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Toxicological information

Genetic toxicity: in vitro

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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 2011-10-31
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Version / remarks:
2005
Qualifier:
according to
Guideline:
other: ICH Guideline S2A:'Genotoxicity: Specific Aspects of Regulatory gentoxocoty tests for Pharmaceuticals (CPMP/ICH/141/95)'
Qualifier:
according to
Guideline:
other: ICH Giudeline S2B: 'A Standard Battery for Genotoxicity Testing of Pharmaceuticals (CPMP/ICH/174/95)'
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
purity: > 98.6%

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
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), a correction factor of 1.014 was used to correct for impurities.
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 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
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:
cytotoxicity
Remarks:
without and with metabolic activation at concentrations of 3160 and 5000 µg/plate, precipitation was noted at 5000 µg/plate.
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


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:
Interpretation of results (migrated information):
negative

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 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 purpose of this study was to evaluate cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, caprolactam-blocked 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 Ames (1983).

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 test item 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 the test item, tested up to a concentration of 3160 µg/plate, that led to cytotoxicity and/or test item precipitationin 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 effect in theSalmonella 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.