Isodecyl acrylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 August 2012 to 25 January 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

Isodecyl acrylate

EC Number:

215-542-5

EC Name:

Isodecyl acrylate

Cas Number:

1330-61-6

Molecular formula:

C13H24O2

IUPAC Name:

2-methyloctyl prop-2-enoate

Test material form:

liquid

Method

Target gene:

Mouse lymphoma L5178Y cells

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The master stock of L5178Y tk+/- (3.7.2C) 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:

mammalian liver post-mitochondrial fraction (S-9), prepared from male Sprague Dawley rats induced with Aroclor 1254

Test concentrations with justification for top dose:

Experiment 1: thirteen concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and 10 to 140 mg/mL in the presence of S-9, were tested.
Experiment 2: twelve concentrations, ranging from 10 to 50 µg/mL in the absence of S-9 and fourteen concentrations, ranging from 20 to 140 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

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9, ranging from 31.25 to 1000 mg/mL (limited by solubility in culture medium).

Vehicle / solvent:

ethanol diluted 100 fold in the treatment medium

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

ethanol

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

benzo(a)pyrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in suspension

DURATION
- Preincubation period: NA
- Exposure duration: 7 days

Cultures were maintained in flasks for a period of 7 days during which the hprt- mutation would be expressed. Sub-culturing was performed as required with the aim of not exceeding 1 x 106 cells/mL and, where possible, retaining at least 6 x 106 cells/flask. From observations on recovery and growth of the cultures during the expression period, the cultures were selected to be plated for viability and 6TG resistance.
At the end of the expression period, cell concentrations in the selected cultures were determined using a Coulter counter and adjusted to give 1 x 105 cells/mL in readiness for plating for 6TG resistance. Samples from these were diluted to 8 cells/mL.
Using a multichannel pipette, 0.2 mL of the final concentration of each culture was placed into each well of 2 x 96-well microtitre plates (192 wells averaging 1.6 cells/well). The plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (8 to 10 days). Wells containing viable clones were identified by eye using background illumination and counted.
At the end of the expression period, the cell densities in the selected cultures were adjusted to 1 x 105 cells/mL. 6TG (1.5 mg/mL) was diluted 100-fold into these suspensions to give a final concentration of 15 µg/mL. Using a multichannel pipette, 0.2 mL of each suspension was placed into each well of 4 x 96-well microtitre plates (384 wells at 2 x 104 cells/well). Plates were incubated at 37±1ºC in a humidified incubator gassed with 5±1% v/v CO2 in air until scoreable (12 to 13 days) and wells containing clones were identified as above and counted.

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.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Not required

Results and discussion

Test results

Key result

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:

Range-finder study :
In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S 9 ranging from 31.25 to 1000 µg/mL (limited by solubility in culture medium). Upon addition of the test article to the cultures, precipitate was observed at the highest three concentrations in the absence and presence of S-9 (250 to 1000 µg/mL). No evidence of precipitation was observed following the 3 hour incubation period therefore all cultures were retained. Complete or extreme toxicity was observed at 62.5 µg/mL and above in the absence of S-9 and at 125 µg/mL and above in the presence of S-9. The highest concentrations to give >10% relative survival (RS) were 31.25 µg/mL in the absence of S-9 and 62.5 µg/mL in the presence of S-9, which gave 83% and 45% RS, respectively.
No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration tested (1000 µg/mL) as compared to the concurrent vehicle controls.

Main experiments :
In Experiment 1, thirteen concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and 10 to 140 µg/mL in the presence of S-9, were tested. The highest seven concentrations tested in the absence of S-9 (45 to 80 µg/mL) were not plated for survival due to excessive toxicity. Seven days after treatment, the highest remaining concentration in the absence of S-9 (40 µg/mL) and the highest five concentrations in the presence of S-9 (90 to 140 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition an intermediate concentration of 30 µg/mL in the presence of S-9 was not plated as there were sufficient non toxic concentrations available for analysis. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 35 µg/mL in the absence of S-9 and 80 µg/mL in the presence of S-9, which gave 2% and 7% RS, respectively. Steep concentration-related toxicity was observed between 30 and 35 µg/mL (37% and 2% RS, respectively) in the absence of S-9 and between 70 and 80 µg/mL (33% and 7%, respectively) in the presence of S-9, therefore both concentrations were analysed in each case.
In Experiment 2, twelve concentrations, ranging from 10 to 50 µg/mL in the absence of S-9 and fourteen concentrations, ranging from 20 to 140 µg/mL in the presence of S-9, were tested. Seven days after treatment, the highest concentration tested in the absence of S-9 (50 µg/mL) and the highest three concentrations (110 to 140 µg/mL) in the presence of S-9 were considered too toxic for selection to determine viability and 6TG resistance. In addition intermediate concentrations of 15 and 27.5 µg/mL in the absence of S-9 and 75, 85 and 90 µg/mL in the presence of S-9 were not plated as there were sufficient non toxic concentrations available for analysis. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 40 µg/mL in the absence of S-9 and 105 µg/mL in the presence of S-9, gave 19% and 20% RS, respectively.

