Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2000-04-06 to 2000-08-31
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2000
Report date:
2000

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
July 1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
July 1999
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: pellets

Method

Target gene:
Not applicable
Species / strain
Species / strain / cell type:
mammalian cell line, other: human lymphocytes
Details on mammalian cell type (if applicable):
Stimulated cultured human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemical classes.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9-mix of Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
Dose range finding test:
10, 33, 100, 133, 1000 µg/mL with and without S9-mix;

Chromosome aberrations:
Without S9-mix: 275, 300, and 530 µg lithium hydroxide/mL culture medium (24 h treatment, 24 h fixation time);
350, 375 and 400 µg lithium hydroxide/mL culture medium (48 h treatment, 48 h fixation time),
With S9-mix: 400, 425 and 450 µg lithium hydroxide/mL culture medium (3 h treatment, 48 h fixation time);
Vehicle / solvent:
DMSO
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Remarks:
MMC-C
Positive control substance:
mitomycin C
Remarks:
without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Remarks:
CP
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix
Details on test system and experimental conditions:
Cytogenetic assay:
Lithium hydroxide was tested in the absence and presence of 1.8 % (v/v) S9-fraction in duplicate in two independent experiments.

Experiment 1:
Lymphocyte cultures (0.4 mL blood of a healthy male donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed in duplicate to selected doses of lithium hydroxide for 3 h in the absence and presence of S9-mix.
After 3 h treatment, the cells exposed to lithium hydroxide were rinsed once with 5 mL of HBSS and incubated in 5 mL of culture medium for another 20-22 h (24 h fixation time).
Based on the mitotic index of the dose range finding test and the first cytogenetic assay appropriate dose levels were selected for the second cytogenetic assay. The independent repeat was performed with the following modification of experimental conditions.

Experiment 2:
Lymphocyte cultures (0.4 mL blood of a healthy male donor was added to 5 mL or 4.8 mL culture medium, without and with metabolic activation respectively and 0.1 mL (9 mg/mL) Phytohaemagglutinin) were cultured for 48 h and thereafter exposed in duplicate to selected doses of lithium hydroxide for 3 h in the absence and presence of S9-mix.
After 3 h treatment, the cells exposed to lithium hydroxide in the presence of S9-mix were rinsed once with 5 mL of HBSS and incubated in 5 mL of culture medium for another 44-46 h (48 h fixation time).
The cells which were treated for 24 and 48 h in the absence of S9-mix were not rinsed after treatment but were worked up immediately after 24 h and 48 h (24 h and 48 h fixation time).

Chromosome preparation:
During the last 3 h of the culture period, cell division was arrested by the addition of the spindle inhibitor colchicine (0.5 µg/mL medium). Thereafter the cell cultures were centrifuged for 5 min at 1300 rpm (150 g) and the supernatant was removed. Cells in the remaining cell pellet were swollen by a 5 min treatment with hypotonic 0.56 % (w/v) potassium chloride solution at 37 °C. After hypotonic treatment, cells were fixed with 3 changes of methanol:acetic acid fixative (3:1 v/v).

Preparation of slides:
Fixed cells were dropped onto cleaned slides which were immersed for 24 h in a 1:1 mixture of 96 % (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the study identification number and group number. Two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5 % (v/v) Giemsa solution in tap water.
Thereafter the slides were rinsed in tap-water and allowed to dry. The dry slides were cleared by dipping them in xylene before they were embedded in MicroMount and mounted with a coverslip.

Mitotic index/dose selection for scoring the cytogenetic assay:
The mitotic index of each culture was determined by counting the number of metaphases per 1000 cells. At least three analysable concentrations were used. Chromosomes of metaphase spreads were analysed of those cultures with an inhibition of the mitotic index of about 50 % or greater whereas the mitotic index of the lowest dose level was approximately the same as the mitotic index of the solvent control. Also cultures treated with an intermediate dose were examined for chromosome aberrations.

