Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C7-C9 odd-numbered alkyl) derivs. and sodium hydroxide and chloroacetic acid

Genetic toxicity: in vitro

Some information in this page has been claimed confidential.

Administrative data

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October, 31 2011 - March 9, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1998)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(2008)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonization (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Federal Register 61:18198-18202, April 24, 1996 (S2A document recommended for adoption at step 4 of the ICH process)

Deviations:

no

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: International Conference on Harmonisation (ICH) of Technical Requirements for Registration of Pharmaceuticals for Human Use. Federal Register 62:16026-16030, November 21, 1997 (S2B document recommended for adoption at step 4 of the ICH process)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Amphoacetates C12

Cas Number:

68608-66-2

IUPAC Name:

Amphoacetates C12

Constituent 2

Constituent 3

Reference substance name:

Sodium glycollate

EC Number:

220-624-9

EC Name:

Sodium glycollate

Cas Number:

2836-32-0

Molecular formula:

C2H4O3.Na

IUPAC Name:

sodium glycolate

Constituent 4

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Oxidane

Test material form:

other: aqueous solution

Details on test material:

- Physical state: aqueous solution
- Appearance: clear colorless liquid
- Composition of test material, percentage of components: see section confidential details on test material

Method

Species / strain

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes (HPBL)

Details on mammalian cell type (if applicable):

Peripheral blood lymphocytes were obtained from a healthy non-smoking 24-year-old adult female on 01 November 2011 for the preliminary toxicity assay and from the same donor on 15 November 2011 for the definitive assay and 03 January 2012 for the confirmatory assay. The donor had no recent history of radiotherapy, viral infection or the administration of drugs.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by Aroclor 1254

Test concentrations with justification for top dose:

The dosing preparations were adjusted for the surfactant content of the substance, using a correction factor of 3.3. Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under yellow light.

Expressed as surfactant content:

Dose range finding test:
Without S9-mix, 4 hr exposure; 20 hr fixation: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/mL
Without S9-mix, 20 hr exposure; 20 hr fixation: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/mL
With S9-mix, 4 hr exposure; 20 hr fixation: 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/mL

First cytogenetic test (definitive assay):
Without S9-mix, 4 hr exposure; 20 hr fixation: 30, 65, 130, 146, 162, 190, 200 and 250 µg/mL
Without S9-mix, 20 hr exposure; 20 hr fixation: 30, 65, 130, 146, 162 and 190 µg/mL
With S9-mix, 4 hr exposure; 20 hr fixation: 30, 65, 130, 146, 162, 190, 200 and 250 µg/mL

Second cytogenetic test (confirmatory assay):
With S9-mix, 4 hr exposure; 20 hr fixation: 30, 65, 125, 140, 155, 170, 185 and 200 µg/mL

Note: expressed as solid content the tested concentrations need to be multiplied by a factor of 1.27

Vehicle / solvent:

- Vehicle used: water (obtained from Gibco, Lot no. 1029809)
- Justification for choice of vehicle: Test compound was stable in water and soluble in culture medium (up to 50 mg/mL)

Controls

Negative solvent / vehicle controls:

yes

Remarks:

(water)

Positive controls:

yes

Positive control substance:

other: see section "Any other information on materials and methods incl. tables"

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
DURATION
- Preincubation period: 44-48 hr
- Exposure duration: 4 hr (with and without S9-mix), 20 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hr
SPINDLE INHIBITOR (cytogenetic assays): Colcemid®
STAIN: Giemsa
NUMBER OF REPLICATIONS: duplicates in two independent experiments
NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture
DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 500 cells
OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A test substance would be considered to induce a positive response when the percentages of cells with aberrations were increased in a dose-responsive manner, with one or more dose levels being statistically significant and clearly outside the historical solvent control data (p ≤ 0.05). However, values that are statistically significant and fall within or just outside the range of historical solvent control values may be judged as not biologically significant. Test substances not demonstrating a statistically significant increase in aberrations would be concluded to be negative. In the event of a positive response only at the high dose level with at least 50% reduction in cell growth relative to the respective solvent control and no evidence of dose response in one or more treatment conditions, the test substance will be considered to induce positive response at a cytotoxic dose level.

