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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
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14 Oct 2011 - 23 Dec 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Identification: Halophosphate
- Substance type: White powder

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.
- 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
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.
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/ first cytogenetic test:
Without and with S9-mix, 3hr exposure: 10, 33 and 100 μg halophosphate/mL
Without S9-mix, 24/48hr exposure: 1, 3, 10, 33, 100, 333 and 1000 μg halophosphate/mL
Second cytogenetic test:
Without S9-mix, 24/48 hr exposure: 10, 33, 100 and 1000 μg/mL
With S9-mix, 3 hr exposure; 48 hr fixation: 10, 33 and 100 μg/mL
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle: A homogeneous suspension could be obtained in DMSO and DMSO is accepted and approved by authorities and international guidelines

Controlsopen allclose all
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9

Migrated to IUCLID6: in Hank's Balanced Salt Solution: 0.5 µg/mL and 1 µg/mL for a 3 h exposure period; 0.2 µg/mL and 0.4 µg/mLfor a 24 h exposure period; 0.1 µg/mL and 0.2 µg/mLfor a 48 h exposure period.
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9

Migrated to IUCLID6: in Hank's Balanced Salt Solution: 10 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in 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

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: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 100 μg/ml and above in all experiments.
- No toxicity was observed up to and including the highest precipitating tested dose
- 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. The effect of the highest concentration of mitomycin C was not evaluated, since the lower concentration gave acceptable results.
Remarks on result:
other: strain/cell type: human cultured lymphocytes
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
A chromosome aberration study with halophosphate was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes with and without metabolic activation in two independent experiments. It is concluded that halophosphate is not clastogenic in human lymphocytes.
Executive summary:

In a chromosome aberration study, cultured peripheral human lymphocytes were exposed to different concentrations of halophosphate (dissolved in DMSO), in the presence and absence of S9-mix according to the most recent OECD and EC guidelines. In the first cytogenetic assay, halophosphate was tested up to precipitating concentration for a 3 h exposure time with a 24 h fixation time (100 μg/ml). In the second cytogenetic assay, halophosphate 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. Halophosphate 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 halophosphate 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 halophosphate does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations nor polyploidy.