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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:
2009-07-03 to 2009-09-24
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study conducted to current standard guidelines. The justification for read-across from MnCl2 to MnSO4 is based on the fact that in an in vitro system, in the case of both substances, the aqueous solution surrounding the test cells will consist simply of Mn2+ cations with the anions not likely to play any part in the toxicity.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material : Manganese chloride (Eramet)
- Molecular formula : MnCl2
- Substance type: Light pink solid flakes
- Physical state: Solid
- Analytical purity: >95%
- Lot/batch No.: 104896-05
- Storage condition of test material: Room temperature in the dark

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
lymphocytes: human, peripheral
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
See table 1 under section Any other information on materials and methods incl. tables
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Minimal Essential Medium (MEM)
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
In the absence of S9 Migrated to IUCLID6: 0.4 and 0.2 µg/mL for the 4(20)-hour and 24-hour exposures respectively in MEM
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In the presence of S9 Migrated to IUCLID6: 5 µg/mL dissolved in dimethyl sulphoxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: Approximately 48 hours incubation at 37 °C 5% CO2 in humidified air.
- Exposure duration: 4 hour exposure (with a further 20 hour incubation once test material had been removed) and 24 hour exposure
- Expression time (cells in growth medium): Typically 17 hours average generation time under experimental exposure conditions.
- Fixation: Mitosis was arrested two hours before the required harvest time. After incubation with demecolcine, the cells were centrifuged, the culture medium was drawn off and discarded, and the cells re-suspended in 0.075 M hypotonic KCl. After 14 minutes (including centrifugation), most of the hypotonic solution was drawn off and discarded. The cells were re-suspended and then fixed by dropping the KCl cell suspension into fresh methanol/glacial acetic acid (3:1 v/v). The fixative was changed at least three times and the cells stored at 4 °C for at least four hours to ensure complete fixation.


SPINDLE INHIBITOR (cytogenetic assays): Demecolcin (Colcemid 0.1 µg/mL) two hours before the required harvest time.
STAIN (for cytogenetic assays): To prepare metaphase spreads, lymphocytes were suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data. When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.


NUMBER OF REPLICATIONS: Duplicate lymphocyte cultures (A and B) were prepared for each dose level.


NUMBER OF CELLS EVALUATED: Where possible the first 100 consecutive well-spread metaphases


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

- Other: Slides were checked microscopically to determine the quality of the metaphases and also the toxicity and extent of precipitation of the test material. These observations were used to select the dose levels for mitotic index evaluation.
Evaluation criteria:
Coding:
Slides were coded using a computerised random number generator and any supplementary slides were coded manually.
Mitotic index:
A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.
Scoring of chromosome damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted. Where there were approximately 30 to 50 % of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the International System for Chromosome Nomenclature (1985) Scott et al and Savage (1976) in the UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogenticist prior to decoding the slides.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.

Results and discussion

Test results
Species / strain:
lymphocytes: human, peripheral
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Test material was noted to precipitate at 315 µg/mL in the 4(20)-hour cultures without S9, and above 630 µg/mL in the 4(20)-hour cultures in the presence of S9. No precipitate was observed at the end of the exposure period in the 24-hour cultures.


RANGE-FINDING/SCREENING STUDIES: The preliminary toxicity test dose range was 4.92 to 1260 µg/mL. The maximum dose was based on the maximum recommended 10 mM concentration. A precipitate of the test material was observed in the cultures at the end of the exposure, at and above 157.5 µg/mL in the 4(20)-hour exposure in the absence of S9 at and above 78.75 µg/mL in the 4(20)-hour exposure in the presence of S9, and at above 315 µg/mL in the 24 hour continuous exposure group. Haemolysis was observed at and above 315 µg/mL at harvesting in all three exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1260 µg/mL in the 4(20)-hour exposure in the presence of metabolic activation and up to 157.5 µg/mL in the 4(20)-hour exposure in the absence of S9. The maximum dose with metaphases present in the 24-hour continuous exposure was 39.38 µg/mL. The test material induced clear evidence of toxicity in all of the exposure groups.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of test and of the activity of the metabolising system. The test material was found to be toxic to lymphocytes, and did not induce any toxicologically significant increases in the frequency of cells with aberrations, in any of the exposure conditions, using a dose range that included dose levels that induced approximately 50% mitotic inhibition.

Please refer to attached document, Appendix 1 for full tabulated results

Applicant's summary and conclusion

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

The test material did not induce any toxicologically significant increases in the frequency of cells with aberrations in either of the 4(20)-hour exposure groups in the absence or presence of a liver enzyme metabolising system or following 24 hours continuous exposure. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.The justification for read-across from MnCl2 to MnSO4 is based on the fact that in an in vitro system, in the case of both substances, the aqueous solution surrounding the test cells will consist simply of Mn2+ cations with the anions not likely to play any part in the toxicity.