Any other information on results incl. tables

Table 1: RS values: Range-Finder Experiment

Treatment (µg/mL)	-S-9 % RS	+S-9 % RS
0	100	100
UTC	118	73
31.25	83	81
62.5	1	45
125	0	0
250 P	NP	0
%RS- Percentage Relative Survival adjusted by post treatment cell counts P - Precipitation observed at time of treatment NP - Not plated for survival UTC - Untreated control

 

Table2: Summary of mutation data

Experiment 1 (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	6.62		0		100	6.75
UTC		100	4.49	NS	UTC		100	8.09	NS
5		104	8.02	NS	10		89	4.32	NS
10		86	5.59	NS	20		91	5.59	NS
20		74	5.31	NS	40		77	3.10	NS
30		37	6.18	NS	50		73	3.35	NS
35		2	3.75	NS	60		67	3.96	NS
					70		33	2.34	NS
					80		7	3.10	NS
Linear trend			NS		Linear trend			NS
NQO					B[a]P
0.15		61	45.16		2		49	49.13
0.2		37	42.84		3		19	74.69
§                     6-TG resistant mutants/10^6viable cells 7 days after treatment %RS                Percent relative survival adjusted by post treatment cell counts NS                   Not significant UTC                 Untreated controls

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	2.38		0		100	9.30
UTC		101	2.86	NS	UTC		96	8.22	NS
10		87	3.24	NS	20		93	6.41	NS
20		71	4.68	NS	40		75	6.86	NS
25		85	1.73	NS	60		71	2.62	NS
30		79	4.88	NS	70		62	6.24	NS
32		84	4.51	NS	80		49	10.68	NS
34		70	2.46	NS	95		32	5.31	NS
36		77	2.68	NS	100		17	5.41	NS
38		42	2.43	NS	105		20	4.37	NS
40		19	3.10	NS
Linear trend			NS		Linear trend			NS
NQO					B[a]P
0.15		65	42.01		2		74	19.21
0.2		49	83.41		3		49	35.80
§                     6-TG resistant mutants/10^6viable cells 7 days after treatment %RS                Percent relative survival adjusted by post treatment cell counts NS                   Not significant UTC                 Untreated controls

 

Applicant's summary and conclusion

Conclusions:

It is concluded that Isodecyl Acrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).

Executive summary:

Isodecyl Acrylate 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 ethanol.

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 31.25 to 1000 mg/mL (limited by solubility in culture medium). The highest concentrations to give >10% relative survival (RS) were 31.25 µg/mL in the absence of S-9 and 62.5 µg/mL in the presence of S-9, which gave 83% and 45% RS, respectively.

Accordingly, for Experiment 1, thirteen concentrations, ranging from 5 to 80 µg/mL in the absence of S-9 and 10 to 140 mg/mL in the presence of S-9, were tested. Seven days after treatment the highest concentrations analysed to determine viability and 6TG resistance were 35 µg/mL in the absence of S-9 and 80 µg/mL in the presence of S-9, which gave 2% and 7% RS, respectively. Steep concentration-related toxicity was observed between 30 and 35 µg/mL (37% and 2% RS, respectively) in the absence of S-9 and between 70 and 80 mg/mL (33% and 7%, respectively) in the presence of S-9, therefore both concentrations were analysed in each case.

In Experiment 2, twelve concentrations, ranging from 10 to 50 µg/mL in the absence of S-9 and fourteen concentrations, ranging from 20 to 140 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 105 µg/mL in the presence of S-9, which gave 19% and 20% RS, respectively.

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

In Experiments 1 and 2 no statistically significant increases in mutant frequency were observed following treatment with Isodecyl Acrylate at any concentration tested in the absence and presence of S-9 and there were no significant linear trends. Due to the steep toxicity profile observed, highly toxic concentrations giving <10% RS were analysed in Experiment 1 in the absence and presence of S-9. However, in the absence of any significant increase in mutant frequency at these concentrations or significant linear trends, the data were considered valid and the integrity of the study unaffected.

It is concluded that Isodecyl Acrylate did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S-9).