Octanethioic acid, S-[3-(triethoxysilyl)propyl] ester, reaction products with 2-methyl-1,3-propanediol and 3-(triethoxysilyl)-1-propanethiol

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An Ames study is available performed according to OECD/EC guidelines and GLP principles. The results showed that the substance was positive without metabolic activation in tester strain TA100.

In addition, an in vitro chromosome aberration test is available performed in Chinese Hamster V79 cells according to OECD/EC guidelines and GLP principles. The results do not indicate that the test item is clastogenic.

 

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21 December 2006 - 14 May 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Storage: at room temperature

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Duplicate cultures were treated at each concentration. The selection of the concentrations was based on the results from the pre-test (see below).

Experiment I:
without metabolic activation: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34 and 36 µg/mL
with metabolic activation: 200, 400, 600, 750, 850 and 1000 µg/mL

Experiment II:
without metabolic activation: 1, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28 and 30 µg/mL
with metabolic activation: 150, 250, 350, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1000 µg/mL

Vehicle / solvent:

Ethanol

The test item could not be dissolved in DMSO, but the test item was found to dissolve in ethanol.
Precipitation of the test item was noted at 400 µg/mL and higher.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

With metabolic activation

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation

Details on test system and experimental conditions:

Exposure period experiment I: 4 hours (with and without metabolic activation)
Exposure period experiment II: 4 hours (with metabolic activation, 20 hours (with metabolic activation)

Seeding of Cultures:
3 or 4 days old stock cultures with higher than 50% confluency were trypsinised at 37°C for 5 min. by adding a trypsin solution in Ca-Mg-free PBS solution. The enzymatic treatment was stopped with complete culture medium. A single cell suspension was prepared. The trypsin concentration for all subculturing steps was 0.2%. The cells were rinsed with Ca-Mg-free PBS solution prior to the trypsin treatment.
The cells were seeded into Quadriperm dishes which contain microscopic slides (at least 2 chambers per dish and test group). Into each chamber 1 x 10^4 - 5x 10^5 cells were seeded with regard to preparation interval. The medium was minimum essential medium supplemented with 10% FCS.

Experiment II: Short time exposure (with metabolic activation), long time exposure (without metabolic activation)
The treatment with metabolic activation:
2 days after seeding of the cells, the culture medium was replaced with serum-free medium containing the test item and 50 µL/mL S9 mix (with metabolic activation). Additional negative and positive controls were performed with and without exogenic metabolic activation.
4 hours after treatment the cultures were washed twice with PBS and cultured in complete medium for the remaining culture time.
In the experiment without metabolic activation, 2 days after seeding the cells were incubated with the test item in complete medium (MEM with 10% FCS) for 20 hours. The cells were prepared at the end of the incubation. Additional negative and positive controls were tested.

All cultures were incubated at 37°C in a humidified atmosphere with 5.0% CO2 (95.0% air).

The cytotoxicity was determined by the mitotic index (% cells in mitosis) by counting the number of mitotic cells per 1000 cells. As an additional parameter the relative cell density was calculated as the mean of twenty cell counts per test group.

Rationale for test conditions:

A pre-test was conducted under identical conditions as described for the main experiment. The following concentrations were tested:
Without S9-mix: 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40 µg/mL
With S9-mix: 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1000 µg/mL

Evaluation criteria:

Criteria for determining a positive result:
- a clear and dose-related increase in the number of cells with aberrations,
- a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (up to 4.5% aberrant cells).
According to 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 (3)(10). [...]
A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group. Although most experiments will give clearly positive or negative results, in some cases the data set will preclude making a definitive judgement about the activity of the test substance.

Statistics:

The non-parametric Fisher's exact test was used.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The following concentrations were selected for microscopic analyses:
Experiment I
without metabolic activation: 10, 12, 14 and 16 µg/mL
with metabolic activation: 400, 600 and 750 µg/mL

Experiment I
without metabolic activation: 26, 28 and 30 µg/mL
with metabolic activation: 750, 800, 850 and 900 µg/mL

Precipitation: Precipitate was noted in all dose groups with metabolic activation. No precipitate of the test item was observed in the dose groups without metabolic activation.

