1,10-decanediyl 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:

January - February 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

1,10-decanediyl bismethacrylate

EC Number:

229-745-1

EC Name:

1,10-decanediyl bismethacrylate

Cas Number:

6701-13-9

Molecular formula:

C18H30O4

IUPAC Name:

decane-1,10-diyl bis(2-methylacrylate)

Test material form:

liquid

Method

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver

Test concentrations with justification for top dose:

In the pre-experiment the concentration range of the test item was 3-5000 µg/plate. No relevant toxic effects were observed. 5000 µg/plate was chosen as maximal concentration.
The following concentrations were tested: 33, 100, 333, 1000, 2500 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:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine, without S9, TA1537, TA98. 2-aminoanthracene : with S9, all strains

Details on test system and experimental conditions:

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

Pre-experiment for toxicity :
To evaluate the toxicity of the test item a pre-experiment was performed with strains TA98 and TA100. 8 concentrations were tested for toxicity and mutation induction with each 3 plates. The experimental conditions in the pre-experiment were the same as described for the experiment 1 (plate incorporation test).
Toxicity of the test item can be evident as a reduction in the number of spontaneous revertants of a clearing of the bacterial background lawn.
Evaluable plates (> 0 colonies) at five concentrations or more in all strains used.

For each strain and dose level, including the controls, three plates were used.

In the preincubation assay, test solution, S9 mix and bacterial suspension were mixed and incubated at 37°C for 60 minutes. After pre-incubation agar was added to each tube. The mixture was poured on minimal agar plates. After solidification the plates were incubated upside down for at least 48 hours at 37 °C in the dark.

Acceptability of the assay :
-regular background growth in the negative and solvent control
-the spontaneous reversion rates in the negative and solvent control are in the arnge of the historical control data
-the psitive control substances should produce a significant increase in mutant colony frequencies

Evaluation criteria:

A test item is considered positive if either a dose related increase in the number of revertants of a biologically relevant increase for at least one test concentration is induced.
A test item producing neither a dose related increase in the number of revertants nor a biologically relevant positive response at any dose of the test points is considered non-mutagenic in this system.
A biologically relevant response is described as : A test item is considered mutagenic if the number of reversions is at least twice the spontaneous reversion rate instrains TA98, TA 100 and TA 102, or trice in strains TA1535, TA1537. Also a dose-dependent increase in the number of revertants is regarded as an indication of possibly existing mutagenic potential of the test item regardless whether the highest dose induced the criteria described above or not.

Statistics:

no

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium, other: TA98, TA100, TA102, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No precipitation of the test item occurred up to the highest investigated dose.
No toxic effects, evident as a reduction in the number of revertants, occurred in the tes groups with and without metabolic activation.
The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.
No substantial increase in revertant colony numbers of any of the five tester srains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.
Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Any other information on results incl. tables

Ames tables

Pre-experiment

Test group	Concentration per plate (µg)	Revertants per plate (mean of 3 plates)
		TA98	TA98	TA 100	TA 100
		- S9	+ S9	- S9	+ S9
Negative control	-	28	38	166	138
Solvent control	-	20	32	166	155
4-NOPD	10	223	-	-	-
Sodium azide	10	-	-	690	-
2-AA	2.5	-	223	-	742
Test item	3	22	29	143	126
Test item	10	29	28	139	132
Test item	33	30	29	143	128
Test item	100	27	32	143	126
Test item	333	27	30	133	118
Test item	1000	18	3	132	130
Test item	2500	24	23	131	133
Test item	5000	24	22	124	130

 

Experiment 1

TA1535 without S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	8	7	11	9	2.1
Solvent control	8	13	8	10	2.9	1.0
Positive control	615	664	664	648	28.3	67.0
33	13	10	7	10	3.0	1.0
100	9	9	10	9	0.6	1.0
333	10	7	8	8	1.5	0.9
1000	6	11	5	7	3.2	0.8
2500	9	5	9	8	2.3	0.8
5000	6	10	8	8	2.0	0.8

 

TA1535 with S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	8	8	11	9	1.7
Solvent control	15	15	9	13	3.5	1.0
Positive control	73	86	64	74	11.1	5.7
33	8	11	17	12	4.6	0.9
100	8	8	12	9	2.3	0.7
333	7	12	10	10	2.5	0.7
1000	11	11	12	11	0.6	0.9
2500	10	7	5	7	2.5	0.6
5000	6	9	7	7	1.5	0.6

 

