3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, reaction products with 2-butanone oxime

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:

2011-09-26 to 2011-11-10

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:

2012

Report date:

2012

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:

2005

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: ICH Guideline S2A:'Genotoxicity: Specific Aspects of Regulatory gentoxocoty tests for Pharmaceuticals (CPMP/ICH/141/95)'

Test guideline 5

Qualifier:

according to guideline

Guideline:

other: ICH Giudeline S2B: 'A Standard Battery for Genotoxicity Testing of Pharmaceuticals (CPMP/ICH/174/95)'

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, reaction products with 2-butanone oxime

EC Number:

500-287-7

EC Name:

3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, reaction products with 2-butanone oxime

Cas Number:

103170-26-9

Molecular formula:

>= C48H81N9O9 (IPDI-Isocyanurate n=3, 2-butanone oxime-blocked)

IUPAC Name:

3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, reaction products with 2-butanone oxime

Test material form:

solid: particulate/powder

Method

Target gene:

mutated gene locus responsible for histidine auxotrophy

Species / strain

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: histidine auxotroph

Metabolic activation:

with and without

Metabolic activation system:

Arochlor 1254 induced rat liver S9; male rats

Test concentrations with justification for top dose:

Experiment I and II:
10.0, 31.6, 100, 316, 1000 and 3160 µg per plate.

Vehicle / solvent:

Test item was completely dissolved in dimethyl sulfoxide (DMSO).

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, Cumene hydroperoxide in DMSO for TA 102

Remarks:

with metabolic activation: 2-aminoanthracene for TA 98, TA 102, TA 1537, Cyclophosphamide fpr TA 100, TA 1535

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,
10 concentrations ranging from 0.316 to 5000 µg/plate were tested. Cytotoxicity and/or test item precipitation were noted in the plate
incorporation test without and with metabolic activation at concentrations of 3160 and 5000 µg/plate.
- Main test: 1st - Standard plate incorporation method, 2nd - Preincubation method
- Metabolic activation assay: Arochlor 1254 induced rat liver S9 fraction, the protein content of the S9 fraction was 34.2 mg/mL S9, cytochrome
P-450: 0.36 nmol/mg protein
ADMINISTRATION
- Dosing: 10.0, 31.6, 100, 316, 1000 and 3160 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
Cumene hydroperoxide in DMSO for TA 102, 10 µg/plate
- with metabolic acivation
2-aminoanthracene in DMSO for TA 98, TA 102, TA 1537, 2 µg/plate
Cyclophosphamide in aqua ad iniectabilia for TA 100, TA 1535, 1500 µg/plate
- negative control: solvent control: DMSO for all strains
- Incubation time: 48 h to 72 h at 37 °C in the dark
- Pre-incubation time: 20 min at 37 °C;

Evaluation criteria:

The 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) compared to the solvent control to at least
2-fold of the solvent control for TA 98, TA 100 and TA 102 and 3-fold of the solvent control for TA 1535 and TA 1537 in both independent
experiments.
- additionally, a significant (p ≤ 0.05) concentration (log value)-related effect (Spearman's rank correlation coefficient) is observed.
- 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.

Statistics:

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

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity and/or test item precipitation were noted in the plate incorporation test without and with metabolic activation at concentrations of 3160 and 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity and/or test item precipitation were noted in the plate incorporation test without and with metabolic activation at concentrations of 3160 and 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity and/or test item precipitation were noted in the plate incorporation test without and with metabolic activation at concentrations of 3160 and 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity and/or test item precipitation were noted in the plate incorporation test without and with metabolic activation at concentrations of 3160 and 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Species / strain:

S. typhimurium TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity and/or test item precipitation were noted in the plate incorporation test without and with metabolic activation at concentrations of 3160 and 5000 µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

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

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

see attchached document

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative

Under the present test conditions 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, reaction
products with 2-butanone oxime tested up to a concentration of 3160 µg/plate, that led to cytotoxicity and/or test item precipitation, caused no
mutagenic effect in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in
the preincubation test each carried out without and with metabolic activation.

Executive summary:

The test item was examined in the 5 Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 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.

Six concentrations ranging from 10.0 to 3160 µg 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, reaction products with 2-butanone oxime per plate were employed in two independent experiments each carried out without and with metabolic activation.

In the plate incorporation test and in the preincubation test, each carried out without and with metabolic activation, test item precipitation and/or cytotoxicity (reduction of the number of revertants by more than 50 %) were noted at the top concentration of 3160 µg/plate in all test strains.

No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for 3-isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, reaction products with 2-butanone oxime, tested up to a concentrationof 3160 µg/plate, that led to cytotoxicity and/or test item precipitation, in any of the 5 test strains in two independent experiments without and with metabolic activation, respectively (plate incorporation and preincubation test).

In conclusion, under the present test conditions the test item tested up to a concentration of 3160 µg/plate, that led to cytotoxicity and/or test item precipitation, caused no mutagenic effects in the Salmonella typhimurium strains TA 98, TA 100, TA 102, TA 1535 and TA 1537 neither in the plate incorporation test nor in the preincubation test each carried out without and with metabolic activation.