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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:
1 December 2009 to 10 August 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Fully GLP- and guideline compliant study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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:
other: Regulation (EC) No 440/2008 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
Details on test material:
- Name of test material (as cited in study report): 3-Hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde,
dimethylolpropanale
- Trade name: Novaltan PF
- Hill formula: C5H10O3.
- Physical state: Colourless, high viscous
- Composition of test material, percentage of components: ca. 82% Dimethylpropanale, ca. 10% Hydroxymethanesulfonic acid, Na-salt (CAS No: 870-72-4), ca. 6% Water, ca. 2% Potassiumformiate (CAS No: 590-29-4).
- Lot/batch No.: CH 138067/001
- Expiration date of the lot/batch: 30 November 2010
- Stability under test conditions: min. 1 year
- Storage condition of test material: Room temperature

Method

Target gene:
not applicable
Species / strain
Species / strain / cell type:
lymphocytes:
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Lyophilized Post-Mitochondrial Supernatant (Moltox, Art. No. 11-01L.2, Lot.: 2361) from male Sprague-Dawley rat livers, induced with Aroclor-1254, in 0.154M KCL
Test concentrations with justification for top dose:
Experiment A (3 hours incubation, no metabolic activation system): 0.044, 0.13, 0.39, 1.18 mg/mL
Experiment A (3 hours incubation, with metabolic activation system): 0.044, 0.13, 0.39, 1.18 mg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: RPMI 1640 medium with L-glutamine (Gibco BRL Life Technologies, UK, article number 21875-034).
For each experiment a stock solution of the test substance was prepared in RPMI medium. To achieve the intended final concentrations of the test substance, the further test substance solutions (one for each tested concentration) were then obtained by diluting these stock solutions with RPMI medium.
All preparations were made freshly before adding them to the cell cultures.

- Justification for choice of solvent/vehicle: Common vehicle.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
see below for details

Migrated to IUCLID6: and cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Cultures were kept at 37 °C and 5% CO2 for ca. 48 hours before further processing
- Exposure duration: 3 hours (one experiment without and one experiment with metabolic activation)
- Expression time (cells in growth medium): 15 hours (plus 2 hours with spindle inhibitor added)
- Fixation time (start of exposure up to fixation or harvest of cells): 20 hours

SPINDLE INHIBITOR (cytogenetic assays): 0.1 mL of Colcemid (Gibco BRL Life Technologies, UK, article no 15210-057, 10 µg/mL in balanced salt solution) per culture.
STAIN (for cytogenetic assays): 10 minutes with Giemsa solution (10% v/v in a buffer of 0.067 M KH2PO4 and 0.067 M Na2HPO4 x 2 H2O in deionised water), rinsed in tap-water and then in deionised water.

NUMBER OF REPLICATIONS: 2 per culture

NUMBER OF CELLS EVALUATED:
Mitotic indices: 2000 lymphocytes per culture
Chromosome aberrations: 100 metaphases per culture (i.e. 200 per concentration, apart from cultures with obviously high numbers of metaphases with aberrations)

DETERMINATION OF CYTOTOXICITY
- Method: The mitotic indices were determined by counting a total of 2000 lymphocytes per cell culture and by recording the number of lymphocytes in any stage of mitosis. This number was then expressed as percentage of mitotic lymphocytes.

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
Only well spread cells with 44 to 47 chromosomes, polyploid and endoreduplicated cells were acceptable for analysis. Structural aberrations were scored according to well defined (and reported) criteria.
Statistics:
The Chi2-Test (two-tailed, p=0.05) was used for the comparison between the negative control and the test substance cultures. If the results were positive, comparisons were made separately between the negative control and each concentration. If conditions for the Chi2-Test were not met, Fisher’s Exact Test was used. Chi2-Test or Fisher’s Exact Test were also used for the comparison between the negative and the positive controls.