Statistics:

Statistical analysis of the percent aberrant cells was performed using the Fisher's Exact test. Fisher's Exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's Exact test at any test substance dose level, the Cochran-Armitage test was used to measure dose-responsiveness.

Results and discussion

Test results

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes (HPBL)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/mL
- Effects of pH: No. The pH of the highest dose level of test substance in treatment medium was approximately 7.5.
- Effects of osmolality: Yes, the osmolality in treatment medium of the highest dose level tested, 5000 μg/mL, was 292 mmol/kg.
The osmolality of the solvent (water) in the treatment medium was 236 mmol/kg. The osmolality of the test substance dose level in the treatment medium exceeded the osmolality of the solvent by more than 20%. The osmolality in treatment medium of the second highest dose level, 1500 μg/mL was measured (270 mmol/kg) and was found to be acceptable.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 500 µg/ml and above in the absence and presence of S9, 4 hr treatment/20 hr fixation and at dose levels of 150 µg/ml and above in the absence of S9 for the continuous treatment of 20 hr

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures were within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, induced appropriate responses.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.

Any other information on results incl. tables

Definitive Chromosome Aberration Assay

- Cytogenetic analysis of the non-activated 4-hour exposure group: At the highest test dose level evaluated microscopically for chromosome aberrations, 250 μg/mL, mitotic inhibition was 55%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 65, 146 and 250 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to solvent control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) group was statistically significant (16.0%) (p ≤ 0.01, Fisher's Exact test).

- Cytogenetic analysis of the S9 -activated 4 -hour exposure group: At the highest test dose level evaluated microscopically for chromosome aberrations, 162 μg/mL, mitotic inhibition was 51%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 30, 65 and 162 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to solvent control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the CP (positive control) group was statistically significant (12.0%) (p ≤ 0.01, Fisher's Exact test).

- Cytogenetic analysis of the non-activated 20-hour exposure group: At the highest test dose level evaluated microscopically for chromosome aberrations, 146 μg/mL, mitotic inhibition was 58%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 30, 65 and 146 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to solvent control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the MMC (positive control) group was statistically significant (12.0%) (p ≤ 0.01, Fisher's Exact test).

Confirmatory Chromosome Aberration Assay

- Cytogenetic analysis of the S9-activated 4-hour exposure group: At the highest test dose level evaluated microscopically for chromosome aberrations, 155 μg/mL, mitotic inhibition was 48%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 65, 125 and 155 μg/mL. The percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to solvent control at any dose level (p > 0.05, Fisher's Exact test). The percentage of structurally aberrant cells in the CP (positive control) group was statistically significant (20.0%) (p ≤ 0.01, Fisher's Exact test).

Applicant's summary and conclusion

Conclusions:

A chromosome aberration study with Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-2-(C11 alkyl) derivs. and sodium hydroxide and chloroacetic acid (aqueous solution; amphoacetates C12) was performed according to OECD 473 guideline and GLP principles, in peripheral human lymphocytes in two independent experiments with and without metabolic activation. It is concluded that the substance is not clastogenic in human lymphocytes.

Executive summary:

In accordance with OECD 473 (1998), EU Method B.10 (2008) and according to GLP principles, a chromosome aberration study with Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-2-(C11 alkyl) derivs. and sodium hydroxide and chloroacetic acid (aqueous solution; amphoacetates C12) was performed in human peripheral blood lymphocytes in two independent experiments with and without metabolic activation. The dosing preparations were adjusted for the surfactant content of the substance and were set based on the observed toxicity in the dose range finding study (no precipitation was observed up to and including the top dose of 5000 µg/mL).

In the first cytogenetic assay, the cells were treated for 4 hr (with and without S9-mix) and for 20 hr (without S9-mix) with the substance at doses ranging from 30 to 250 µg/mL. In the second cytogenetic assay, the cells were treated for 4 hr (with S9 -mix) with the substance at doses ranging from 30 to 200 µg/mL.

Toxicity was reached at the dose levels selected for scoring. In all exposure groups, the percentage of cells with structural or numerical aberrations in the test substance-treated group was not significantly increased relative to solvent control at any dose level (p > 0.05, Fisher's Exact test). The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

No effects of the substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

Based on the results of this study, it is concluded that the substance does not disturb mitotic processes and cell cycle progression and is not clastogenic in human lymphocytes.