TA1537 without S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	6	5	6	6	0.6
Solvent control	10	7	4	7	3.0	1.0
Positive control	52	51	52	52	0.6	7.4
33	8	5	7	7	1.5	1.0
100	8	5	4	6	2.1	0.8
333	5	8	5	6	1.7	0.9
1000	5	7	8	7	1.5	1.0
2500	6	4	4	5	1.2	0.7
5000	4	5	5	5	0.6	0.7

 

TA1537 with S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	16	11	7	11	4.5
Solvent control	5	7	5	6	1.2	1.0
Positive control	107	96	84	96	11.5	16.9
33	12	7	7	9	2.9	1.5
100	8	8	6	7	1.2	1.3
333	6	6	6	6	0	1.1
1000	8	7	4	6	2.1	1.1
2500	5	5	5	5	0	0.9
5000	8	4	4	5	2.3	0.9

 

 

TA98 without S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	27	27	31	28	2.3
Solvent control	23	20	18	20	2.5	1.0
Positive control	241	212	217	223	15.5	11.0
33	29	32	30	30	1.5	1.5
100	28	29	25	27	2.1	1.3
333	24	29	27	27	2.5	1.3
1000	17	18	19	18	1.0	0.9
2500	25	22	26	24	2.1	1.2
5000	23	24	26	24	1.5	1.2

 

TA98 with S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	38	39	38	38	0.6
Solvent control	34	32	29	32	2.5	1.0
Positive control	379	430	402	404	25.5	12.7
33	29	27	30	29	1.5	0.9
100	31	31	34	32	1.7	1.0
333	29	32	28	30	2.1	0.9
1000	21	23	25	23	2.0	0.7
2500	22	23	24	23	1.0	0.7
5000	19	23	24	22	2.6	0.7

 

TA100 without S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	156	161	180	166	12.7
Solvent control	166	160	171	166	5.5	1.0
Positive control	721	702	648	690	37.9	4.2
33	143	148	138	143	5.0	0.9
100	143	142	145	143	1.5	0.9
333	132	135	131	133	2.1	0.8
1000	139	128	130	132	5.9	0.8
2500	130	133	129	131	2.1	0.8
5000	125	121	127	124	3.1	0.8

 

TA100 with S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	147	132	135	138	7.9
Solvent control	158	157	149	155	4.9	1.0
Positive control	713	757	756	742	25.1	4.8
33	130	129	124	128	3.2	0.8
100	127	121	131	126	5.0	0.8
333	122	118	115	118	3.5	0.8
1000	125	129	135	130	5.0	0.8
2500	132	129	139	133	5.1	0.9
5000	132	128	131	130	2.1	0.8

 

TA102 without S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	207	194	194	198	7.5
Solvent control	195	182	201	193	9.7	1.0
Positive control	973	915	1026	971	55.5	5.0
33	233	198	205	212	18.5	1.1
100	229	195	178	201	26.0	1.0
333	210	210	183	201	15.6	1.0
1000	190	190	177	186	7.5	1.0
2500	193	205	177	192	14.0	1.0
5000	217	203	185	202	16.0	1.0

 

TA102 with S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	192	149	149	163	24.8
Solvent control	161	194	238	198	38.6	1.0
Positive control	749	785	806	780	28.8	3.9
33	184	198	237	206	27.5	1.0
100	196	196	167	186	16.7	0.9
333	147	165	181	164	17.0	0.8
1000	172	182	198	184	13.1	0.9
2500	156	139	117	137	19.6	0.7
5000	116	107	117	113	5.5	0.6

 

 

Experiment 2

TA1535 without S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	9	10	8	9	1.0
Solvent control	13	11	9	11	2.0	1.0
Positive control	738	754	677	723	40.6	65.7
33	13	11	7	10	3.1	0.9
100	14	10	13	12	2.1	1.1
333	10	10	11	10	0.6	0.9
1000	8	10	10	9	1.2	0.8
2500	10	7	7	8	1.7	0.7
5000	6	8	8	7	1.2	0.7

 

TA1535 with S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	10	13	13	12	1.7
Solvent control	13	10	9	11	2.1	1.0
Positive control	120	128	119	122	4.9	11.5
33	10	11	11	11	0.6	1.0
100	13	9	8	10	2.6	0.9
333	8	7	9	8	1.0	0.8
1000	5	13	6	8	4.4	0.8
2500	11	12	8	10	2.1	1.0
5000	13	9	10	11	2.1	1.0

 

