Trilithium hexafluoroaluminate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The mutagenic activity of Lithium cryolite was investigated in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay according to the OECD Testing Guideline 471 and under GLP.

Lithium cryolite was tested with four histidine-requiring strains of S. typhimurium (TA1535, TA1537, TA98 and TA100) and with a tryptophan-requiring strain if E. coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

Lithium cryolite did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

The negative control values were within the laboratory historical control data ranges.

The strain-specific positive control values were at least three times the concurrent vehicle control group mean indicating that the test conditions were adequate.

Based on the results of this study it is concluded that Lithium cryolite is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

The ability of Lithium cryolite to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiment) was investigated in a study performed according to the OECD Testing Guideline 473 and under GLP.

In the first cytogenetic assay, Lithium cryolite was tested up to 300 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Lithium cryolite precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Lithium cryolite was tested up to 300 μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 470 μg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. In the presence of S9-mix Lithium cryolite was tested up to 500 μg/ml for a 3 h exposure time with a 48 h fixation time. Lithium cryolite precipitated in the culture medium at this dose level.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. 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.

In the first cytogenetic assay, Lithium cryolite did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix.

In the second cytogenetic assay, at the 3 h exposure period, Lithium cryolite did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the presence of S9-mix.

In the absence of S9-mix, both at the 24 and 48 h prolonged exposure periods, Lithium cryolite induced a statistically significant, dose dependent increase in the number of cells with chromosome aberrations both when gaps were included and excluded. Since the type of aberrations included exchanges (48 h exposure period), observed when gaps were included and excluded and moreover the number of cells with chromosome aberrations was high above the historical control data range, the increases were considered biologically relevant.

No effects of Lithium cryolite on the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Lithium cryolite does not induce numerical chromosome aberrations. It was observed that only at the prolonged exposure period of 48 h there was an increase in the number of polyploid cells. This might indicate that Lithium cryolite does disturb mitotic processes and cell cycle progression under the experimental conditions described.

Finally, it is concluded that this test is valid and that Lithium cryolite is clastogenic in human lymphocytes under the experimental conditions described in this report. The clastogenic activity is confined to prolonged incubations of 24 and 48 hours.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 23 September 2013 (Study Plan completion) to 05 November 2013 (Quality Assurance statement)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes were routinely prepared from adult male Wistar rats, which were obtained from Charles River, Sulzfeld, Germany

Test concentrations with justification for top dose:

Dose range finding test with tester strain WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate.
Mutation assay: 100, 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

Preparation of test solutions started with solutions of 50 mg/ml dilutions in dimethyl sulfoxide (DMSO, SeccoSolv, Merck, Darmstadt, Germany) applying treatment with ultrasonic waves resulting in a homogeneous white suspension. The lower test concentrations were prepared by subsequent dilutions in DMSO. At concentrations of 0.33 mg/ml and higher Lithium cryolite formed a suspension in DMSO.
At concentrations of 0.1 mg/ml and lower the test substance was fully soluble.
Test substance concentrations were used within 2 hours after preparation.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

The vehicle of the test substance, which was DMSO.

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48h +/-4h in the dark at 37 °C +/- 1°C
- Expression time (cells in growth medium): cells were counted just after the exposure

NUMBER OF REPLICATIONS: triplicate

NUMBER OF CELLS EVALUATED: 0.1 mL of fresh bacterial culture (10E9 cells/mL)

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of Lithium cryolite, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were examined.

OTHER EXAMINATIONS:
precipitation of the test substance

Evaluation criteria:

The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually and evidence of test article precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: yes for two strains, see details on results

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES and FIRST MUTATION EXPERIMENT:
Lithium cryolite was tested in tester strain WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537, TA98 and TA100 in the absence and presence of S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate.

