1,10-decanediyl bismethacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames test (OECD 471) is available on 1,10 decanediyl bis methacrylate. In this study, the test item did not induce gene mutations by base pair changes of frameshifts in the genome of the strains used.

No in vitro study on mammalian cells are available on 1,10 decanediyl bis methacrylate. However data are available on an analogue substance of 1,10 decanediyl bis methacrylate : 1,10-decanediol diacrylate. On this analogue substance, HPRT test (OECD 476) and in vitro micronucleus test (OECD 487) showed negative results in presence and in absence of metabolic activation.

Bacterial reverse mutation assay (Sokolowski 2010):

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) using 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.

In vitro mammalian cell micronucleus test (Brient 2013) / read-across :

The objective of this study was to evaluate the potential of 1,10-decanediol diacrylate to induce an increase in the frequency of micronucleated cellsin the mouse cell line L5178YTK+/-. This study conducted in compliance with OECD Guideline No. 487 and the principles of Good Laboratory Practices.

After a preliminary toxicity test, the test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

-First experiment: 3 h treatment + 24 h recovery (without and with S9 mix),

-Second experiment :24 h treatment + 20 h recovery (without S9 mix), and3 h treatment + 24 h recovery (with S9 mix).

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells and quality of the cells on the slides has also been taken into account.

For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per concentration). 

The test item was dissolved in dimethylsulfoxide (DMSO).

 Experiments without S9 mix: Following the first experiment,a severe toxicity was induced at the highest tested dose-level of 20 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 10 µg/mL induced a slight but acceptable toxicity, as shown by a 37% decrease in the PD. Following the second experiment,a severe toxicity was induced at the highest tested dose-level of 40 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 20 µg/mL induced no toxicity, as shown by no noteworthy decrease in the PD.

In the first experiment, a statistically significant increase in the frequency of micronucleated cells was noted at the highest dose-level of 10 µg/mL. However, no dose-response relationship was noted, and only one replicate of the two cultures used for this dose-level showed a frequency of micronucleated cells above the corresponding vehicle control historical data range. These results were thus considered to be equivocal, and the second experiment without S9 mix was performed following a long treatment period. During the second experiment, no statistically significant increase in the frequency of micronucleated cells was noted. Consequently, the increase observed during the first experiment was not reproduced, and was thus not considered to be biologically relevant.

Experiments with S9 mix: Following the first experiment,a marked toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 77% decrease in the PD. The immediately lower dose-level of 40 µg/mL induced a slight but acceptable toxicity, as shown by a 38% decrease in the PD. Following the second experiment, a slight toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 28% decrease in the PD.

In the first experiment, a dose-response relationship was noted, but no statistically significant increase in the frequency of micronucleated cells was observed. In the second experiment performed in the same experimental conditions, some increases in the frequency of micronucleated cells were noted at both higher doses (40 and 80 µg/mL). However, these increases were not statistically significant, and the corresponding frequencies of micronucleated cells remained within the historical data of the vehicle control. Consequently, these increases did not meet the criteria for a positive response and were thus considered as non-biologically relevant.

Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, in the absence or in the presence of a rat metabolising system.

 

In vitro mammalian cell gene mutation assay (Massip 2013) / Read-across :

1,10-decanediol diacrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used for all experiments.

In Experiment 1, thirteen concentrations, ranging from 2.5 to 150 µg/mL in the absence of S-9 and from 25 to 400 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 40mg/µL in the absence of S-9 and 330 µg/mL in the presence of S-9, limited by toxicity, which gave 17% and 12% RS, respectively.

In Experiment 2 twelve concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and from 50 to 400 µg/mL in the presence of S-9, were tested.The highest concentrations analysed to determine viability and 6TG resistance were 30 µg/mL in the absence of S-9 and 270 µg/mL in the presence of S-9, which gave 14% and 12% RS, respectively.

In Experiments 1 and 2 no statistically significant increases in MF were observed following treatment with1,10-decanediol diacrylate at any concentration tested in the absence and presence of S-9 and there were no significant linear trends.

It is concluded that 1,10-decanediol diacrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations in two independent experiments, in the absence and presence of a rat liver metabolising system (S-9)

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:

January - February 2001

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

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

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

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

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.

