Alkane-alpha,omega-diyl bis{[(triethoxysilyl)propyl]carbamate}_E2

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-01-23 to 2015-02-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study; GLP study without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

march 2005. OPPTS changed its name to OCSPP,

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Test material form:

other: liquid

Details on test material:

Reaction mass of 2,2,4-trimethylhexane-1,6-diylbis{[3-(triethoxysilyl)propyl]carbamate} and 2,4,4-trimethylhexane-1,6-diylbis{[3-(triethoxysilyl)propyl]carbamate} manufactured by Evonik Degussa GmbH. Batch No. IPES-Addukt 7W of 2014-06-23, purity: 95.05.%.

Method

Target gene:

mutated gene loci resposible for histidine auxotropy

Species / strain

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Details on mammalian cell type (if applicable):

- obtained from Trinova Biochem according to Dr. Bruce N. AMES,

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254 induced rat liver S9; male rats, obtained from Trinova Biochem

Test concentrations with justification for top dose:

Plate incorporation test: 31.6, 100, 316, 1000, 3160 or 5000 µg per plate;
Preincubation test: 31.6, 100, 316, 1000, 3160 or 5000 µg per plate;

Vehicle / solvent:

The test item was completely dissolved in dimethylsulfoxide (DMSO). A correction factor of 1.05 was used to correct for the purity of 95.05%. The
vehicle DMSO served as the negative control

Controls

Untreated negative controls:

no

Remarks:

solvent test will be used as negative reference item

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Remarks:

for details see below

Positive control substance:

other: without metabolic activation: sodium azide in aqua ad iniectabilia for TA 1535 and TA 100, 2-nitroflurene in DMSO for TA 98, 9-amino-acridine in ethanol abs. for TA 1537, Mitomycin C in DMSO for TA 102

Remarks:

with metabolic activation: 2-aminoanthracene in DMSO for TA 100, TA 1535, Benzo(a)pyrene in DMSO for TA 98, TA 102, TA 1537

Details on test system and experimental conditions:

Bacterial Reverse Mutation Test
SYSTEM OF TESTING
- Pre-Experiment: plate incorporation cytotoxicity test (+/- metabolic activation) with strain TA 100,
The test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg µg test item/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. Hence, 5000 µg test item/plate was chosen as top concentration for the main study in the plate incorporation test and in the
preincubation test.
- Main test: 1st - Standard plate incorporation method, 2nd - Preincubation method
- Metabolic activation assay: Arochlor 1254 induced rat liver S9 fraction.
ADMINISTRATION
- Dosing:
* Plate incorporation test: 31.6, 100, 316, 1000, 3160 or 5000 µg per plate;
* Preincubation test: 31.6, 100, 316, 1000, 3160 or 5000 µg per plate;
- Data : 2 independent experiments with and without metabolic activation
- Number of replicates: 3 per concentration and experiment
- Positive and negative control groups and treatment:
- without metabolic activation:
* sodium azide in aqua ad iniectabilia for TA 1535 and TA 100, 10 µg/plate
* 2-nitroflurene in DMSO for TA 98, 10 µg/plate
* 9-amino-acridine in ethanol abs. for TA 1537, 100 µg/plate
* Mitomycin C in DMSO for TA 102, 10 µg/plate
- with metabolic acivation
* 2-aminoanthracene in DMSO for TA 100 and TA 1535, 2 µg/plate
* Benzo(a)pyrene in DMSO for TA 98, TA 102 and 1537, 10 µg/plate
- negative control: the vehicle DMSO was used as negative reference item (all test strains).
- Incubation time: 48 h to 72 h at 37 °C in the dark
- Pre-incubation time: 20 min at 37 °C;

NUMBER OF REPLICATIONS: 3 per concentration and experiment

NUMBER OF CELLS EVALUATED: approximately 10E8 viable cells in the late exponential or early stationary phase

DETERMINATION OF CYTOTOXICITY
- Method: No signs of cytotoxicity were noted up to the top concentration of 5000 µg test item/plate in the plate incorporation test and in the
preincubation test, each carried out without and with metabolic activation in all test strains.

Evaluation criteria:

A test item is considered to show a positive response if
- the number of revertants is significantly increased (p  0.05, U-test according to MANN and WHITNEY, see section 6, reference 3) compared to the
solvent control to at least 2-fold of the solvent control for TA98, TA100, TA1535 and TA1537 and 1.5-fold of the solvent control for TA102 in both independent experiments.
Or
- a concentration-related increase over the range tested in the number of the revertants per plate is observed. The Spearman's rank correlation
coefficient may be applied.
- positive results have to be reproducible and the histidine independence of the revertants has to be confirmed by streaking random samples on
histidine-free agar plates.
Biological relevance of the results should be considered first.
A test item for which the results do not meet the above mentioned criteria is considered as non-mutagenic in the AMES test.

Acceptance Criteria
The results of the negative and positive control cultures have to be within the range of the historical data generated by LPT.

Statistics:

According to the OECD Guideline 471, a statistical analysis of the data is not mandatory

Results and discussion

Test results

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

GENTOXIC EFFECTS:
- With metabolic activation: negativ
- Without metabolic activation: negativ

CYTOTOXICITY EFFECTS:
No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with metabolic
activation up to the top concentration of 5000 µg test item/plate in any test strain.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

see attchached document

Applicant's summary and conclusion

Conclusions:

In conclusion, under the present test conditions test item tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.

Executive summary:

The purpose of this study was to evaluate the test substance for mutagenic activity (gene mutation) in bacteria without and with the addition of a mammalian metabolic activation system as originally described by AMES et al. (1973, 1975) and revised by MARON and (1983).

The potential of the test item to induce gene mutation was examined in 5 Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 in two independent experiments, each carried out without and with metabolic activation (a microsomal preparation derived from Aroclor 1254-induced rat liver). The first experiment was carried out as a plate incorporation test and the second as a preincubation test.

The test item was completely dissolved indimethylsulfoxide (DMSO).A correction factor of 1.05 was used to correct for the purity of 95.05%. The vehicle DMSO served as the negative control.

Preliminary test

Test item was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA100. Ten concentrations ranging from 0.316 to 5000 µg test item/plate were tested.No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate. Hence, 5000 µg test item/plate was chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

Main study

Six concentrations,31.6, 100, 316, 1000, 3160 or 5000 µg test item/plate were employed in the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation.

Cytotoxicity

No signs of cytotoxicity were noted in the plate incorporation test and in the preincubation test, each carried out without and with

metabolic activation up to the top concentration of 5000 µg test item/plate in any test strain.

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for test item, tested up to aconcentration of 5000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

The positive control items showed a significant increase in the number of revertant colonies of the respective test strain and

confirmed the validity of the test conditions and the sensitivity of the test system.

In conclusion, under the present test conditions test item tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in the Salmonella typhimurium strains TA98, TA100, TA102, TA1535 and TA1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.