Precipitate: Precipitation of Lithium cryolite on the plates was observed at the start of the incubation period at concentrations of 1000 µg/plate and upwards but not at the end of the incubation period.
Toxicity: Cytotoxicity was only observed in the tester strains TA1535 in the presence S9-mix and TA1537 in absence of S9-mix, where an extreme reduction of the revertant colonies was observed at the test substance concentration of 5000 µg/plate. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all other tester strains in the absence and presence of S9-mix.
Mutagenicity : In the first mutation experiment, no increase in the number of revertants was observed upon treatment with Lithium cryolite under all conditions tested.

MUTATION EXPERIMENT 2:
Based on the results of the dose range finding test, Lithium cryolite was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in two mutation assays. The first mutation experiment was performed with the strains TA1535, TA1537, TA100 and TA98 and the second mutation experiment was performed with the strains TA1535, TA1537, TA98, TA100 and WP2uvrA.

Precipitate: Precipitation of Lithium cryolite on the plates was observed at the start of the incubation period at the concentration of 1000 µg/plate and upwards and no precipitate was observed at the end of the incubation period.

Toxicity : Cytotoxicity was observed in tester strain TA1535 in the presence of S9-mix, TA98 in absence and presence of S9-mix and TA100 in the presence of S9-mix, where an extreme reduction of the revertant colonies or no revertant colonies were observed at the test substance concentration of 5000 µg/plate. There was no reduction in the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all other tester strains in the absence and presence of S9-mix.
Mutagenicity : In the second mutation experiment, no increase in the number of revertants was observed upon treatment with Lithium cryolite under all conditions tested.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

List of deviations

Protocol deviations:

1. Selection of an adequate range of doses for the mutation experiments was based on a dose range finding test with tester strain WP2uvrA (in the absence and presence of S9-mix). Tester strain TA100 was tested in the first mutation experiment.

Evaluation: The performing of the dose range finding test with tester strain WP2uvrA and the testing of tester strain TA100 in the first mutation experiment had no effect on the results of the study.

2. In the second experiment, the positive control substances of the following tester strains: TA1535 (absence of S9-mix) and TA100 (presence of S9-mix) and in the first experiment WP2uvrA (absence and presence of S9-mix) showed responses (mean plate count) which were not within the laboratory historical range.

Evaluation: These values were more than 3 times greater than the concurrent solvent control values, therefore this deviation in the mean plate count of the positive controls had no effect on the results of the study.

The study integrity was not adversely affected by the deviations.

 

Standard operating procedures deviations:

Any deviations from standard operating procedures were evaluated and filed in the study file. There were no deviations from standard operating procedures that affected the integrity of the study.

Conclusions:

Based on the results of this study it is concluded that Lithium cryolite is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The mutagenic activity of Lithium cryolite was investigated in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay according to the OECD Testing Guideline 471 and under GLP.

 

Lithium cryolite was tested with four histidine-requiring strains of S. typhimurium (TA1535, TA1537, TA98 and TA100) and with a tryptophan-requiring strain if E. coli (WP2uvrA). The test was performed in two independent experiments in the presence and absence of S9-mix (rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone).

 

At concentrations of 0.33 mg/ml and higher Lithium cryolite formed a suspension in dimethyl sulfoxide whereas at 0.1 mg/ml and lower it was fully soluble.

 

In the dose range finding test, Lithium cryolite was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix in tester strain WP2uvrA. Lithium cryolite did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. Results of this dose range finding test were reported as part of the first experiment of the mutation assay.

 

Based on the results of the dose range finding test, Lithium cryolite was tested in the first mutation assay at a concentration range of 100 to 5000 µg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537, TA98 and TA100. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in the tester strains TA1537 in the absence of S9-mix and TA1535 in the presence of S9-mix at the highest tested concentration.

In an independent repeat of the assay with additional parameters, Lithium cryolite was tested at the same concentration range as the first assay in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Cytotoxicity, as evidenced by a decrease in the number of revertants, was observed in the tester strains TA1535 and TA100 in the presence of S9-mix and TA98 in the absence and presence of S9-mix at the highest tested concentration.

Lithium cryolite did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

The negative control values were within the laboratory historical control data ranges.

 

The strain-specific positive control values were at least three times the concurrent vehicle control group mean indicating that the test conditions were adequate.

