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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:
weight of evidence
Study period:
01MAY2015 - 16JUL2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted September 26, 2014
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
Official Journal of the European Union No. L142, 31 May 2008
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): PEMP product
- Substance type: organic
- Physical state: Colourless transparent liquid
- Storage condition of test material: In refrigerator (2-8°C)

Method

Species / strain
Species / strain / cell type:
lymphocytes: Cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture 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.
- Culture medium
Culture medium consisted of 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.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.
Test concentrations with justification for top dose:
Dose range finding test:
Without S9-mix, 3hr exposure; 24 hr fixation: 17, 52 and 164 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 1.7, 5.4, 17, 52 and 164 µg/mL
With S9-mix, 3hr exposure; 24 hr fixation: 17, 52 and 164 µg/mL

First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time: 17, 52 and 164 µg/mL
With S9-mix, 3 h exposure, 24 h fixation time: 17, 52 and 164 µg/mL

Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 17, 52, 89 and 164 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 17, 52, 89 and 164 µg/mL
Vehicle / solvent:
- Solvent used: DMSO
- Justification for choice of solvent/vehicle: Choice of solvent was based on a solubility test.
Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
Mitomycin C: at 0.5 and 0.75 μg/ml (3 h exposure period), 0.2 and 0.3 μg/ml (24 h exposure period) and 0.1 and 0.15 μg/ml (48 h exposure period).
Remarks:
without S9
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Remarks:
Cyclophosphamide: 10 μg/ml (3 h exposure period; 24 h fixation time).
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): 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 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Results and discussion

Test results
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Precipitating concentration = 164 μg/mL
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No

- Precipitation: Precipitation in the exposure medium was observed at dose levels of 164 µg/ml and above
- No toxicity was observed up to and including the highest precipitating tested dose

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.

Applicant's summary and conclusion

Conclusions:
A chromosome aberration study with PEMP product was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in two independent experiments. It is concluded that the test substance is not clastogenic in human lymphocytes.
Executive summary:

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of PEMP product (dissolved in DMSO), in the presence and absence of S9-mix according to OECD/ EC guidelines and GLP principles. In the first cytogenetic assay, PEMP product was tested up to precipitating concentration for a 3 h exposure time with a 24 h fixation time (≥ 164 μg/ml). In the second cytogenetic assay, PEMP product was tested up to precipitating concentrations for a 24 h and 48 h continuous exposure time with a 24 h and 48 h fixation time in the absence of S9-mix. PEMP product did not induce a 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 independently repeated experiments. No effects of PEMP product 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 PEMP product does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations nor polyploidy.