Reaction product of trimethylhexamethylenediisocyanate and (3-mercaptopropyl)trimethoxysilane

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A bacterial reverse mutation (Ames) test was performed according to OECD guideline 471. The test substance was not mutagenic.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

The experimental start date was 04 May 2018 and the experimental completion date was 22 May 2018.

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:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BZA13264
- Expiration date of the lot/batch: 23 June 2018
- Manufacturingt date: 23 April 2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature under nitrogen
- Not stable at higher temperatures

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No correction was made for the purity/composition of the test item. A solubility test was performed based on visual assessment. The test item formed a clear colourless solution in DMSO. Test item concentrations were used within 2 hours after preparation.
- Final dilution of a dissolved solid, stock liquid or gel: dilutions of the test item where prepared using DMSO as vehicle.

FORM AS APPLIED IN THE TEST (if different from that of starting material) : diluted in DMSO.

Target gene:

Histidine locus and tryptophan locus

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Trinova Biochem GmbH
- Suitability of cells: The Salmonella typhimurium strains were checked at least every year to confirm their histidine-requirement, crystal violet sensitivity, ampicillin resistance (TA98 and TA100), UV-sensitivity and the number of spontaneous revertants.

MEDIA USED
- Type and identity of media: Agar plates (ø 9 cm) contained 25 mL glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Salmonella typhimurium strains also contained 12.5 μg/plate biotin (Merck) and 15 μg/plate histidine (Sigma).

Additional strain / cell type characteristics:

other:

Remarks:

deep rough, mutation in the galactose metabolism, mutation in nitrate reductase, defective biotin synthesis, loss of the excision repair system

Species / strain / cell type:

E. coli WP2 uvr A

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells:
Trinova Biochem GmbH
- Suitability of cells: The Escherichia coli strain was checked to confirm the tryptophan requirement, UV-sensitivity and the number of spontaneous revertants at least every year.

MEDIA USED
- Type and identity of media: Agar plates (ø 9 cm) contained 25 mL glucose agar medium. Glucose agar medium contained per liter: 18 g purified agar (Oxoid LTD) in Vogel-Bonner Medium E, 20 g glucose (Fresenius Kabi, Bad Homburg, Germany). The agar plates for the test with the Escherichia coli strain contained 15 μg/plate tryptophan (Sigma).

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) prepared from male Sprague Dawley rats that had been injected intraperitoneally with Aroclor 1254 (500 mg/kg body weight).

Test concentrations with justification for top dose:

Dose-range finding test: Selection of an adequate range of doses was based on a dose-range finding test with the strains TA100 and WP2uvrA, both with and without S9-mix. Eight concentrations, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate were tested in triplicate. The highest concentration of Thio Isocyanate Adduct used in the subsequent mutation assays was 5000 μg/plate or the level at which the test item inhibited bacterial growth.

First experiment - direct plate assay: The dose-range finding study with two tester strains is reported as a part of the direct plate assay. In the second part of this experiment, the test item was tested at 5.4, 17, 52, 164, 512, and 1000 ug/plate. Initially in tester strains TA1535 and TA98 no dose level with precipitation and/or cytotoxicity was tested in the presence of S9-mix. Therefore an additional experiment was performed at 1600 and 5000 ug/plate with these tester strain in presence of S9-mix to complete the data.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191 ; 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (direct plate incorporation and preincubation)
- Cell density at seeding (if applicable): 1 mL (10 EXP 9 cells/mL) added to 3.6 mL medium with test item.

DURATION
- Preincubation period: when applicable, 30 +/- 2 minutes
- Exposure duration: 48 +/- 4 h

NUMBER OF REPLICATIONS: 3

- OTHER: Colony counting: The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Rationale for test conditions:

Recommended test system in international guidelines

Evaluation criteria:

In addition to the criteria stated below, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98 is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 1000 µg/plate in the absence of S9-mix and at 1600 µg/plate and upwards in the presence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 1000 µg/plate in the absence of S9-mix and at 1600 µg/plate and upwards in the presence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 1000 µg/plate in the absence of S9-mix and at 1600 µg/plate and upwards in the presence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 1000 µg/plate in the absence of S9-mix and at 1600 µ/plate and upwards in the presence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At 1000 µg/plate in the absence of S9-mix and at 1600 µg/plate and upwards in the presence of S9-mix

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Evidence of test item precipitate on the plates is recorded by addition of the following precipitation definition:
Slight precipitate: Distinguished by noticeable precipitate on the plate. However, the precipitate does not influence automated counting of the plate.
Moderate precipitate: Distinguished by a marked amount of precipitate on the plate, requiring the plate to be hand counted.
Heavy precipitate: Distinguished by a large amount of precipitate on the plate, making the required hand count difficult.
In the dose-range finding study, the test item precipitated on the plates at the highest dose level tested (1600 µ/plate and upwards at the start of th incubation period and at 5000 µg/plate at the end of the incubation period). In the pre-incubation assay, precipitation of the test substance was observed at the start of the incubation period at 1600 and 5000 µg/plate and no precipitate was observed at the end of the incubation period.

