1,4-Cyclohexanedicarboxamide, N1,N1,N4,N4-tetrakis(2-hydroxyethyl)-, trans-

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:

2011-06-24 to 2011-08-01

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:

2011

Report date:

2011

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:

(1r,4r)-N1,N1,N4,N4-tetrakis(2-hydroxyethyl)cyclohexane-1,4-dicarboxamide

EC Number:

700-597-4

Cas Number:

1215841-86-3

Molecular formula:

C16H30N2O6

IUPAC Name:

(1r,4r)-N1,N1,N4,N4-tetrakis(2-hydroxyethyl)cyclohexane-1,4-dicarboxamide

Constituent 2

Reference substance name:

1,4-Cyclohexanedicarboxamide, N1,N1,N4,N4-tetrakis(2-hydroxyethyl)-, trans

IUPAC Name:

1,4-Cyclohexanedicarboxamide, N1,N1,N4,N4-tetrakis(2-hydroxyethyl)-, trans

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

CAS name: 1,4-Cyclohexanedicarboxamide, N1,N1,N4,N4-tetrakis(2-hydroxyethyl)-, trans
Chemical characterization: Trans-N,N,N',N'-Tetrakis(2-hydroxyethyl)-cyclohexyl-1,4-diamide
Characteristics: Whitish, solid, powder, hydroscopic
Contents:
91.53 % Trans-N,N,N',N'-Tetrakis(2-hydroxyethyl)-cyclohexyl-1,4-diamide

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:
31.6, 100, 316, 1000, 3160, 5000 µg/plate

Vehicle / solvent:

aqua ad iniectabilia, Batch no. 0443A242; B. Braun Melsungen AG

Controls

Untreated negative controls:

no

Remarks:

solvent test will be used as negative reference item

Negative solvent / vehicle controls:

yes

Remarks:

aqua ad iniectabilia

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, Cyclophosphamide

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, no signs of cytotoxicity
- Main test: 1st - Standard plate incorporation method, 2nd - Preincubation method
- Metabolic activation assay: Arochlor 1254 induced rat liver S9 fraction, collected from 20 - 30 rats
ADMINISTRATION
- Dosing: 31.6, 100, 316, 1000, 3160, 5000 µg/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
2-nitroflurene in DMSO for TA 98
9-amino-acridine in ethanol abs. for TA 1537
Cumene hydroperoxide in DMSO for TA 102
- with metabolic acivation
2-aminoanthracene in DMSO for TA 98, TA 102, TA 1537
Cyclophosphamide in aqua ad iniectabilia for TA 100, TA 1535
- negative control: solvent control: aqua ad iniectabilia 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:

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

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

Under the present test conditions trans-N1,N1,N4,N4-Tetrakis(2-hydroxyethyl)1,4-cyclohexanedicarboxamide tested up to a concentration of 5000 µg/plate, 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 substance trans-N1,N1,N4,N4-Tetrakis(2-hydroxyethyl)1,4-cyclohexanedicarboxamide was examined for its potential to induce gene mutations in the plate incorporation test and the pre-incubation test using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100 and TA 102, with and without the addition of a metabolising system (Arochlor 1254 induced rat liver S9 mix)

The study was carried out according to OECD method 471 in compliance with the current OECD Principles of Good Laboratory Practice. Dose levels covering a total range of 31.6 to 5000 µg/plate, in triplicate both with and without the addition of a metabolising system were employed. The test item was completely dissolved inaqua ad iniectabilia. A factor of 1.093 was used to correct for impurities.

Preliminary test

trans-N1,N1,N4,N4-Tetrakis(2-hydroxyethyl)1,4-cyclohexanedicarboxamide was examined in two preliminary cytotoxicity tests (plate incorporation test without and with metabolic activation) in test strain TA 100. Ten concentrations ranging from 0.316 to 5000 µg/plate were tested. No signs of cytotoxicity were noted up to the top concentration of 5000 µg/plate.

Hence, 5000 µg of the test item/plate was chosen as top concentration for the main study in the plate incorporation test and in the preincubation test, respectively.

Main study

Six concentrations ranging from 31.6 to 5000 µg trans-N1,N1,N4,N4-Tetrakis(2-hydroxyethyl)1,4-cyclohexanedicarboxamide/plate were employed in two independent experiments each carried out without and with metabolic activation.

Cytotoxicity

No signs of cytotoxicity were noted in the plate incorporation test or in the preincubation test, each carried out without and with metabolic activation, up to the top concentration of 5000 µg/plate in all test strains.

Mutagenicity

No mutagenic effect (no increase in revertant colony numbers as compared with control counts) was observed for the test item, tested up to aconcentrationof 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).

In conclusion, under the present test conditions trans-N1,N1,N4,N4-Tetrakis(2-hydroxyethyl)1,4-cyclohexanedicarboxamide tested up to a concentration of 5000 µg/plate, caused no mutagenic effect in theSalmonella typhimuriumstrains 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.