Observations:
No biologically relevant increase of the aberration rates was noted after treatment with the test item with and without metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

No increase in the frequencies of polyploid cells was found.

The results are included below in tabular form (including historical control data).

Conclusions:

Based on the results of an in vitro chromosome aberration test in Chinese Hamster V79 cells according to OECD/EC guidelines and GLP principles, the test item is considered to be non-clastogenic.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 January 2007 - 04 June 2007

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:

adopted 21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Stability: stable, if solvent or vehicle is anhydrous
Storage: at room temperature

Target gene:

histidine (S. typhimurium) or tryptophan (E.coli)

Species / strain / cell type:

E. coli WP2 uvr A

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9-Mix

Test concentrations with justification for top dose:

Pre-test (TA 98 and TA 100): 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (reported as part of main experiment I);
Experiment I: 31.6, 100, 316, 1000, 2500 and 5000 µg/plate;
Experiment II: 31.6, 10, 316, 1000, 2500 and 5000 µg/plate (with S9), 1.00, 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate (TA 1535 and TA 1537 without S9);
Experiment III: 250, 350, 750, 1500, 3000 and 5000 µg/plate (TA 100 without S9);
Experiment IV: 110, 130, 150, 175, 250 and 300 µg/plate (TA 100 with S9)

Vehicle / solvent:

Solvent: DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other:

Remarks:

With metabolic activation

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

E.coli WP2 uvrA

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other:

Remarks:

TA98, TA1537, without metabolic activation

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA100, TA1535, without metabolic activation

Details on test system and experimental conditions:

For each strain and dose level three plates were used.
Evaluation of cytotoxicity was done based on characterization of the background lawn and colony morphology.

Evaluation criteria:

A test item is considered mutagenic if:
- a clear and dose-related increase in the number of revertants over at least two increasing concentrations occurs without concomitant cytotoxicity in at least one of these groups, and/or
- a biologically relevant positive response for at least one of the non-cytotoxic dose groups occurs.

A biologically relevant increase is described as follows:
- if in tester strains TA100 and E.coli WP2 uvrA the number of reversions is at least twice as high
- if in tester strains TA1535, TA1537 and TA98 the number of reversions is at least three times higher
as compared to the reversion rate of the solvent control.

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

Statistics:

A statistical evaluation was not performed.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 250 µg/ plate and above

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle 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:

no cytotoxicity

Vehicle 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

Remarks:

at 1000 µg/ plate and higher, without metabolic activation

Vehicle 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

Remarks:

at 2500 µg/ plate and higher, without metabolic activation

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 316 µg/ plate and higher, without metabolic activation

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The results are attached below in tabular format.

The reference mutagens induced a clear increase of revertant colonies verifying the validity of the experimental set-up.

No biologically revertant increases in revertant colony numbers were noted in tester strains TA98, TA1535, TA1537 and E.coli WP2 uvrA in absence or presence of metabolic activation.

Biologically relevant increases of revertant colony numbers were observed without metabolic activation in tester strain TA100 in three independent experiments. The threshold of 3.2 was reached at a dose of 5000 µg/ plate in the second experiment. Moreover, a dose-response relationship was found in each experiment.

Conclusions:

In an Ames study performed according to OECD/EC guidelines and GLP principles, the substance was found to be positive without metabolic activation in tester strain TA100.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

An in vivo Micronucleus study is available, which shows that the substance is not clastogenic in vivo. As the substance was tested positive in an AMES study, a test proposal for an in vivo COMET assay is included.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 2000/32 L 136 Annex 4C, B 12

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann
- Age at start of treatment: minimum 7 weeks
- Weight at study initiation: male: 30.7 - 38.2g; female: 23.2 - 32.3g
- Assigned to test groups randomly: yes
- Housing: 5 animals of identical sex per cage (Macrolon Type III; Hereto)
- Diet: Altromin 1324 maintenance diet for rats and mice, TPF
- Water: tap water, ad libitum
- Acclimation period: adequate acclimatisation period

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25°C
- rel. Humidity (%): 55±10%
- Photoperiod (hrs dark / hrs light): artificial light 6:00 - 18:00

Route of administration:

intraperitoneal

Vehicle:

Cotton Seed Oil

Details on exposure:

The dosing formulations were prepared 1 hour before treatment (positive control formulation was prepared on the same day). All animals received a single volume of 10 mL/ kg bw.

