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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:
Experimental phases: 4 August 2005 to 05 October 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of the relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2006

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
Details on test material:
- Name of test material (as cited in study report): NLP#10 Teracol*5903
- Physical state: Viscous liquid
- Analytical purity: > 99 %
- Lot/batch No.: SG25080735
- Storage condition of test material: Room temperature under nitrogen over silica gel in the dark

Method

Target gene:
Not applicable.
Species / strain
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitabilityThe volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Experiment 1:
Without S9-mix: 0*, 156.25, 312.5, 625*, 1250*, 2500*, 3750 (* Dose levels selected for metaphase analysis)
With S9-mix: 0*, 156.25, 312.5, 625, 1250*, 1875*, 2500* (* Dose levels selected for metaphase analysis)
Experiment 2:
Without S9-mix: 0*, 156.25, 312.5*, 625*, 1250*, 1875, 2500 (* Dose levels selected for metaphase analysis)
With S9-mix: 0*, 156.25, 312.5, 625*, 1250*, 1875, 2500* (* Dose levels selected for metaphase analysis)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was selected as the solvent because the test material was readily soluble in it at the required concentrations.
Controlsopen allclose all
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: (CP)
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: (MMC)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period:
48 hrs

- Exposure duration:
Experiment 1 - 4 hrs with and without S9. Experiment 2 - 24 hrs without S9, 4 hrs with S9.

- Expression time (cells in growth medium): 20 hrs for 4 hrs exposure.

- Selection time (if incubation with a selection agent): Not applicable.

- Fixation time (start of exposure up to fixation or harvest of cells): 24 hrs.

SPINDLE INHIBITOR (cytogenetic assays): Demecolcine

STAIN (for cytogenetic assays): When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.

NUMBER OF REPLICATIONS: Duplicate cultures

NUMBER OF CELLS EVALUATED: 100/culture


DETERMINATION OF CYTOTOXICITY
- Method:
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 was approximately 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 simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing. Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy: Frequency of polyploid cells
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 dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
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
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Refer to information on results and attached tables.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
EXAMPLE
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: There was no significant change in pH when the test material was dosed into media.
- Effects of osmolality: The osmalality did not increase by more than 50 mOsm.
- Evaporation from medium: Not applicable.
- Water solubility: Not applicable, test material suspended in MEM
- Precipitation:
Premlinary toxictiy test: No precipitate up to 5000 ug/mL
Experiment 1: No precipitate
Experiment 2: No precipitate
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test

The dose range for the Preliminary Toxicity Test was 19.53 to 5000 µg/mL. The maximum dose was the maximum recommended dose level. No precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure period, up to 5000 ug/mL in all exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 2500 µg/ml in both the 4(20) hours exposure groups in the presence and absence of metabolic activation. In the 24-hour continuous exposure group the maximum level with metaphases was 1250 ug/mL. The mitotic index data are presented in Table 1 (see attached background material). The test material demonstrated evidence of toxicity in all of the exposure groups although the toxicity curve was more pronounced in the 24-hour continuous exposure group. The dose ranges for the main experiments were based on toxicity of the test material and intermediate dose levels used to try and achieve an ideal 50 % reduction in mitotic index.

COMPARISON WITH HISTORICAL CONTROL DATA: All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
EXPERIMENT 1:
The qualitative assessment of the slides determined that the test material was not as toxic in the with metabolic activation group as that observed in the preliminary toxicity test and that there were sufficient scorable metaphases present at the maximum dose level of test material used, 2500 ug/mL, in the presence of metabolic activation. In the absence of metabolic activation there was a steep toxicity curve and there were no scorable metaphases at the dose level 3750 ug/mL. The maximum dose level with scorable metaphases was 2500 ug/mL.

The mitotic index data are given in Table 2 (see attached background material). They confirm the qualitative observations in that there was a dose-related inhibition of mitotic index observed. An ideal 50 % mitotic inhibition was not achieved in either exposure group but 31 % inhibition was achieved at 2500 ug/mL in the absence of metabolic activation. In the presence of metabolic activation there was a 43 % mitotic inhibition at 2500 ug/mL. Adequate toxicity was therefore considered to have been achieved in both exposure groups.

EXPERIMENT 2:
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at the maximum test material dose level of 2500 µg/mL in the presence of metabolic activation. In the absence of metabolic activation the maximum test material dose level with metaphases suitable for scoring was 1250 µg/mL.

The mitotic index data are given in Table 3 (see attached background material). They confirm the qualitative observations in that there was a dose-related inhibition of mitotic index was observed and that 55 % mitotic inhibition was acheived at 2500 ug/mL in the presence of metabolic activation. In the absence of metabolic activation there was 60 % mitotic inhibition at 1250 ug/mL. Therefore an ideal toxicity was achieved in both exposure groups.

CHROMOSOME ABERRATION RESULTS
EXPERIMENT 1:
The chromosome aberration data are given in Table 4 and Table 5 (see attached background material). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test material did not induce any toxicologically significant increases in the frequency of cells with aberrations either in the absence or presence of metabolic activation. However there was an increase in the frequency of cells with aberrations in the with metabolic activation group at 2500 ug/mL only. Additional metaphases were scored at this dose level to confirm the conclusion. It was observed that 2.8 % of cells had aberrations excluding gaps which is within the laboratory's historical range for with metabolic activation groups. It should be noted that there were several cells with multiple aberrations.

The polyploid cell frequency data are given in Table 8 (see attached background material).. The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups

EXPERIMENT 2:
The chromosome aberration data are given in Table 6 and Table 7 (see attached background material). All of the vehicle control cultures had frequencies of cells with chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.

The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or presence of metabolic activation. The increase in aberrant cells at 2500 ug/mL in the presence of metabolic activation in Experiment 1 was not reproduced in Experiment 2 and was therefore considered to be spurious and of no toxicological significance.

The polyploid cell frequency data are given in Table 8 (see attached background material). The test material did not induce a statistically significant increase in the numbers of polyploid cells at any dose level in either of the exposure groups.

Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

For the tables of results mentioned above, please refer to the attached background material section:

Table 1: Mitotic Index - Preliminary Toxicity Test

Table 2: Mitotic Index - Experiment 1

Table 3: Mitotic Index - Experiment 2

Table 4: Results of Chromosome Aberration Test - Experiment 1 Without Metabolic Activation (S9)

Table 5: Results of Chromosome Aberration Test - Experiment 1 With Metabolic Activation (S9)

Table 6: Results of Chromosome Aberration Test - Experiment 2 Without Metabolic Activation (S9)

Table 7: Results of Chromosome Aberration Test - Experiment 2 With Metabolic Activation (S9)

Table 8:  Mean Frequency of Polyploid Cells (%)

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.