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

 

 

 

 

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.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 April 2013 - 18 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline No. 487 (In vitro mammalian cell micronucleus test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Target gene:

Not applicable (not a gene mutation assay).

Species / strain / cell type:

other: L5178Y TK+/- mouse lymphoma cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 medium containing 10% (v/v) heat-inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatments, were as follows:
. 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL in the first experiment without S9 mix,
. 0.31, 0.63, 1.25, 2.5, 5, 10, 20 and 40 µg/mL in the second experiment without S9 mix,
. 0.63, 1.25, 2.5, 5, 10, 20, 40 and 80 µg/mL in both experiments with S9 mix.

Vehicle / solvent:

- Vehicle used: the vehicle was dimethylsulfoxide (DMSO), batch No. K42474850 145.
- Justification for choice according to solubility assays performed at CiToxLAB France: highest recommended dose-level of 5000 µg/mL was achievable using a test item solution at 500 mg/mL under a treatment volume of 1% (v/v) in the culture medium.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: mitomycin C, colchicine (without S9 mix); cyclophosphamide (with S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
-First experiment: 3 h treatment + 24 h recovery (without and with S9 mix),
-Second experiment : 24 h treatment + 20 h recovery (without S9 mix), and 3 h treatment + 24 h recovery (with S9 mix).

NUMBER OF CELLS EVALUATED: 2000 mononucleated cells/dose

DETERMINATION OF CYTOTOXICITY
- Method: population doubling

Evaluation criteria:

A test item was considered to have clastogenic and/or aneugenic potential, if all the following criteria were met:
- a dose-related increase in the frequency of micronucleated cells was observed,
- for at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,
- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.

The biological relevance of the results was considered first.
If the criteria of a positive response are only partially met, results will be evaluated on a case by case basis, taking into account other parameters such as reproducibility between experiments. If results remain inconclusive, or when the highest analyzable dose-level does not exhibit about 55% toxicity (in case of toxic items), additional confirmatory experiments may be needed.

Statistics:

no

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Experiments without S9 mix
Cytotoxicity
Following the first experiment,a severe toxicity was induced at the highest tested dose-level of 20 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 10 µg/mL induced a slight but acceptable toxicity, as shown by a 37% decrease in the PD.
Following the second experiment,a severe toxicity was induced at the highest tested dose-level of 40 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 20 µg/mL induced no toxicity, as shown by no noteworthy decrease in the PD.
 
Micronucleus analysis
The dose-levels selected for micronucleus analysis were as follows:
. 2.5, 5 and 10 µg/mL for the 3-hour treatment, the higher being too cytotoxic,
. 5, 10 and 20 µg/mL for the 24-hour treatment, the higher being too cytotoxic.
 
In the first experiment, a statistically significant increase in the frequency of micronucleated cells was noted at the dose-level of 10 µg/mL. However, no dose-response relationship was noted, and only one replicate of the two cultures used for this dose-level showed a frequency of micronucleated cells above the corresponding vehicle control historical data range. These results were thus considered to be equivocal, and the second experiment without S9 mix was performed following a long treatment period. During the second experiment, no statistically significant increase in the frequency of micronucleated cells was noted. Consequently, the increase observed during the first experiment was not reproduced, and was thus not considered to be biologically relevant.

Experiments with S9 mix
Cytotoxicity
Following the first experiment,a marked toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 77% decrease in the PD. The immediately lower dose-level of 40 µg/mL induced a slight but acceptable toxicity, as shown by a 38% decrease in the PD.
Following the second experiment, a slight toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 28% decrease in the PD.
 
Micronucleus analysis
The dose-levels selected for micronucleus analysis were as follows:
. 10, 20 and 40 µg/mL for the first experiment, the higher being too cytotoxic,
. 20, 40 and 80 µg/mL for the second experiment, the latter showing a precipitate at the end of the treatment period.
 
In the first experiment, a dose-response relationship was noted, but no statistically significant increase in the frequency of micronucleated cells was observed. In the second experiment performed in the same experimental conditions, some increases in the frequency of micronucleated cells were noted at 40 and 80 µg/mL. However, these increases were not statistically significant, and the corresponding frequencies of micronucleated cells remained within the historical data of the vehicle control. Consequently, these increases did not meet the criteria for a positive response and were thus considered as non-biologically relevant.