Duration of treatment / exposure:

Single dose

Post exposure period:

Sampling of the peripheral blood was done at 44 (all groups, including solvent control and positive control) and 68 hours (solvent control and high dose group only) after treatment.

Dose / conc.:

400 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 (main study)
6 males and 3 females (pre-experiment)

Control animals:

yes, concurrent vehicle

Positive control(s):

40 mg/kg bw cyclophosphamide dissolved in physiological saline. Intraperitoneal injection at 10 mL/kg bw

Tissues and cell types examined:

Blood from tail vein.

Details of tissue and slide preparation:

Blood was immediately fixed in ultracold methanol. At least 16 hours after fixation blood cells were washed in Hank's balanced salt solution, centrifuged at 600 g for 5 minutes. Blood cell populations were discriminated against using CD71 and CD61 and DNA content of micronuclei was determine dby the use of a DNA specific strain (propidium iodid).

Evaluation criteria:

Evaluation was performed using FACScan BD flow cytometer. At least 10,000 immature erythrocytes/animals were scored for the incidence of micronucleated immature erythrocytes.
To assess cytotoxicity, the ratio between immature and mature erythrocytes was determined and expressed as relative PCE (proportion of polychromatic (immature) erythrocytes among total erythrocytes.

Criteria for determining a positive result:
- dose related increase in the number of micronucleated cells and/or
- biologically relevant increase in the number of micronucleated cells for at least one of the dose groups

A test item is considered negative if there is no biologically relevant and/or statistically significant increase in the number of micronucleated cells at any dose level.

Statistics:

The nonparametric Mann-Whitney Test was used.

The data generated were considered acceptable, if:
- at the commencement of the study, the weight variation of animals should be minimal and not exceed ± 20% of the mean weight of each sex,
- the background frequency of micronucleated cells is in the normal range as reported in the literature or within the laboratory's historical range,
- the test system is sensitive to the known mutagen as judged by the results in the concurrent positive control animals.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

Reduction rel. PCE

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The results are included in tabular form in the attached document.

The results of the positive control group and the solvent control group were within the range of the corresponding historical control data.

The relative PCE values observed in the male and female group were reduced compared to the corresponding negative control. Although the reduction was not statistically significant, the outcome was considered to be a good indication of systemic exposure of the mice.

The mean values of micronuclei observed after treatment were within the range of the negative control, except for the high dose group samples 68 hours after treatment. The values for males and females were slightly increased compared to the negative control (0.26% (0.25%) in the male (female) controls vs. 0.35% (0.30%) for the high dose males (females). As the increase was minimal and not statistically significant this was not considered to be toxicologically significant.

Historical control data ranges are attached below.

Results pre-experiment to find MTD:

Two males died within 72 hours after application of the test item. They showed toxic symptoms as reduction of spontaneous activity, rough fur, constricted opistosoma, palpebral closure and loss of weight. 

All other animals survived.

The females and three males did not show signs of toxicity. 

One male mouse showed toxic symptoms including reduction of spontaneous activity, rough fur, constricted opisthosoma, palpebral closure and loss of weight. 

 

Conclusions:

Based on the results of an in vivo Micronucleus study performed according to OECD/EC guideline and GLP principles, the test substance is considered to be non-cytogenic.

Endpoint conclusion

Endpoint conclusion:

no study available (further information necessary)

Justification for classification or non-classification

Based on the available data, the test substance is not classified as a mutagen according to Regulation (EC) No 1272/2008, however it is proposed to conduct an in vivo study to clarify this endpoint.