Dilithium azelate

Genetic toxicity: in vitro

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:

02 February 2006 and 23 August 2006.med between 12 May 2008 and 28 August 2008.

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted 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. The study report was conclusive, done to a valid guideline and the study was conducted under GLP conditions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids C18-(unsaturated) lithium salts

Cas Number:

502962-81-4

IUPAC Name:

Fatty acids C18-(unsaturated) lithium salts

Test material form:

solid

Details on test material:

- Batch number: Not reported
- Expiry date: Not reported

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:

Preliminary toxicity test:
0, 19.5, 39, 78.1, 156.25, 312.5, 625, 1250, 2500 and 5000 µg/ml

Chromosome Aberration Test - Experiment 1: The final concentration of fatty acids C18 (unsaturated) lithium salts (µg/ml) in the controls and treatments of the 4(20)-hour without S9 group were 0*, 20, 40, 80, 160*, 240*, 320*, MMC 0.4* and for the 4(20)-hour with S9 group were 0*, 40, 80*, 160*, 240*, 320, 480, CP 5* (* = Dose levels selected for metaphase analysis).
Chromosome Aberration Test - Experiment 2: The final concentration of fatty acids C18 (unsaturated) lithium salts (µg/ml) in the controls and treatments of the 24-hour without S9 group were 0*, 20, 40, 80*, 120*, 160*, 240*, MMC 0.2* and for the 4(20)-hour with S9 group were 0*, 40, 80*, 160*, 240*, 320, 480, CP 5*(* = Dose levels selected for metaphase analysis).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not stated in report

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

0 µg/ml

Negative solvent / vehicle controls:

yes

Remarks:

solvent treatment groups were used as the vehicle control

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

In the presence of S9 at 5 µg/ml in both experiments

Controls 2

Untreated negative controls:

yes

Remarks:

0 µg/ml

Negative solvent / vehicle controls:

yes

Remarks:

solvent treatment groups were used as the vehicle control

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

In the absence of S9 used at 0.4 and 0.2 µg/ml in both experiments.

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.

SELECTION AGENT (mutation assays): - not applicable

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:

Exhibited a very steep dose-response curve

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

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:
Preliminary toxicity test: A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 625-1250 µg/ml, in the 4(20)-hour pulse exposure groups and at and above 78.1 µg/ml in the continuous exposure group.

Chromosome aberration Test - Experiments 1 and 2: Precipitate observations taken in the Preliminary Toxicity Test were considered to be representative for the study.

RANGE-FINDING/SCREENING STUDIES:
Preliminary Toxicity Test
Haemolysis of the red blood cells was observed at and above 312.5 µg/ml in all three exposure groups. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present at up to156.25 and 312.5 µg/ml in the 4(20)-hour exposures in the absence and presence of metabolic activation (S9) respectively. The maximum dose with metaphases present in the 24-hour continuous exposure was 156.25 µg/ml. The test material induced clear evidence of toxicity in all of the exposure groups and the toxic dose response curve was very steep.
The selection of the maximum dose level was based on toxicity for the all of the exposure groups.


COMPARISON WITH HISTORICAL CONTROL DATA:
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.


ADDITIONAL INFORMATION ON CYTOTOXICITY:

EXPERIMENT 1: The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were scorable metaphases present at 320 µg/ml in the absence of metabolic activation (S9). In the presence of S9 the maximum dose level of the test material with scorable metaphases was 240 µg/ml.


EXPERIMENT 2:
The qualitative assessment of the slides determined that there were scorable metaphases present at the maximum test material dose level of 240 µg/ml in both the absence and presence of S9.

Remarks on result:

other: all strains/cell types tested

Any other information on results incl. tables

Chromosome Aberration Test - Experiment 1

Mitotic index data confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 38% mitotic inhibition was achieved at 320 µg/ml in the absence of S9. In the presence of S9 only 18% mitotic inhibition was achieved at 240 µg/ml but complete mitotic inhibition was observed at 320 and 480 µg/ml.

The maximum dose level selected for metaphase analysis was the maximum dose level in each case where scorable metaphase cells were evident.

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 aberrations either in the absence or presence of metabolic activation.

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.

 Chromosome Aberration Test - Experiment 2

Mitotic index data confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed and that 63% mitotic inhibition was achieved at 240 µg/ml in the absence of S9. In the presence of S9 22% mitotic inhibition was achieved at 240 µg/ml and total inhibition at the next dose level of 320 µg/ml. The maximum dose level selected for metaphase analysis was 240 µg/ml in both exposure groups.

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 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.

Applicant's summary and conclusion

Conclusions:

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.

Executive summary:

Introduction. 

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 2000/32/EC. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods. 

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at three or four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

Results. 

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 did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments. The test material was toxic and exhibited a very steep dose-response curve. The dose range used included a dose level that induced approximately 50% mitotic inhibition or was the maximum sub-toxic dose level.

Conclusion. 

The test material was considered to be non-clastogenic to human lymphocytes in vitro.