Results and discussion

Test results
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- None

COMPARISON WITH HISTORICAL CONTROL DATA:
Yes, historical control data (on negative and positive controls) given in the report. The positive control substances caused in each experiment clearly higher numbers of metaphases with structural aberrations (statistically significant) than found in the negative controls, without as well as with the use of a metabolic activation system, thus demonstrating that the test systems were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
see "any other information on results incl. tables"
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Mitotic index

At the highest concentration of 1.18 mg/mL marked cytotoxicity was noted regardless whether a metabolic activation system was used or not.

Experiment without a metabolic activation system, 3 hours of incubation:

Test substance concentration
(mg/mL)

0.044

0.13

0.39

1.18

Mitotic index
(% of respective negative control)

114.5

115.3

96.5

54.0

Bold figures: These concentrations were analysed

 

Experiment with a metabolic activation system, 3 hours of incubation:

Test substance concentration
(mg/mL)

0.044

0.13

0.39

1.18

Mitotic index
(% of respective negative control)

103.0

73.4

82.0

23.1

Bold figures: These concentrations were analysed

Numerical aberrations

No statistically significant differences in the number of metaphases with numerical aberrations were noted in any experiment at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.

 

Gaps

In the experiment without a metabolic activation system the number of gaps was statistically significantly higher at all test substance concentrations compared to the corresponding negative control and there was no clear concentration-response relationship.

In the experiment with a metabolic activation system no statistically significant increase in the number of gaps was noted at any concentration analysed compared to the concurrent negative controls.

Structural aberrations

In the experiment without a metabolic activation system the number of metaphases with structural aberrations was statistically significantly higher at all test substance concentrations compared to the corresponding negative control and there was a clear concentration-response relationship. The numbers were also clearly beyond the data of historical negative controls.

In the experiment with a metabolic activation system the number of metaphases with structural aberrations was not statistically significantly increased at any concentration analysed compared to the concurrent negative controls. Nevertheless, at the highest concentration evaluated (0.39 mg/mL) the number of metaphases with structural aberrations was beyond the range of the historical negative controls and within the range of historical positive controls. The numbers of the other test substance concentrations (0.044 mg/mL and 0.13 mg/mL) were within the range of historical negative controls.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
positive

The overall results of the study indicate clastogenic properties of the test substance at a treatment length of 3 hours without the use of a metabolic activation system. There is also relevant evidence that the test substance has mutagenic properties at a treatment length of 3 hours with the use of a metabolic activation system.
Executive summary:

Objective

The study was performed to determine possible mutagenic properties of"3-hydroxy-2-(hydroxymethyl)-2-methylpropionaldehyde"(“Novaltan PF”) by means of anin vitromammalian chromosome aberration test in human lymphocytes, according to Regulation (EC) No 440/2008 method B.10. and to OECD Guideline 473.

Methods

A total of two experiments was performed and analysed: one of them without and one with the use of a metabolic activation system (liver microsomes from Aroclor 1254 induced rats, with a co-factor solution). A concentration range betweennominal 0.044 mg/mL and 1.18 mg/mL mg test substance per mL medium was tested. 1.18 mg/mL as the highest test substance concentration is 0.01M and was chosen in accordance with the EC directive and the OECD guideline.

Primary lymphocyte cultures were established from whole blood freshly obtained from one male donor. After 48 hours of incubation, the test substance was added. In all experiments, regardless whether a metabolic activation system was used or not, the test substance was washed out three hours later and the cultures were cultivated for another 17 hours. Colcemid was added two hours before the end of the cultivation period, and then cells were fixed and slides prepared.

The test substance was dissolved in RPMI medium and for each concentration of the test substance two cultures were established. One negative control (RPMI medium) and one positive control (methanesulfonic acid methyl ester, MMS, for cultures without metabolic activation system and cyclophosphamide, CP, for cultures with a metabolic activation system) were set up concurrently in each experiment.

The concentrations of the test substancein the experiments performed were 0.044, 0.13, 0.49 and 1.18 mg/mL.

In general, apart from cultures with obviously high numbers of metaphases with aberrations, 100 metaphases per culture (i.e. 200 per concentration) were analysed for structural and numerical chromosomal aberrations. The slides were coded before analysis. The mitotic indices were calculated from 2000 lymphocytes per culture for an assessment of cytotoxicity.

