Isooctyl 3-mercaptopropionate

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:

21 November 2008 - 03 April 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Isooctyl 3-mercaptopropionate

EC Number:

250-157-6

EC Name:

Isooctyl 3-mercaptopropionate

Cas Number:

30374-01-7

Molecular formula:

C11H22O2S

IUPAC Name:

2-methylheptyl 3-sulfanylpropanoate

Details on test material:

- Name of test material (as cited in study report): Isooctyl mercaptopropionate
- Physical state: liquid l colourless
- Analytical purity: 99.88%
- Purity test date: 2008-10-08
- Lot/batch No.: 25051
- Expiration date of the lot/batch: 2009-10-07
- Stability under test conditions: confirmed by sponsor
- Storage condition of test material: at room temperature

Method

Target gene:

thymidine kinase

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction was prepared at BSL BIOSERVICE GmbH. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and ß-naphtoflavone (100 mg/kg bw) for three consecutive days by oral route.

Test concentrations with justification for top dose:

with metabolic activation: 0.05, 0.10, 0.20, 0.25, 0.28, 0.30, 0.32, 0.34 µL/mL
without metabolic activation: 0.001, 0.003, 0.005, 0.008, 0.01, 0.02, 0.05, 0.10 µL/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the cells and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

-S9

Migrated to IUCLID6: 500 µg/mL in medium

Controls 2

Positive control substance:

methylmethanesulfonate

Remarks:

-S9

Migrated to IUCLID6: 10 µg/mL in saline

Controls 3

Positive control substance:

benzo(a)pyrene

Remarks:

+S9

Migrated to IUCLID6: 2.5 µg/mL in medium

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 15 min
- Exposure duration: 4 h
- Expression time (cells in growth medium): 6 days
- Selection time (if incubation with a selection agent): 11-14 days

SELECTION AGENT (mutation assays): trifluorothymidine (5 µg/mL)


DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth;

Evaluation criteria:

There are several criteria for determining a positive result:
- clear and dose-related increase in the mutant frequency,
- biologically relevant response (at least a 2-fold increase of mutant frequencies related to the respective negative control values and higher than the historical range of negative controls) for at least one of the dose groups,
- combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (slow growth colonies) indicated by a low large/small colonies ratio (1.5 times the ratio of clastogenic controls MMS and/or B[a]P) is an indication for potential clastogenic effects and/or chromosomal aberrations.

Statistics:

According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results. A statistical evaluation of
the results is not regarded as necessary.
A test item is considered to be negative if there is no biologically relevant increase in the induction of mutant cells above concurrent control levels, at any dose level.

Results and discussion

Test results

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 examined

Positive controls validity:

valid

Additional information on results:

In the main experiment with metabolic activation, the quotient of large/small colonies of the negative controls were found to be 3.11 and
3.38, the quotients of large/small colonies of the solvent controls were found to be 2.62 and 3.59, the quotient of large/small colonies of the
positive control was found to be 1.04. The quotient of the highest dose groups were found to be 2.49 (0.30 µL/mL), 3.28 (0.32 µL/mL) and 1.07(0.34 µl/mL). In the highest concentration an increased number of small colonies was found. Here a quotient of 1.07 was evaluated. Since no
mutagenicity was found in this dose group the finding is considered to be a secondary effect of the attended toxicity (RTG 5.51) and not biologically relevant.
In the main experiment without metabolic activation, the quotient of large/small colonies of the negative controls were found to be 3.27 and
2.50, the quotients of large/small colonies of the solvent controls were found to be 1.98 and 6.06, the quotient of large/small colonies of the
positive control was found to be 0.86. The quotient of the highest dose groups were found to be 2.10 (0.02 µL/mL), 2.69 (0.05µL/mL) and 1.95
(0.10 µL/mL). No clastogenic effects of the test item were indicated.
The positive controls MMS (10 µg/mL) and B[a]P (2.5 µg/mL) induced a significant increase of the mutant frequency and a biologically significant
increase of small colonies, thus proving the ability of the test system to indicate potential clastogenic effects.

Any other information on results incl. tables

Table 1: Experiment I - 4 h exposure - With Metabolic Activation

Concentration [µL/ mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 cells	Mutation factor	Quotient large / small colonies
0 (DMSO)	100	100	212.0	1	3.11
0.05	103.05	104.13	193.24	0.91	–
0.10	102.44	111.80	176.04	0.83	–
0.20	107.32	111.98	145.99	0.69	–
0.25	101.83	93.20	222.17	1.05	–
0.28	108.54	90.72	174.38	0.82	–
0.30	98.78	61.18	253.99	1.20	2.49
0.32	101.22	26.45	228.37	1.08	3.28
0.34	90.85	5.51	258.01	1.22	1.07
B[a]P, 2.5 µg/mL	78.05	61.71	1199.02	5.66	1.04

B[a]P  Benzo[a]pyrene

 

 

Table 2: Experiment I - 4 h exposure - Without Metabolic Activation

Concentration [µL/ mL]	Cloning efficiency [%]	Relative Total Growth [%]	Mutants per 1E+06 cells	Mutation factor	Quotient large / small colonies
0 (DMSO)	100	100	186.15	1	4.02
0.001	105.49	103.66	144.31	0.78	–
0.003	106.71	106.70	131.23	0.70	–
0.005	101.22	103.04	184.74	0.99	–
0.008	110.37	109.43	152.04	0.82	–
0.01	107.32	102.13	152.66	0.82	–
0.02	101.22	96.72	160.67	0.86	2.10
0.05	104.88	99.66	150.39	0.81	2.69
0.10	104.27	41.05	234.02	1.26	1.95
EMS, 500µg/mL	86.59	55.28	1375.31	7.39	–
MMS, 10µg/mL	83.54	30.41	1344.14	7.22	0.86

EMS   Ethyl methane sulphonate

MMS  Methyl methane sulphonate

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

Isooctyl mercaptopropionate is considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

Executive summary:

The test item Isooctyl mercaptopropionate was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.
The selection of the concentrations was based on data from the pre-experiment. In the main experiment 0.34 uL/mL (with metabolic
activation) and 0.10 pL/mL (without metabolic activation) were selected as the highest concentrations. The test item was investigated in the main experiment at the following concentrations:
with metabolic activation:
0.05, 0.10, 0.20, 0.25, 0.28, 0.30, 0.32, 0.34 wL/mL
and without metabolic activation:
0.001, 0.003, 0.005, 0.008, 0.01, 0.02, 0.05, 0.10 pL/mL
Growth inhibition was observed in the main experiment with and without metabolic activation.
In the main experiment with metabolic activation the relative total growth (RTG) was 5.51% for the highest test item concentration (0.34 pL/mL) evaluated. The highest test item concentration evaluated without metabolic activation was 0.10 wL/mL with a RTG of 41.05%.
In the main experiment with and without metabolic activation no biologically relevant increase of mutants was found after treatment with the test item Isooctyl mercaptopropionate. No dose-response relationship was observed.
In addition, colony sizing did not indicate potential clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Isooctyl mercaptopropionate is
considered to be non-mutagenic in the mouse lymphoma thymidine kinase locus using the cell line LS178Y.