C8-18 alkylampho(di)propionates

Genetic toxicity: in vitro

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:

July 16, 2012 - January 23, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in accordance with OECD 473 (1998), EU Method B.10 (2008) and according to GLP principles.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

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

Test material form:

other: aqueous solution

Details on test material:

- Physical state: aqueous solution
- Appearance: amber 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 23-year-old adult female on 17 July 2012 for the preliminary toxicity assay and from the same donor on 30 July 2012 for the definitive assay and on 3 December 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 solid content of the substance, using a correction factor of 2.5934. Test substance dilutions were prepared immediately before use and delivered to the test system at room temperature under yellow light.

Expressed as solid 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, 60, 120, 170, 200, 250, 350 and 500 µg/mL
Without S9-mix, 20 hr exposure; 20 hr fixation: 30, 60, 120, 170, 200, 250, 350 and 500 µg/mL
With S9-mix, 4 hr exposure; 20 hr fixation: 30, 60, 120, 170, 200, 250, 350 and 500 µg/mL

Second cytogenetic test (confirmatory assay):
With S9-mix, 4 hr exposure; 20 hr fixation: 75, 150, 300, 350, 400 and 500 µg/mL

Vehicle / solvent:

- Vehicle used: water (Lot No. 1119693, Exp. Date 28 Feb 2014 and Lot No. 1169199, Exp. Date May 2014; obtained from Gibco)
- Justification for choice of vehicle: Test compound was stable in water and soluble in culture medium (up to 50 mg solids/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 (RPMI 1640 with 15% FBS)
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:

The number and types of aberrations per cell, the percentage of structurally and numerically aberrant cells and the frequency of structural aberrations per cell was calculated and reported. Chromatid and isochromatid gaps are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell.
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

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: Yes. The pH of the highest dose level of test substance in treatment medium was 8.0. In order to maintain neutral pH in the treatment medium, the pH was adjusted using 3 drops of 1N HCl. The final pH at 5000 μg/mL was 7.0. The pH of the 2nd highest dose level (1500 μg/mL) was also measured and was found to be 7.5.
- Effects of osmolality: No. The osmolality in treatment medium of the highest dose level tested, 5000 μg/mL, was 279 mmol/kg.
The osmolality of the solvent (water) in the treatment medium was 256 mmol/kg. The osmolality of the test substance dose level in the treatment medium is acceptable because it did not exceed the osmolality of the solvent by more than 20%.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 500 µg/ml and above in all 3 exposure groups.

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, 350 μg/mL, mitotic inhibition was 54%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 120, 200 and 350 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 (26.0%) (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, 350 μg/mL, mitotic inhibition was 56%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 120, 200 and 350 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 (22.0%) (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, 200 μg/mL, mitotic inhibition was 52%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 60, 120 and 200 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 (28.0%) (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, 300 μg/mL, mitotic inhibition was 56%, relative to the solvent control. The dose levels selected for analysis of chromosome aberrations were 75, 150 and 300 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 (19.0%) (p ≤ 0.01, Fisher's Exact test).

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

A chromosome aberration study with the substance 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:

A chromosome aberration study with the substance was performed in accordance with OECD 473 (1998), EU Method B.10 (2008) and according to GLP principles

in human peripheral blood lymphocytes in two independent experiments, with and without metabolic activation. The dosing preparations were adjusted for the solid 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 500 µg/mL. In the second cytogenetic assay, the cells were treated for 4 hr (with S9 -mix) with the substance at doses ranging from 75 to 500 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). Adequate negative and positive controls were included.

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.