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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:
26 April 2007 to 17 July 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP, no deviations

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 1997
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
adopted 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): 3-Mercaptopropionic acid
- Physical state: Clear colourless liquid
- Analytical purity: 99.5% (GC)
- Purity test date: 2007-04-18
- Lot/batch No.: 19185
- Expiration date of the lot/batch: 2008-04-17
- Stability under test conditions: no data
- Storage condition of test material: ~4°C in the dark

Method

Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: MEM
- Properly maintained: not applicable, primary culture
- Periodically checked for Mycoplasma contamination: not applicable, primary culture
- Periodically checked for karyotype stability: not applicable, primary culture
- Periodically "cleansed" against high spontaneous background: not applicable, primary culture
Metabolic activation:
with and without
Metabolic activation system:
S9 was prepared from the livers of male Sprague-Dawley rats weighing ~250 g. These had received three daily oral doses of a mixture of phenobarbitone (80 mg/kg) and ß-naphthoflavone (100 mg/kg), prior to S9 preparation on the fourth day.
Test concentrations with justification for top dose:
4-h exposure: 33.1 to 1060 µg/mL
24-h exposure: 33.1 to 530 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: culture medium
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:
without S9

Migrated to IUCLID6: 0.4 and 0.2 µg/mL for 4- and 24-h exposure
Positive control substance:
cyclophosphamide
Remarks:
with S9

Migrated to IUCLID6: 5 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 4, 24 h
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h


SPINDLE INHIBITOR (cytogenetic assays): Colcemid 0.1 µg/mL
STAIN (for cytogenetic assays): 5% Gurrs Giemsa


NUMBER OF REPLICATIONS: 2


NUMBER OF CELLS EVALUATED: 200 per concentration


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear concentration-relationship. For modest increases in aberration frequency a concentration response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
Fisher's Exact test.

Results and discussion

Test results
Species / strain:
lymphocytes: human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Remarks:
10 mM
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
The concentration range for the Preliminary Toxicity Test was 16.5 to 1060 µg/mL. The maximum concentration was the 10 mM concentration. No precipitate of the test material was observed at the end of the exposure period in any of the three exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 1060 µg/mL in the 4(20)-hour exposures both in the presence and absence of metabolic activation (S9). The maximum concentration with metaphases present in the 24-hour continuous exposure was 1060 µg/mL, though it should be noted that frequency of metaphases was very low. The test material induced some evidence of toxicity.


COMPARISON WITH HISTORICAL CONTROL DATA: within historical reference range
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 the test and of the activity of the metabolising system.  The test material was moderately toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a concentration range that included a concentration level that induced approximately 50% mitotic inhibition.

MPA produced some evidence of toxicity at 1060 µg/mL in the preliminary  toxicity test, as well as in the chromosome aberration test.

Table 1: Experiment 1 - 4 h treatment, 20 h fixation - Without Metabolic Activation

Concentration
[µg/ mL]

Mitotic index [%]

Polyploid cells [%]

Aberrant cells (%)

incl. gaps

excl. gaps

0

100

0.0

0.5

0.0

265

91

0.0

1.5

0.5

530

75

0.5

0.0

0.0

1060

71

0.0

2.0

1.5

MMC, 0.4

60

0.0

35.3

33.3***

MMC: Mitomycin C

*** p<0.001

 

Table 2: Experiment 1- 4 h treatment, 20 h fixation - With Metabolic Activation

Concentration
[µg/ mL]

Mitotic index [%]

Polyploid cells [%]

Aberrant cells (%)

incl. gaps

excl. gaps

0

100

0.0

1.0

0.5

265

100

0.0

0.5

0.5

530

69

0.0

0.5

0.5

1060

70

0.0

1.5

1.5

CP, 5

30

0.0

38.8

33.0***

CP: Cyclophosphamide

*** p<0.001

 

Table 3: Experiment 2- 20 h treatment, 20 h fixation - Without Metabolic Activation

Concentration
[µg/ mL]

Mitotic index [%]

Polyploid cells [%]

Aberrant cells (%)

incl. gaps

excl. gaps

0

100

0.5

0.5

0.5

66.25

104

0.0

2.0

0.5

132.5

82

0.0

2.5

0.5

265

36

0.0

3.0

2.0

MMC, 0.4

50

0.0

17.0

16.0***

MMC: Mitomycin C

*** p<0.001

 

 

Table 4: Experiment 2- 4 h treatment, 20 h fixation - With Metabolic Activation

Concentration
[µg/ mL]

Mitotic index [%]

Polyploid cells [%]

Aberrant cells (%)

incl. gaps

excl. gaps

0

100

0.0

0.0

0.0

265

178

0.0

2.0

1.5

530

142

0.0

0.5

0.5

1060

161

0.0

2.5

0.5

CP, 5

54

0.0

28.0

24.0***

CP: Cyclophosphamide

*** p<0.001

Applicant's summary and conclusion

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

The test material is non-clastogenic to human lymphocytes in vitro, with and without metabolic activation.