RANGE-FINDING/SCREENING STUDIES: Thio Isocyanate Adduct was initially tested in the tester strains TA100 and WP2uvrA as a dose-range finding test with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix.
Based on the results of the dose-range finding test, a dose-range was selected for the first mutation assay with tester strains TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 5.4, 17, 52, 164, 512, and 1000 μg/plate.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Measurement of cytotoxicity used: decrease in the number of revertants and/or reduction of the bacterial background lawn.

Remarks on result:

other:

Remarks:

Precipitation observed at the start of the incubation period at 1600 µ/plate and upwards and at 5000 µg/plate at the end of the incubation period

Historical control data of the solvent control:

	TA1535		TA1537		TA98		TA100		WP2uvrA
S-9 mix	-	+	-	+	-	+	-	+	-	+
Range	3-29	3-27	3-20	3-23	8-41	8-55	61-176	60-160	10-59	9-67
Mean	10	11	6	6	16	22	110	106	26	33
SD	3	4	2	3	5	7	17	20	6	8
n	2458	2426	2402	2352	2416	2458	2473	2398	2237	2217

  

Historical control data of the positive control items:

	TA1535		TA1537		TA98		TA100		WP2uvrA
S-9 mix	-	+	-	+	-	+	-	+	-	+
Range	128-1530	73-1206	58-1407	54-1051	365-1978	250-1977	439-1993	408-2379	93-1958	111-1359
Mean	901	239	817	340	1355	903	905	1249	1059	444
SD	174	115	354	160	230	357	163	371	506	144
n	2400	2296	2051	2337	2357	2367	2402	2354	2153	2232

 

 Direct plate assay – Mean number of revertant colonies:

	TA1535		TA1537		TA98
Dose (µg/plate)      S9-mix	-	+	-	+	-	+
Positive control	1306	399	830	384	1220	1227
Solvent control	13	12	5	10	13	16
5.4	12	14	4	4	12	23
17	10	12	4	4	12	12
52	6	12	3	5	6	16
164	10	9	4	3	8	13
512	13	11	1	4	12	13
1000	9	10	2	2	13	12

 

Pre-incubation assay – Mean number of revertant colonies:

	TA1535		TA1537		TA98		TA100		WP2uvrA
Dose (µg/plate)             S9-mix	-	+	-	+	-	+	-	+	-	+
Positive control	1144	238	175	243	1342	718	784	1931	275	567
Solvent control	18	16	14	13	28	42	107	133	31	45
5.4	18	13	8	8	24	31	124	116	-	-
17	11	15	13	12	35	33	116	130	37	49
52	13	12	9	13	28	32	115	105	38	51
164	14	14	6	10	29	35	103	161	35	39
512		13				37		106	36	35
1000									22	36

 

Conclusions:

Based on the results of this study it is concluded that Thio Isocyanate Adduct is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The objective of the study was to determine the potential of Thio Isocyanate Adduct and/or its metabolites to induce reverse mutations at the histidine locus in several strains of Salmonella typhimurium (S. typhimurium; TA98, TA100, TA1535, and TA1537), and at the tryptophan locus of Escherichia coli (E. coli) strain WP2uvrA in the presence or absence of an exogenous mammalian metabolic activation system (S9).

The test was performed in two independent experiments, at first a direct plate assay was performed and secondly a pre-incubation assay.

In the dose-range finding study, the test item was initially tested up to concentrations of 5000 μg/plate in the strains TA100 and WP2uvrA in the direct plate assay. Thio Isocyanate Adduct precipitated on the plates at the highest dose level tested. Cytotoxicity, as evidenced by a decrease in the number of revertants and/or a reduction of the bacterial background lawn, was observed in both tester strains in the absence and presence of S9-mix. Results of this dose-range finding test were reported as part of the first mutation assay.

In the first mutation experiment, the test item was tested up to concentrations of 1000 μg/plate in the strains TA1535, TA98 and TA1537. Except for tester strains TA1535 and TA98 in the presence of S9-mix where the test item was tested up to concentrations of 1600 μg/plate. Thio Isocyanate Adduct did not precipitate on the plates at this dose level.

Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

In the second mutation experiment, the test item was tested up to concentrations of 1600 μg/plate in the tester strains TA1535, TA1537, TA98 and TA100 and up to concentrations of 5000 μg/plate in tester strain WP2uvrA in the pre-incubation assay. Thio Isocyanate Adduct did not precipitate on the plates at this dose level. Cytotoxicity, as evidenced by a decrease in the number of revertants, reduction of the bacterial background lawn and/or the presence of microcolonies, was observed in all tester strains in the absence and presence of S9-mix.

Acceptable responses were obtained for the negative and strain-specific positive control items indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment.

In conclusion, based on the results of this study it is concluded that Thio Isocyanate Adduct is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Thio Isocyanate Adduct has been tested for mutagenicity to bacteria, in a study that was conducted according to the OECD TG 471 and in compliance with GLP. The test item did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in a follow-up experiment. Based on the results of this study it is concluded that Thio Isocyanate Adduct is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Justification for classification or non-classification

Based on the available in vitro genotoxicity data, Thio Isocyanate Adduct is not classified for mutagenicity according to Regulation (EC) No 1272/2008.