Analysis of slides for chromosome aberrations:
To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. An adhesive label with study identification number and code was stuck over the marked slide. At least 100 metaphase chromosome spreads per culture were examined by light microscopy for chromosome aberrations. In case the number of aberrant cells, gaps excluded, was >= 25 in 50 metaphases no more metaphases were examined. Only metaphases containing 46 chromosomes were analysed. The number of cells with aberrations and the number of aberrations were calculated.
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
b) a statistically significant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) in crease in the number of cells with chromosome aberrations.

The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, inclusive or exclusive gaps) for each treatment group was compared to that of the solvent control using Chi-square statistics:

X*2 = (N-1)x(ad-bc)*2/(a+b)(c+d)(a+c)(b+d)

where b = the total number of aberrant cells in the control cultures,
d = the total number of non aberrant cells in the control cultures,
n0 = the total number of cells scored in the control cultures,
a = the total number of aberrant cells in treated cultures to be compared with the control,
c = the total number of non aberrant cells in treated cultures to be compared with the control,
n1 = the total number of cells scored in the treated cultures,
N = sum of n= and n1

If P [ X*2 > (N-)x(ad-bc)*2/(a+b)(c+d)(a+c)(b+d)] (two-tailed) is small (P < 0.05) the hypothesis that the incidence of cells with chromosome aberrations is the same for both the treated and the solvent group is rejected and the number of aberrant cells in the test group is considered to be significantly different from the control group at the 95 % confidence level.

Results and discussion

Test results
Key result
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
at 1000 µg/mL
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Dose range finding test:
Lithium hydroxide precipitated in the culture medium at a concentration of 1000 µg/mL, therefore a concentration of 1000 µg/mL was used as the highest concentration of lithium hydroxide.
In the dose range finding test, blood cultures were treated with 10, 33, 100, 333 and 1000 µg lithium hydroxide per mL culture medium with and without S9-mix.
The pH of a concentration of 1000 mg lithium hydroxide/mL was 11.83 (compared to 8.15 in the solvent control).

Cytogenetic assay:
Based on the results of the dose range finding test the following dose levels were selected for the cytogenetic assay:

Experiment 1A:
Without S9-mix: 100, 180, 333, 420 and 560 µg lithium hydroxide/mL culture medium (3 h treatment time, 24 h fixation time);
With S9-mix: 100, 333, 420 and 560 µg lithium hydroxide/mL culture medium (3h treatment, 24 h fixation time);
Lithium hydroxide precipitated in the culture medium at a concentration of 560 µg/mL, therefore a concentration of 560 µg/mL was used as the highest concentration of lithium hydroxide in the first cytogenetic assay.
Since the highest dose level of 560 µg lithium hydroxide/mL was too cytotoxic to the cells (mitotic index of 21 % both in the absence and in the presence of S9-mix) and no dose level resulting in a mitotic index of 50 % could be selected in both the absence and presence of S9-mix, an additional experiment was performed with the following dose levels:

Experiment 1B:
With and without S9-mix: 300, 350, 400, 450, 500 and 550 µg lithium hydroxide/mL culture medium (3 h treatment, 24 h fixation time);
Because of the high cytotoxicity in cultures treated with 350 µg/mL lithium hydroxide and upwards in the presence and absence of S9-mix, the test was not used for evaluation but a third experiment was performed with the following dose levels: see Experiment C

Experiment 1C:
With and without S9-mix: 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 µg lithium hydroxide/mL culture medium (3 h treatment, 24 h fixation time).

Despite the narrow concentration range used, the mitotic index of cultures treated with 375 and 400 µg/mL lithium hydroxide (without S9-mix) drastically decreased from 128 % to 0 %. In the presence of S9-mix, cytotoxicity was observed at a concentration of 375 µg/mL lithium hydroxide and upwards.
The pH of the concentrations 275, 300, 325, 350, 375, 400 and 425 µg/mL was 9.61, 9.69, 9.66, 9.68, 9.66, 9.80 and 10.19, respectively. Possibly these high pH values also play a role in the cytotoxicity of lithium hydroxide.