Results

Cytotoxicity

At the highest concentration of 1.18 mg/mL marked cytotoxicity was noted regardless whether a metabolic activation system was used or not.

Experiment without a metabolic activation system, 3 hours of incubation:

Test substance concentration
(mg/mL)

0.044

0.13

0.39

1.18

Mitotic index
(% of respective negative control)

114.5

115.3

96.5

54.0

Bold figures: These concentrations were analysed

 

Experiment with a metabolic activation system, 3 hours of incubation:

Test substance concentration
(mg/mL)

0.044

0.13

0.39

1.18

Mitotic index
(% of respective negative control)

103.0

73.4

82.0

23.1

Bold figures: These concentrations were analysed

Numerical aberrations

No statistically significant differences in the number of metaphases with numerical aberrations were noted in any experiment at any concentration analysed compared to the concurrent negative controls, regardless whether a metabolic activation system was used or not.

Gaps

In the experiment without a metabolic activation system the number of gaps was statistically significantly higher at all test substance concentrations compared to the corresponding negative control and there was no clear concentration-response relationship.

In the experiment with a metabolic activation system no statistically significant increase in the number of gaps was noted at any concentration analysed compared to the concurrent negative controls.

Structural aberrations

In the experiment without a metabolic activation system the number of metaphases with structural aberrations was statistically significantly higher at all test substance concentrations compared to the corresponding negative control and there was a clear concentration-response relationship. The numbers were also clearly beyond the data of historical negative controls.

In the experiment with a metabolic activation system the number of metaphases with structural aberrations was not statistically significantly increased at any concentration analysed compared to the concurrent negative controls. Nevertheless, at the highest concentration evaluated (0.39 mg/mL) the number of metaphases with structural aberrations was beyond the range of the historical negative controls and within the range of historical positive controls. The numbers of the other test substance concentrations (0.044 mg/mL and 0.13 mg/mL) were within the range of historical negative controls.

As experiment A without metabolism gave positive results the performance of an experiment B with without a metabolic activation system with an extended treatment length of 20 hours, equivalent to about 1.5 normal cell cycle lengths was not required. Due to the clear positive result in the experiment without metabolism and due to the relevant evidence that the test substance did also induce structural chromosomal aberrations in the experiment with metabolism a confirmation of the experiment with metabolism was not considered to be necessary.

Positive controls

The positive control substances caused in each experiment clearly higher numbers of metaphases with structural aberrations (statistically significant) than found in the negative controls, without as well as with the use of a metabolic activation system, thus demonstrating that the test systems were adequate and that the metabolic activation system functioned properly.

 

Conclusion

In each experiment the test substance caused marked cytotoxic effects (reduction to about 50% or less, compared to concurrent negative controls) at the highest concentration tested. The highest tested concentration was 0.01M and thus in accordance with the EC directive and the OECD guideline.

Under the conditions of this study the test substance induced structural chromosomal aberrations in cultured human lymphocytes after a treatment length of 3 hours in the absence of a metabolic activation system. The conclusion is based on a statistically significant increase of metaphases with structural aberrations at all test substance concentrations (1.18, 0.39 and 0.13 mg/mL) with a clear concentration-response relationship and on the fact that these figures were far beyond the range of historical negative controls. Gaps are not necessarily an indicator for clastogenic effects, but at all test substance concentrations the number of gaps was also statistically increased.

There was also relevant evidence that the test substance did induce structural chromosomal aberrations in cultured human lymphocytes in the experiment with metabolism, although there was no statistically significant increase compared to the controls. The conclusion is mainly based on an important increase of metaphases with structural aberrations at the highest test substance concentration (0.39 mg/mL) and on the fact that this figure was beyond the range of historical negative controls and within the range of historical positive controls.

The overall results of the study indicate clastogenic properties of the test substance at a treatment length of 3 hours without the use of a metabolic activation system. There is also relevant evidence that the test substance has mutagenic properties at a treatment length of 3 hours with the use of a metabolic activation system.