2-({[3-(isocyanatomethyl)-3,5,5-trimethylcyclohexyl]carbamoyl}oxy)ethyl prop-2-enoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In conclusion, under the present test conditions, the test item tested up to a cytotoxic concentration of 1000 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 (LPT, 2016).

Link to relevant study records

Reference

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:

2016-06-22 to 2016-07-07

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:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

The test item was completely dissolved in dimethylsulfoxide (DMSO) . The vehicle dimethylsulfoxide (DMSO) served as the negative control. Fresh preparations of the test item were used for the treatment in all experimental parts.

Target gene:

mutated gene loci resposible for histidine auxotropy

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: 3.16, 10.0, 31.6, 100, 316 and 1000 µg per plate
Preincubation test: 3.16, 10.0, 31.6, 100, 316 and 1000 µg per plate

Vehicle / solvent:

The test item was completely dissolved in dimethylsulfoxide (DMSO) . The vehicle dimethylsulfoxide (DMSO) served as the negative control.
Batch no. STBF8930V; SIGMA-ALDRICH Chemie GmbH, 82024 Taufkirchen, Germany

Untreated negative controls:

yes

Remarks:

solvent test will be used as negative reference item

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

benzo(a)pyrene

mitomycin C

other: 2-aminoanthracene

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/plate were tested. Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted starting at concentrations of 1000 µg/plate. Hence, 1000 µg test item per plate were 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: 3.16, 10.0, 31.6, 100, 316 and 1000 µg per plate
* Preincubation test: 3.16, 10.0, 31.6, 100, 316 and 1000 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 highly purufied water 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 highly purifies water 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: Overnight cultures were grown in a water bath for 15 h at 37°C in Oxoid 2 nutrient broth. The final cell density was approximately 10E8 - 10E9 cells/mL.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn.

Rationale for test conditions:

The study was performed in compliance with:
- Regulation (EC) No. 440/2008 method B.13/14: Mutagenicity: Reverse Mutation Test Using Bacteria, adopted May 30, 2008;
- OECD Guideline for Testing of Chemicals, No. 471: Bacterial Reverse Mutation Test, adopted July 21, 1997;

Evaluation criteria:

A test item is considered to show a positive response if
- the number of revertants is significantly increased (p 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.

The range of spontaneous reversion frequencies per plate is based on Kirkland:
TA98: 20 - 60
TA100: 100 - 200
TA102: 240 - 320
TA1535: 10 - 35
TA1537: 3 - 20

The numbers may be slightly different on plates with S9 and may vary slightly from experiment to experiment.
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the vehicle control and/or a scarce background lawn.


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

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 test item/plate

Vehicle controls validity:

valid

Untreated negative 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 test item/plate

Vehicle controls validity:

valid

Untreated negative 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 test item/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

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 test item/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1000 µg test item/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

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

CYTOTOXICITY EFFECTS:
In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 1000 µg test item/plate in all test strains.

see attchached document

Conclusions:

In conclusion, under the present test conditions, test item tested up to a cytotoxic concentration of 1000 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 test item to induce gene mutations 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 in dimethylsulfoxide (DMSO). 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/plate were tested. Pronounced cytotoxicity (scarce background lawn and reduction of the number of revertants by more than 50%) was noted starting at concentrations of 1000 µg/plate. Hence, 1000 µg test item per plate were chosen as top concentration for the main study in the plate incorporation test and in the preincubation test.

 

Main study

Six concentrations ranging from 3.16 to 1000 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

In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, cytotoxicity (scarce background lawn and reduction of the number of revertants) was noted at the top concentration of 1000 µg test item/plate in all test strains.

 

Mutagenicity

No increase in revertant colony numbers as compared with control counts was observed for test item, tested up to a cytotoxic concentration of 1000 µg/plate, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation test 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 cytotoxic concentration of 1000 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.

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the in vitro genotoxicity study the test item must not be classifed according to the criteria of EC Directive 67/548/EECand EC Regulation 1272/2008.