Conclusions:

Under the experimental conditions of the study, 1,10-decanediol diacrylate did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, in the absence or in the presence of a rat metabolising system.

Executive summary:

The objective of this study was to evaluate the potential of 1,10-decanediol diacrylate to induce an increase in the frequency of micronucleated cellsin the mouse cell line L5178YTK+/-. This study conducted in compliance with OECD Guideline No. 487 and the principles of Good Laboratory Practices.

 

Methods

After a preliminary toxicity test, the test item was tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254, as follows:

-First experiment: 3 h treatment + 24 h recovery (without and with S9 mix),

-Second experiment : 24 h treatment + 20 h recovery (without S9 mix), and 3 h treatment + 24 h recovery (with S9 mix).

Each treatment was coupled to an assessment of cytotoxicity at the same dose-levels. Cytotoxicity was evaluated by determining the PD (Population Doubling) of cells and quality of the cells on the slides has also been taken into account.

For each main experiment (with or without S9 mix), micronuclei were analyzed for three dose-levels of the test item, for the vehicle and the positive controls, in 1000 mononucleated cells per culture (total of 2000 mononucleated cells per concentration).

 

The test item was dissolved in dimethylsulfoxide (DMSO).

 

Results

 

The mean PD and mean frequencies of micronucleated cells for the vehicle control cultures were as specified in the acceptance criteria. Positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

 

Since the test item was found to be cytotoxic and poorly soluble in the preliminary test, the selection of the highest dose-level to be used in the main experiments was based on the level of precipitate/emulsion and/or cytotoxicity, according to the criteria specified in the international guidelines.

 

With a treatment volume of 1% (v/v) in culture medium, the dose-levels used for treatments, were as follows:

. 0.16, 0.31, 0.63, 1.25, 2.5, 5, 10 and 20 µg/mL in the first experiment without S9 mix,

. 0.31, 0.63, 1.25, 2.5, 5, 10, 20 and 40 µg/mL in the second experiment without S9 mix,

. 0.63, 1.25, 2.5, 5, 10, 20, 40 and 80 µg/mL in both experiments with S9 mix.

 

A precipitate was observed at the end of the treatments performed with S9 mix at the highest tested dose-level of 80 µg/mL.

Experiments without S9 mix

Cytotoxicity

Following the first experiment,a severe toxicity was induced at the highest tested dose-level of 20 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 10 µg/mL induced a slight but acceptable toxicity, as shown by a 37% decrease in the PD.

Following the second experiment,a severe toxicity was induced at the highest tested dose-level of 40 µg/mL, as shown by a 100% decrease in the PD. The immediately lower dose-level of 20 µg/mL induced no toxicity, as shown by no noteworthy decrease in the PD.

 

Micronucleus analysis

The dose-levels selected for micronucleus analysis were as follows:

. 2.5, 5 and 10 µg/mL for the 3-hour treatment, the higher being too cytotoxic,

. 5, 10 and 20 µg/mL for the 24-hour treatment, the higher being too cytotoxic.

 

In the first experiment, a statistically significant increase in the frequency of micronucleated cells was noted at the dose-level of 10 µg/mL. However, no dose-response relationship was noted, and only one replicate of the two cultures used for this dose-level showed a frequency of micronucleated cells above the corresponding vehicle control historical data range. These results were thus considered to be equivocal, and the second experiment without S9 mix was performed following a long treatment period. During the second experiment, no statistically significant increase in the frequency of micronucleated cells was noted. Consequently, the increase observed during the first experiment was not reproduced, and was thus not considered to be biologically relevant.

Experiments with S9 mix

Cytotoxicity

Following the first experiment,a marked toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 77% decrease in the PD. The immediately lower dose-level of 40 µg/mL induced a slight but acceptable toxicity, as shown by a 38% decrease in the PD.

Following the second experiment, a slight toxicity was induced at the highest tested dose-level of 80 µg/mL, as shown by a 28% decrease in the PD.

 

Micronucleus analysis

The dose-levels selected for micronucleus analysis were as follows:

. 10, 20 and 40 µg/mL for the first experiment, the higher being too cytotoxic,

. 20, 40 and 80 µg/mL for the second experiment, the latter showing a precipitate at the end of the treatment period.

