curves. TC50 = 51.3±5.4 µmol/L (about 24 µg/mL).

Table 1: Induction of micronuclei in V79 cells after exposure to UDMA

UDMA [µg/mL]	Micronuclei per 1000 cells (mean ± SD)*
	Experiment 1	Experiment 2
0	12.7 ± 1.5	9.7 ± 1.2
11.75	8.5 ± 2.1	7.3 ± 5.0
23.5	17.0 ± 5.3	10.3 ± 2.1
35.25	21.0 ± 3.6	17.7 ± 4.9
EMS# 620 µg/mL	234.1 ± 39.5	-

* Frequencies of micronuclei (MN) are mean values (± standard deviation) scored in 1000 cells of each of 3 separate slides (n=3)

# The numbers of micronuclei induced by ethylmethane sulfonate (EMS; positive control) are mean values (± SD) of 29 slides (n=29) in 10 independent experiments.

Conclusions:

Interpretation of results:
ambiguous without metabolic activation UDMA slightly increased the numbers of micronuclei at cytotoxic concentration levels (>= 24 µg/mL).
other: no experiment with metabolic activation

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Additional information

Gene mutation in bacteria

A bacterial gene mutation assay with the test substance was performed in accordance with OECD Guideline 471 and in compliance with GLP (Paulus, 2009). In two independent experiments, the Salmonella typhimurium strains TA 97a, TA 98, TA 100, TA 102 and TA 1535 were exposed to the test substance dissolved in DMSO using either the preincubation or the plate incorporation method. Test substance concentrations of 50, 150, 500, 1501 and 5004 µg/plate were selected for the plate incorporation test with and without metabolic activation. In the second experiment, 312, 624, 1247, 2493 and 4986 µg/plate were selected for the preincubation method with and without metabolic activation. No signs of cytotoxicity were observed up to and including the limit concentration. Up to 5000 µg/plate, the test substance did not induce an increase in the mutation frequency of the tester strains in the presence and absence of a metabolic activation system. The determined vehicle values for the spontaneous revertants of the controls and all positive control values were within the range of historical data. Under the conditions of this experiment, the test substance did not show mutagenicity in the selected S. typhimurium strains in the presence and absence of metabolic activation.

 

In vitro cytogenicity

An in vitro micronucleus assay was performed with the test substance (Schweikl, 2001). In two independent experiments, Chinese hamster lung fibroblasts were exposed to the test substance dissolved in DMSO at concentrations of 11.75, 23.5, 35.25 µg/mL for 24 h in the absence of metabolic activation. Cytotoxicity of the test substance was observed and the TC50 value was assessed to be 24 µg/mL. At cytotoxic concentration levels of the test substance (≥ 24 µg/mL) the numbers of micronuclei were slightly increased in the absence of metabolic activation. Ethyl methanesulphonate was used as positive control and produced a distinct increase in micronuclei frequency indicating that the test conditions were adequate. Under the conditions of this experiment, the potential of the test substance to induce micronuclei is equivocal.

 

In vitro mutagenicity in mammalian cells

An in vitro HPRT assay was performed with the test substance (Schweikl, 1998). In three replicate cultures Chinese hamster lung fibroblasts were exposed to the test substance dissolved in DMSO at concentrations of 11.75, 23.5, 35.25 µg/mL for 24 h in the absence of metabolic activation. Cytotoxicity of the test substance was observed at concentrations ≥ 23.5 µg/mL. No mutagenic activity of UDMA was detected. Ethyl methanesulphonate was used as positive control and produced a distinct increase in mutant frequency indicating that the test conditions were adequate. Thus, under the conditions of this experiment, the test substance did not show mutagenicity in V79 cells without metabolic activation.

Due to the positive result in the in vitro micronucleus test without metabolic activation at cytotoxic concentrations a micronucleus test in vivo should be conducted to conclude on genotoxic potential of the test substance.

Justification for classification or non-classification

The available data on genetic toxicity are not sufficient for classification according to Regulation (EC) No 1272/2008.