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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 10, 2017 to June 17, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
26 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
other: in vitro mammalian chromosome aberration test

Test material

Constituent 1
Chemical structure
Reference substance name:
(Z)-N-[2-(2-hydroxyethoxy)ethyl]-9-octadecenamide
Cas Number:
20429-33-8
Molecular formula:
C22H43NO3
IUPAC Name:
(Z)-N-[2-(2-hydroxyethoxy)ethyl]-9-octadecenamide
Test material form:
solid
Details on test material:
Physical appearance: light yellow paste
Storage conditions: at room temperature
Specific details on test material used for the study:
Stability at higher temperatures: yes, maximum temperature: 30°C
light yellow paste

Method

Species / strain
Species / strain / cell type:
lymphocytes: Cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: blood samples were collected by venepuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Suitability of cells: cells from healthy, adult, non smoking humans were used as recommended in international guidelines (e.g. OECD).
- Average Generation Time: 13.7 h (dose-range finding), 15.8 h (first cytogenetic assay), 13.4 (second cytogenic assay) and 14.2 h (additional second cytogenetic assay)
- Sex, age and number of blood donors: approx. 22 years of age
- Whole blood treated with heparin was used


MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 medium supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) and 30 U/ml heparin.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw).
Test concentrations with justification for top dose:
Dose range finding test:
With and without S9-mix for all exposures: 78, 156, 313, 625, 1250 and 2500 µg/mL
The highest tested concentration was determined by the solubility of the test substance in the culture medium.

First cytogenetic test:
Without S9 (3h exposure time, 24h fixation time): 25, 100, 200, 225, 250, 275 and 300 µg/mL (25, 200 and 250 µg/mL were selected for scoring)
With S9 (3h exposure time, 24h fixation time): 25, 100, 200, 225, 250, 275, 300 and 350 µg/mL (25, 250 and 300 µg/mL were selected for scoring)
The test substance precipitated in the culture medium from 250 µg/mL and upwards.

Second cytogenetic test:
Without S9 (24 h exposure time, 24 h fixation time and 48 h exposure time, 48 h fixation time): 10, 50, 100, 125, 150, 175, 200 and 250 µg/mL (no cytotoxic dose levels could be selected for scoring)
In the 24 h exposure time, the test substance precipitated in the culture medium at 250 µg/mL

Additional second cytogenetic test:
Without S9 (24 h exposure time, 24 h fixation time): 50, 100, 150, 180, 200, 220 and 240 µg/mL (50, 100 and 180 µg/mL and 150, 175 and 200 µg/mL were selected for scoring in the 24 h exposure and 48 h exposure times, respectively)
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent: A solubility test was performed based on visual assessment. The test substance was dissolved in dimethyl sulfoxide of spectroscopic quality.
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9
Details on test system and experimental conditions:
Two independent cytogenetic assays were performed, preceeded by a dose-range finding assay.

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration experiment 1: 3 h (with and without S9)
- Exposure duration experiment 2: 24, 48 h (without S9)
- Fixation time: 24 h (for 3 or 24 hour exposure period) and 48 h (for 48 hour exposure period)

ENVIRONMENTAL CONDITIONS:
- Humidity: 38-91%
- Temperature: 34.5-37.1 °C

SPINDLE INHIBITOR (cytogenetic assays): colchicine

STAIN (for cytogenetic assays): Giemsa

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96%
(v/v) ethanol (Merck)/ether (Merck) and cleaned with a tissue. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry.

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 1000

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE: 150 in each replicate

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity: In the first assay, the test substance was difficult to dissolve in aqueous solutions, the highest concentration analysed was determined by the solubility in the culture medium.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
EVALUATION CRITERIA:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) At least one of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) The increase is dose related when evaluated with an Cochran Armitage trend test.
c) Any of the results are outside the 95% control limits of the historical control data range.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if:
a) None of the test concentrations exhibits a statistically significant (Fisher’s exact test, one-sided, p < 0.05) increase compared with the concurrent negative control.
b) There is no concentration-related increase when evaluated with an Cochran Armitage trend test.
c) All results are inside the 95% control limits of the negative historical control data range.

ACCEPTABILITY CRITERIA:
a) The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
b) The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
c) The positive control substance induces a statistically significant increase in the number of cells with chromosome aberrations. The positive control data will be analysed by the Fisher’s exact test (one-sided, p < 0.05).
Statistics:
Fisher's exact test, one-sided, was used to compare the incidence of abberant cells cells with the concurrent negative control.
Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
In the second assay the highest concentration (250 μg/ml) was too toxic for scoring (83%)
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No

RANGE-FINDING/SCREENING STUDIES:
- The test substance precipitated in the test medium at and above a dose level of 313 µg/mL. At and above this concentration, no metaphases were observed and cell lysis occurred.

- Precipitation in the main tests: In the first cytogenetic test, the test substamce precipitated in the culture medium from 250 µg/mL and upwards. In the second cytogenetic test, in the 24 h exposure time, the test substance precipitated in the culture medium at 250 µg/mL. No precipitation was observed in the additional second test.

DOSE LEVELS SELECTED FOR SCORING:
- First cytogenetic test:
Without S9: 25, 200 and 250 µg/mL
With S9: 25, 250 and 300 µg/mL

- Second cytogenetic test:
At the 24 h exposure time, 24 h fixation time, no cytotoxic dose levels could be selected for scoring since at the concentration of 200 μg/ml not enough cytotoxicity was observed (44%), whereas the next higher concentration of 250 μg/ml was too toxic for scoring (83%). Therefore, an additional second cytogenetic assay was performed with a 24 h exposure time and 24 h fixation time only.

- Additional second cytogenetic test:
24 hour exposure time, 24 hour fixation time: 50, 100 and 180 µg/mL
48 hour exposure time, 48 hour fixation time: 150, 175 and 200 µg/mL


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

Applicant's summary and conclusion

Conclusions:
Under the study conditions, it is concluded that the test substance is not clastogenic in human lymphocytes.
Executive summary:

A study was conducted to determine the potential of the test substance to induce structural chromosome aberrations in cultured human lymphocytes according to OECD Guideline 473, in compliance with GLP. Two independent experiments were performed. In the first cytogenetic assay, the test substance was tested up to 250 μg/mL and 300 μg/mL for a 3 h exposure time with a 24 h fixation time in the absence and presence of S9-mix, respectively. The test substance precipitated in the culture medium at these dose levels. In the second cytogenetic assay, test substance was tested up to 180 μg/mL for a 24 h continuous exposure time with a 24 h fixation time and up to 200 μg/mL for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. The negative control used in the test system was the vehicle for the test substance DMSO. Mitomycin C and Cyclophosphamide served as positive controls. The test substance did not induce any statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two experiments. No effects of the test substance on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that the test substance does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations. The number of cells with chromosome aberrations found in the solvent control cultures was within the 95 % control limits of the distribution of the historical negative control database. Tests conducted with the positive control chemicals, confirmed that the test conditions were adequate and that the metabolic activation system functioned properly. Under the study conditions, it is concluded that the test substance is not clastogenic in human lymphocytes (Buskens, 2017).

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