 

In the first experiment, a dose-response relationship was noted, but no statistically significant increase in the frequency of micronucleated cells was observed. In the second experiment performed in the same experimental conditions, some increases in the frequency of micronucleated cells were noted at 40 and 80 µg/mL. However, these increases were not statistically significant, and the corresponding frequencies of micronucleated cells remained within the historical data of the vehicle control. Consequently, these increases did not meet the criteria for a positive response and were thus considered as non-biologically relevant.

 

Conclusion

Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, in the absence or in the presence of a rat metabolising system.

 

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 April 2013 to 20 August 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2°C) mouse lymphoma cells originated from Dr Donald Clive, Burroughs Wellcome Co. Cells supplied to Covance Laboratories Ltd. were stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of mutants and confirmed to be mycoplasma free. For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated in a humidified atmosphere of 5 ± 1% v/v CO2 in air. When the cells were growing well, subcultures were established in an appropriate number of flasks.

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

The mammalian liver post-mitochondrial fraction (S-9) used for metabolic activation were from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 88.13 to 2820 µg/mL (equivalent to 10 mM at the highest concentration tested).

In Experiment 1 thirteen concentrations, ranging from 2.5 to 150 µg/mL in the absence of S-9 and from 25 to 400 µg/mL in the presence of S-9, were tested.
In Experiment 2 twelve concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and from 50 to 400 µg/mL in the presence of S-9, were tested.

Positive controls
4-nitroquinoline 1-oxide (NQO), stock solution: 0.015 and 0.020 mg/mL and final concentration: 0.15 and 0.20 µg/mL, no metabolic activation
Benzo[a]pyrene (B[a]P), stock solution: 0.200 and 0.300 mg/mL and final concentration: 2.00 and 3.00 µg/mL with metabolic activation

Vehicle / solvent:

DMSO diluted 100 fold in the treatment medium

Untreated negative controls:

yes

Remarks:

DMSO diluted 100 fold in the treatment medium

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Remarks:

For positive control concentrations see test concentration section

Details on test system and experimental conditions:

DURATION
- Preincubation period: Not applicable
- Exposure duration: 3-hour exposure followed by 7-day incubation period

Evaluation criteria:

For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. The mutant frequency at one or more concentrations was significantly greater than that of the negative control (p < 0.05).
2. There was a significant concentration relationship as indicated by the linear trend analysis (p < 0.05).
3. The effects described above were reproducible.
The test article was considered positive in this assay if all of the above criteria were met.
The test article was considered negative in this assay if none of the above criteria were met.

Statistics:

Not applicable

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 88.13 to 2820 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to provide >10% relative survival (RS) in the presence of S-9 was 176.3 µg/mL, which gave 96% RS. Extreme or complete toxicity was observed at the remaining higher concentrations analysed in the presence of S-9 (352.5 to 705 µg/mL). In the absence of S-9, complete toxicity (0% RS) was observed at all concentrations analysed (88.13 to 705 µg/mL).
Following consultation with the Study Monitor, an extended range of closely spaced concentrations was tested in the absence of S-9 in Experiment 1.
In Experiment 1, thirteen concentrations, ranging from 2.5 to 150 µg/mL in the absence of S-9 and from 25 to 400 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 40 µg/mL in the absence of S-9 and 330 µg/mL in the presence of S-9, limited by toxicity, which gave 17% and 12% RS, respectively.
In Experiment 2, twelve concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and from 50 to 400 µg/mL in the presence of S-9, were tested. The highest concentrations analysed to determine viability and 6TG resistance were 30 µg/mL in the absence of S-9 and 270 µg/mL in the presence of S-9, which gave 14% and 12% RS, respectively.
In both Experiments 1 and 2, no statistically significant increases in mutant frequency were observed following treatment with 1,10-decanediol diacrylate at any concentration tested, in the absence or presence of S-9, and there were no significant linear trends. Mutant frequencies in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals. The study was therefore accepted as valid.