Since it was not possible to determine a concentration which caused the appropriate 50 % inhibition of the mitotic index, the following doses were selected for scoring of chromosome aberrations:
From experiment 1A:
With and without S9-mix: 333, 420 and 560 µg lithium hydroxide/mL (3 h treatment, 24 h fixation time);

From experiment 1C:
Without S9-mix: 325, 350, and 375 µg lithium hydroxide/mL culture medium (3 h treatment time, 24 h fixation time);
With S9-mix: 325, 350, 375, and 400 µg lithium hydroxide/mL culture medium (3 h treatment, 24 h fixation time);
For cultures with S9-mix four doses were selected, since only one of the duplicate cultures contained scorable metaphases at concentrations of 375 and 400 µg/mL lithium hydroxide.

Based on the results of the dose range finding test and experiments 1A, 1B and 1C the following dose levels were selected to perform an independent repeat:

Experiment 2:
Without S9-mix: 275, 300, 325, 350, 375, 400 and 425 µg lithium hydroxide/mL culture medium (24 and 48 h treatment time, 24 and 48 h fixation time);
With S9-mix: 350, 375, 400, 425, 450, 475, 500 and 525 µg lithium hydroxide/mL culture medium (3 h treatment time, 48 h fixation time).
Based on these observations the following doses were selected for scoring of chromosome aberrations:
Without S9-mix: 275, 300 and 350 µg lithium hydroxide/mL culture medium (24 h treatment time, 24 h fixation time);
350, 375 and 400 µg lithium hydroxide/mL culture medium (48 h treatment time, 48 h fixation time),
With S9-mix: 400, 425 and 450 µg lithium hydroxide/mL culture medium (3 h treatment time, 48 h fixation time).

Evaluation of the results:
The ability of lithium hydroxide to introduce chromosome aberrations in human peripheral lymphocytes was investigated. The test was carried out in duplicate in three independent experiments.
The number of cells with chromosome aberrations found in the solvent control cultures were within the laboratory historical control data range {min = 0, max = 5 (mean = 0.8, standard deviation = 1.0) aberrant cells per 100 metaphases in the absence of S9-mix; gaps excluded and min = 0 max = 5 (mean = 0.8, standard deviation = 0.9) aberrant cells per 100 metaphases in the presence of S9-mix, gaps excluded}.
The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Experiments 1A and 1C:
Due to the steepness of the dose response curve for cytotoxicity of lithium hydroxide it was not possible to determine the number of chromosomal aberrations at a mitotic index of 50 %. Therefore, chromosome aberrations were scored from two independent experiments (experiment 1A and 1C) at different concentrations. As a result of extreme cytotoxicity, only 102 and 103 metaphases could be scored in the absence and presence of S9-mix, respectively, in experiment 1A at a concentration of 560 µg/mL lithium hydroxide. At the other concentrations tested, 200 metaphases were scored per concentration. In experiment 1C in the presence of S9-mix at the highest concentrations of 375 and 400 µg/mL only one of the two duplicate cultures could be scored due to extreme cytotoxicity.
Both in the absence and presence of S9-mix lithium hydroxide did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations in both experiments 1A and 1C.

Experiment 2:
In the absence of S9-mix, at the 24 hours continuous treatment time, lithium hydroxide induced statistically significant increases in the number of cells with chromosome aberrations at the lowest tested concentration of 275 µg/mL (only when gaps were included) and at the highest cytotoxic concentration of 350 µg/mL both when gaps were included and excluded. At the intermediate concentration of 300 µg/mL lithium hydroxide did not induce a statistically significant increase in the number of cells with chromosome aberrations.
Since the increase of chromosome aberrations at 275 µg/mL was observed only when gaps were included and furthermore the increase was within the historical control data range, it was not considered biologically relevant.