TA1537 without S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	7	5	7	6	1.2
Solvent control	5	7	6	6	1.0	1.0
Positive control	47	49	65	54	9.9	8.9
33	6	6	8	7	1.2	1.1
100	6	6	6	6	0.0	1.0
333	5	7	7	6	1.2	1.1
1000	8	6	6	7	1.2	1.1
2500	4	7	5	5	1.5	0.9
5000	5	5	4	5	0.6	0.8

 

TA1537 with S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	12	11	11	11	0.6
Solvent control	12	8	9	10	2.1	1.0
Positive control	57	55	55	56	1.2	5.8
33	7	9	10	9	1.5	0.9
100	12	9	12	11	1.7	1.1
333	9	8	13	10	2.6	1.0
1000	9	11	10	10	1.0	1.0
2500	9	0	10	6	5.5	0.7
5000	11	11	11	11	0.0	1.1

 

 

TA98 without S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	25	24	32	27	4.4
Solvent control	31	21	23	25	5.3	1.0
Positive control	220	203	234	219	15.5	8.8
33	34	26	23	28	5.7	1.1
100	30	30	21	27	5.2	1.1
333	19	25	30	25	5.5	1.0
1000	21	30	23	25	4.7	1.0
2500	27	27	21	25	3.5	1.0
5000	19	24	26	23	3.6	0.9

 

TA98 with S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	30	44	40	38	7.2
Solvent control	45	44	34	41	6.1	1.0
Positive control	586	591	548	575	23.5	14.0
33	52	38	46	45	7.0	1.1
100	47	47	42	45	2.9	1.1
333	44	38	46	43	4.2	1.0
1000	35	33	36	35	1.5	0.8
2500	40	30	32	34	5.3	0.8
5000	37	38	34	36	2.1	0.9

 

TA100 without S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	97	112	129	113	16.0
Solvent control	129	107	100	112	15.1	1.0
Positive control	832	902	856	863	35.6	7.7
33	99	100	106	102	3.8	0.9
100	101	107	113	107	6.0	1.0
333	91	102	100	98	5.9	0.9
1000	116	109	101	109	7.5	1.0
2500	90	97	104	97	7.0	0.9
5000	62	84	66	71	11.7	0.6

 

TA100 with S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	81	78	113	91	19.4
Solvent control	132	125	131	129	3.8	1.0
Positive control	691	711	698	700	10.1	5.4
33	119	111	124	118	6.6	0.9
100	104	104	114	107	5.8	0.8
333	98	83	84	88	8.4	0.7
1000	83	100	95	93	8.7	0.7
2500	100	82	84	89	9.9	0.7
5000	89	90	78	86	6.7	0.7

 

TA102 without S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	210	220	212	214	5.3
Solvent control	199	157	189	182	21.9	1.0
Positive control	1040	984	1090	1038	53.0	5.7
33	212	214	183	203	17.3	1.1
100	228	214	207	216	10.7	1.2
333	205	215	198	206	8.5	1.1
1000	184	196	182	187	7.6	1.0
2500	196	199	166	187	18.2	1.0
5000	174	156	163	164	9.1	0.9

 

TA102 with S9

Concentration µg/plate	Plate 1	Plate 2	Plate 3	Mean revertant/ plate	s.d	factor
Negative control	275	191	191	219	48.5
Solvent control	191	201	229	207	19.7	1.0
Positive control	805	723	822	783	52.9	3.8
33	218	234	214	222	10.6	1.1
100	262	251	217	243	23.5	1.2
333	230	224	218	224	6.0	1.1
1000	219	215	202	212	8.9	1.0
2500	225	214	218	219	5.6	1.1
5000	218	228	223	223	5.0	1.1

 

 

 

 

Applicant's summary and conclusion

Conclusions:

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes of frameshifts in the genome of the strains used.
The test item is considered to be non-mutagenic in ths Salmonella typhimurium reverse mutation assay.

Executive summary:

The study was performed to investigate the potential of the test item to induce gene mutations according to the plate incorporation test (experiment 1) and the pre-incubation test (experiment 2) sing the Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and TA102.

The assay was performed in two independent experiments both with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. The test item was tested at the following concentrations: 33, 100, 333, 1000, 2500 and 500 µg/plate.

No toxic effects, evident as a reduction in the number of revertants, occurred in the tes groups with and without metabolic activation.

The plates incubated with the test item showed normal background growth up to 5000 µg/plate with and without S9 mix in all strains used.

No substantial increase in revertant colony numbers of any of the five tester srains was observed following treatment with the test item at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes of frameshifts in the genome of the strains used.

The test item is considered to be non-mutagenic in ths Salmonella typhimurium reverse mutation assay.