Table 1: %RS Values –Range-Finder Experiment

Treatment (µg/mL)	-S-9 % RS	+S-9 % RS
0	100	100
88.13	0	122
176.3	0	96
352.5 P	0	3
705.0 P, PP	0	0
1410 P, PP	NP	NP
2820 P, PP	NP	NP

P Precipitation observed at time of treatment

PP Precipitation observed following treatment incubation period

NP Not plated

 

Table 2: Summary of Mutation Data

Experiment 1 (3-hour treatment in the absence and presence of S-9)

Treatment (mg/mL)		-S-9			Treatment (mg/mL)		+S-9
		%RS	MF§				%RS	MF§
0		100	5.38		0		100	2.79
2.5		103	1.29	NS	50		101	2.39	NS
5		101	2.34	NS	100		77	1.71	NS
10		73	3.45	NS	150	$$, P	51	(4.76)
20		59	2.46	NS	200	P	39	2.45	NS
30		31	2.91	NS	230	P	29	4.18	NS
40		17	4.28	NS	250	P	22	4.00	NS
					270	$$, P	18	(6.36)
					300	P	10	2.18	NS
					330	P	12	1.32	NS
Linear trend			NS		Linear trend			NS
NQO					B[a]P
0.15		63	31.36		2		79	21.52
0.2		43	47.63		3		57	56.34

 

Experiment 2 (3-hour treatment in the absence and presence of S-9)

Treatment (µg/mL)		-S-9			Treatment (µg/mL)		+S-9
		%RS	MF§				%RS	MF§
0		100	3.31		0		100	2.06
5		78	3.20	NS	50		92	5.28	NS
7.5		76	1.52	NS	75		84	1.55	NS
10		64	1.86	NS	100		65	2.83	NS
20		31	1.88	NS	150		43	2.67	NS
25		18	2.15	NS	200		23	3.36	NS
30		14	3.01	NS	250	P	14	3.23	NS
					270	P	12	2.00	NS
Linear trend			NS		Linear trend			NS
NQO					B[a]P
0.15		71	54.02		2		58	23.88
0.2		61	47.44		3		22	53.43

§ 6-TG resistant mutants/10⁶viable cells 7 days after treatment

%RS Percent relative survival adjusted by post treatment cell counts

$$ Treatment excluded from analysis due to excessive heterogeneity for mutation

Data in parentheses indicates marked heterogeneity observed

P Precipitation noted at time of treatment only

NS Not significant

 

Conclusions:

It is concluded that 1,10-decanediol diacrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations in two independent experiments, in the absence and presence of a rat liver metabolising system (S-9).

Executive summary:

1,10-decanediol diacrylate was assayed for the ability to induce mutation at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol. The study consisted of a cytotoxicity Range-Finder Experiment followed by two independent experiments, each conducted in the absence and presence of metabolic activation by an Aroclor 1254-induced rat liver post-mitochondrial fraction (S-9). The test article was formulated in anhydrous analytical grade dimethyl sulphoxide (DMSO).

A 3 hour treatment incubation period was used for all experiments.

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 88.13 to 2820 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration to provide >10% relative survival (RS) in the presence of S-9 was 176.3 µg/mL, which gave 96% RS. Extreme or complete toxicity was observed at the remaining higher concentrations analysed in the presence of S-9 (352.5 to 705 µg/mL). In the absence of S-9 complete toxicity (0% RS) was observed at all concentrations analysed (88.13 to 705 µg/mL). Following consultation with the Study Monitor, an extended range of closely spaced concentrations was tested in the absence of S-9 in Experiment 1.

In Experiment 1 thirteen concentrations, ranging from 2.5 to 150 µg/mL in the absence of S-9 and from 25 to 400 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 40mg/µL in the absence of S-9 and 330 µg/mL in the presence of S-9, limited by toxicity, which gave 17% and 12% RS, respectively.

In Experiment 2 twelve concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and from 50 to 400 µg/mL in the presence of S-9, were tested.The highest concentrations analysed to determine viability and 6TG resistance were 30 µg/mL in the absence of S-9 and 270 µg/mL in the presence of S-9, which gave 14% and 12% RS, respectively.

Negative (vehicle) and positive control treatments were included in each Mutation Experiment in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

In Experiments 1 and 2 no statistically significant increases in MF were observed following treatment with1,10-decanediol diacrylate at any concentration tested in the absence and presence of S-9 and there were no significant linear trends.

It is concluded that 1,10-decanediol diacrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to toxic concentrations in two independent experiments, in the absence and presence of a rat liver metabolising system (S-9).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the negative results in all three regulatory in vitro genotoxicity tests, no classification for 1,10-decanediyl bis methacrylate is required for genotoxicity according to the Regulation EC n°1272/2008.