Isooctyl 3-mercaptopropionate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

IOMP was negative, with and without activation, in an Ames test and in a Mouse Lymphoma Assay.

 

 

Link to relevant study records

Reference

Reference 1

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

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

Target gene:

thymidine kinase

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.

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

Positive control substance:

methylmethanesulfonate

Remarks:

-S9

Migrated to IUCLID6: 10 µg/mL in saline

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.

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.

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

 

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.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Nov 2008 - 12 Jan 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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Artbeitsschutz, Arbeitsmedizin und Sicherheitstechnik

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (S. typhimurium)
trp operon (E. coli)

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

- Type and identity of media: S. typhimurium medium (Nutrient Broth) containing 8.0 g Nutrient Broth and 5.0 g NaCl
- Properly maintained: yes

Characteristics (TA 98):
his D 3052; rfa-; uvrB-; R-factor: frame shift mutations

Strain obtained from MOLTOX, INC., NC 28607, USA.

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

- Type and identity of media: E. coli medium (Luria Bertani) containing 10.0 g Tryptone, 10.0 g NaCl and 5.0 g Yeast extract.
- Properly maintained: yes

Characteristics (TA 1537):
his C 3076; rfa-; uvrB-: frame shift mutations

Strain obtained from DSMZ Sales, Braunschweig, Germany.

Metabolic activation:

with and without

Metabolic activation system:

S9 from Wistar rats induced with Phenoparbital (80 mg/kg bw) and beta-Naphtoflavone (100 mg/kg bw) for three days

Test concentrations with justification for top dose:

Experiment 1 (plate incorporation test):
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μl/plate in addition 0.0100 μl/plate only for tester strain TA 100

Experiment 2 (Pre-incubation test):
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μl/plate

Vehicle / solvent:

DMSO: This solvent was compatible with the survival of the bacteria and the S9 activity.

Untreated negative controls:

yes

Remarks:

Solvent controls, consisting of solvent or vehicle alone, as well as untreated controls were treated in the same way as the treatment groups.

Negative solvent / vehicle controls:

yes

Remarks:

Solvent controls, consisting of solvent or vehicle alone, as well as untreated controls were treated in the same way as the treatment groups.

True negative controls:

no

Positive controls:

yes

Remarks:

with and without metabolic activation

Positive control substance:

other: Without S9: sodium azide (TA 100 and 1535), 4-nitro-o-phenylene-diamine (TA98 and 1537), Methyl methane sulfonate (E. coli); with S9: 2-aminoanthracene (TA98, 100, 1515, 1537 and E. coli)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and pre-incubation

DURATION
- Preincubation period: 60 minutes at 37 °C
- Exposure duration: at least 48 hours in the dark

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test item is considered as mutagenic if:
- a clear and dose-related increase in the number of revertants occurs and/or
- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:
- if in tester strains TA 100 and E. coli WP2 uvr A the number of reversions is at least twice as high
- if in tester strains TA 1535, TA 1537 and TA 98 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control.

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 producing neither a dose related increase in the number of revertants nor a reproducible biologicallyrelevant positive response at any of the dose groups is considered to be non-mutagenic in this system.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1.0 µl/plate and higher (+S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Pre-Experiment for Toxicity: The toxicity of the test item was determined with tester strain TA 98 and TA 100 in a pre-experiment. Eight concentrations were tested for toxicity and induction of mutations with three plates each. The experimental conditions in this pre-experiment were the same as described for the plate incorporation test (Experiment 1). Toxicity may be detected by a clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control. The test item was tested in the pre-experiment at the following concentrations: 0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µl/plate.

No precipitation of the test article was observed in any tester strain used in Experiment 1 and 2 (with and without metabolic activation). Toxic effects of the test article were noted in all tester strains evaluated in Experiment 1 and 2.

COMPARISON WITH HISTORICAL CONTROL DATA: Positive and negative controls of the recent study are within the HCD-range for positive (-S9 / +S9) and negative (-S9 / +S9) controls.

The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

Conclusions:

Interpretation of results: negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Isooctyl mercaptopropionate did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, Isooctyl mercaptopropionate is considered to be non-mutagenic in this bacterial reverse mutation assay.

Executive summary:

The test item Isooctyl mercaptopropionate was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA.
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic
activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:
Experiment I:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
in addition 0.0100 μL/plate only for tester strain TA 100
Experiment II:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
Toxic effects of the test item were noted in all tester strains used in experiment I and II:
• In experiment I and II toxic effects of the test item were observed at concentrations of 0.0316 μL/plate and higher (without metabolic
activation) and at concentrations of 0.316 μL/plate and higher (with metabolic activation), depending on the particular tester strain.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Isooctyl mercaptopropionate at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Isooctyl
mercaptopropionate did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, Isooctyl mercaptopropionate is considered to be non-mutagenic in this bacterial reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Bacterial reverse mutation assay

The test item Isooctyl mercaptopropionate was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and tester strain E. coli WP2 uvrA.
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic
activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:
Experiment I:
0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
in addition 0.0100 μL/plate only for tester strain TA 100
Experiment II:
0.00316, 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 μL/plate
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
Toxic effects of the test item were noted in all tester strains used in experiment I and II:
• In experiment I and II toxic effects of the test item were observed at concentrations of 0.0316 μL/plate and higher (without metabolic
activation) and at concentrations of 0.316 μL/plate and higher (with metabolic activation), depending on the particular tester strain.
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Isooctyl mercaptopropionate at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
The reference mutagens induced a distinct increase of revertant colonies indicating the validity of the experiments.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Isooctyl
mercaptopropionate did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, Isooctyl mercaptopropionate is considered to be non-mutagenic in this bacterial reverse mutation assay.

Gene mutation assay in mammalian cells

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.

Justification for classification or non-classification

Based in the available data, no classification for mutagenicity is required