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Diss Factsheets

Toxicological information

Genetic toxicity: in vitro

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Administrative data

in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
17 August to 2 November 1999
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study according to international guidelines.

Data source

Reference Type:
study report
Report date:

Materials and methods

Test guideline
according to guideline
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Ceraphyl RMT
- Lot/batch No.: T4649
- Appearance: Yellow liquid
- Received: 09 August 1999
- Expiration date of the lot/batch: 23 April 2000 (extended to 22 April 2001)
- Storage condition of test material: At room temperature


Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
Blood collected from healthy male non-smokers. Blood was stored refrigerated and pooled prior to use. Whole blood cultures were established in sterile disposable centrifuge tubes by placing 0.4 ml heparinised blood into 9.0 mL Heps-buffered RPMI medium containing 20% (v/v) foetal calf serum and 50 µg/mL Gentamycin. Phytohaemagglutinin (PHA, reagent grade) was included at a concentraion of approximately 10 µg/mL of culture to stimulate the lymphocytes to divide. Blood cultures wee incubated for approx. 48 hours at 37 dC and rocked continuously.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Araclor induced rat S-9
Test concentrations with justification for top dose:
Experiment 1: 4.844, 6.921, 9.887, 14.12, 20.18, 28.82, 41.18, 58.82, 84.04, 120.1, 171.5, 245, 350, 500 µg/mL
Experiment 2: 37.54, 50.06, 66.74, 88.99, 118.7, 158.2, 210.9, 281.3, 375, 500 µg/mL
Vehicle / solvent:
Dimethylsulfoxide (DMSO)
Untreated negative controls:
Negative solvent / vehicle controls:
True negative controls:
Positive controls:
Positive control substance:
Details on test system and experimental conditions:
Experiment 1 (with or without metabolic activation): 3 h exposure + 17 h
Experiment 2 : with metabolic activation: 3 h exposure + 17 h recovery, without metabolic activation: 20 h exposure + 0 h recovery.
Approx. 2 h prior to harvest addition of colchicine to arrest dividing cells in metaphase. Then centrifugation at 300 g for 10 minutes. Removal of supernatant end resuspending in 4 mL hypotonic (0.075 M) KCl and incubated for 15 min to allow cell swelling. Then fixing of cells using ice-cold methanol/glacial acetic acid (3:1 v/v). Subsequently, centrifugation of fixative + cells, and repeating this procedure until the cell pellets were clean.
Preparation of metaphase spreads:
Lymphocytes were kept for at least 12 h in fixative before preparation of slides. Chromosome spreading was enhanced by adding 45% (v/v) aqueous acetic acid and additonally by flaming the slides if necessary.After drying the slides they were stained in 4% (v/v) filtered Giemsa stain in pH 6.8 buffer. Then the slides were rinsed, dried and mounted with coverslips.
Cytogenetic analysis:
Slides were examined "blind" for mitotic index (MI) or percentage of cells in mitosis based on at least 1000 cells counted per dose. Labels did not include the treatment details like dosage. Only cells with 44-46 chromosomes were analysed for structural aberrations. Cells with deviating numbers were observed separately. Classification of structural aberrations was based on the scheme described by ISCN.
Treatment of data:
After completion of the microscopic analysis, data were decoded and the aberrant cells in each culture were categorised as follows:
1. cells with structural aberrations including gaps;
2. cells with structural aberrations excluding gaps;
3. polyploid, endoreduplicated or hyperdiploid cells.
The totals for cat. 2 in the negative control were compared with the current laboratory negative control ranges for acceptability reasons.
Evaluation criteria:
A test article is considered as positive if:
1. the proportions of cells with structural aberrations at one or more concentration exceeds the normal range in both replicates, and
2. a statistical significant increase in the proportion of cells with structural aberrations (excl. gaps) occurs at these doses.
Statistical analysis by Fisher's exact test. The proportions of aberrant cells in each replicate were also used to establish acceptable heterogenecity between replicates by means of a binomial disperion test. Probability values of p ≤ 0.05 were to be accepted as significant.

Results and discussion

Test resultsopen allclose all
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
(> 500 µg/ml)
Vehicle controls validity:
Untreated negative controls validity:
Positive controls validity:
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Cytotoxicity / choice of top concentrations:
(> 500 µg/ml)
Remarks on result:
other: other: Experiment 1
Migrated from field 'Test system'.

Applicant's summary and conclusion

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Ceraphyl RMT did not induce chromosome structural aberrations in cultured human peripheral blood lymphocytes when tested up to its solubility limit in culture medium in either the absence or presence of S-9.