Scoring of the additional 200 metaphases at the concentration of 350 µg/mL lithium hydroxide verified the statistically significant increase. However, the observed increase was within or just on the border of our historical control data range (min = 0, max = 5 aberrant cells per 100 metaphases, gaps excluded), and was observed at a very toxic concentration. In addition, higher concentrations tested at the prolonged treatment time of 48 hours in the absence of metabolic activation did not induce significant increases in the number of cells with chromosome aberrations. Furthermore, the irregular toxicity profile and the non-physiological test conditions (pH > 9) may be considered as confounding factors. Therefore, the observed increase in the number of aberrant cells at the concentration of 350 µg/mL is considered not biologically relevant.

At the continuous treatment time of 48 hours exposure of cells to 350, 375 or 400 µg/mL lithium hydroxide did not induce a significant increase in the number of cells with chromosome aberrations.

In the presence of S9-mix, lithium hydroxide did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations.

Conclusion:
Finally, it is concluded that this test is to be considered valid and that lithium hydroxide is not clastogenic under the experimental conditions of this test.

Applicant's summary and conclusion

Conclusions:
The effect of lithium hydroxide on the induction of chromosome aberrations in culture peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix) was investigated.
It is concluded that this test should be considered valid and that lithium hydroxide is not clastogenic under the experimental conditions of this test.

Executive summary:

A Chromosome Aberration test with lithium nitrate was not available. Consequently, read-across was applied using study results obtained from lithium hydroxide as it is a characteristically similar compound.

The effect of lithium hydroxide on the induction of chromosome aberrations in culture peripheral human lymphocytes in the presence and absence of a metabolic activation system (Aroclor-1254 induced rat liver S9-mix) was investigated according to OECD Guideline 473 and EU method B.10.  In the absence of S9-mix lithium hydroxide was tested up to 560 µg/mL for a 3 h treatment time with a 24 h fixation time in experiment 1A and up to 375 µg/mL in experiment 1C. In the second experiment lithium hydroxide was tested up to 350 µg/mL for a 24 hours continuous treatment time and up to 400 µg/mL for a 48 hours continuous treatment time.  In the presence of 1.8 % (v/v) S9-fraction lithium hydroxide was tested up to 560 µg/mL for a 3 h treatment time with a 24 h fixation time in experiment 1A and up to 400 µg/mL in experiment 1C. In the second experiment Lithium Hydroxide was tested up to 450 µg/mL for a 3 h treatment time with a 48 h fixation time.  Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.  Experiment 1A and 1C: Both in the absence and presence of S9-mix lithium hydroxide did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations in both experiments 1A and 1C.  Experiment 2: In the absence of S9-mix, at the 24 hours continuous treatment time, lithium hydroxide induced statistically significant increases in the number of cells with chromosome aberrations at the lowest tested concentration of 275 µg/mL (only when gaps were included) and at the highest cytotoxic concentration of 350 µg/mL both when gaps were included and excluded. At the intermediate concentration of 300 µg/mL lithium hydroxide did not induce a statistically significant increase in the number of cells with chromosome aberrations. Since the increase of chromosome aberrations at 275 µg/mL was observed only when gaps were included and furthermore the increase was within the historical control data range and revealed no dose-response-relationship, the increase was not considered biologically relevant.  Scoring of the additional 200 metaphases at the concentration of 350 µg/mL lithium hydroxide verified the statistically significant increase. However, the observed increase within or just on the border of the historical control data range (min = 0, max = 5 aberrant cells per 100 metaphases, gaps excluded), and is observed at a very toxic concentration. In addition, higher concentrations tested at the prolonged treatment time of 48 hours in the absence of metabolic activation did not induce significant increases in the number of cells with chromosome aberrations. Furthermore, the irregular toxicity profile and the non-physiological test conditions (pH > 9) may be considered confounding factors. Therefore, the observed increase in the number of aberrant cells at the concentration of 350 µg/mL is considered not biologically relevant. At the continuous treatment time of 48 hours exposure of cells to 350, 375 or 400 µg/mL Lithium Hydroxide did not induce a significant increase in the number of cells with chromosome aberrations.  In the presence of S9-mix, lithium hydroxide did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations.  Finally, it is concluded that this test is considered valid and that lithium hydroxide is not clastogenic under the experimental conditions of this test. (NOTOX, 2000)