 

Based on the results of this study it is concluded that Lithium cryolite is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 26 February 2014 (Study Plan completion) to 26 March 2014 (signature of the statement of GLP compliance)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

see "any other information on materials and methods incl. tables"

Test concentrations with justification for top dose:

Dose range finding test :
At a concentration of 333 μg/mL the test item precipitated in the culture medium.
- at the 3 h exposure time, with and without S9-mix: 3, 10, 33, 100 and 333 μg test item/mL culture medium
- at the 24 h and 48 h continuous exposure time, without S9-mix: 3, 10, 33, 100, 333, 1000 and 1620 μg test item/mL culture medium. Test item was tested beyond the limit of solubility to obtain adequate toxicity data.

First cytogenetic assay:
Based on the results of the dose range finding test the following dose levels were selected for the first cytogenetic assay:
- at 3 h exposure time, 24 h fixation time, with and without S9-mix: 3, 10, 30, 75, 100, 2001 and 3001 μg/mL culture medium.

Second cytogenetic assay:
Depending on the experimental conditions: from 3 to 1000 µg of test item / mL culture medium. See the section “additional information on results” for details.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO

Untreated negative controls:

other: solvent control

Negative solvent / vehicle controls:

yes

Remarks:

vehicle for the test substance : dimethyl sulfoxide

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Remarks:

Solvent fro positive controls: Hanks’ Balanced Salt Solution (HBSS) (Invitrogen Corporation, Breda, The Netherlands), without calcium and magnesium.

Details on test system and experimental conditions:

BACKGROUND OF THE TEST SYSTEM:
Whole blood samples obtained from healthy male subjects were treated with an anti-coagulant (heparin) and cultured in the presence of a mitogen (phytohaemagglutinin). These stThe stimulated lymphocytes were exposed to Lithium cryolite both in the absence and presence of a metabolic activation system (S9-mix).imulated human lymphocytes were used because they are sensitive indicators of clastogenic activity of a broad range of chemicals.
At predetermined intervals after exposure of the stimulated human lymphocytes to Lithium cryolite, cell division was arrested in the metaphase stage of the cell cycle by addition of the metaphase-arresting chemical colchicine. Cells were harvested, stained and metaphase cells were analysed for the presence of structural chromosome aberrations such as breaks, gaps, minutes, dicentrics and exchange figures. Results from cultures treated with Lithium cryolite were compared with control (vehicle) treated cultures.
Chromosome aberrations were generally evaluated in the first post-exposure mitosis (i.e. 24 hours after exposure). However, since the appearance of the first post-exposure mitosis could be considerably delayed due to toxic insult to the cells, cells were also harvested 48 hours after exposure to cover the interval in which maximum aberration frequency was expected.

TEST SUBSTANCE PREPARATION
Lithium cryolite was suspended in dimethyl sulfoxide of spectroscopic quality. The stock solution was treated with ultrasonic waves to obtain a homogeneous suspension. Lithium cryolite concentrations were used within 2.5 hours after preparation.
The final concentration of the solvent in the culture medium was 1.0% (v/v).

POSITIVE CONTROLS:
Without metabolic activation (+S9-mix):Mitomycin C (MMC-C; CAS no. 50-07-7, Sigma, Zwijndrecht, The Netherlands) was used as a direct acting mutagen at a final concentration of 0.5 and 0.75 μg/ml for a 3 h exposure period, 0.2 and 0.3 μg/ml for a 24 h exposure period and 0.1 and 0.15 μg/ml for a 48 h exposure period.
With metabolic activation (+S9-mix): Cyclophosphamide (CP; CAS no. 50-18-0. Baxter B.V., Utrecht, The Netherlands) was used as an indirect acting mutagen, requiring metabolic activation, at a final concentration of 10 μg/ml for a 3 h exposure period (24 h fixation time).
Solvent for positive controls: Hanks’ Balanced Salt Solution (HBSS) (Invitrogen Corporation, Breda, The Netherlands), without calcium and magnesium.
All reference stock solutions were stored in aliquots at ≤-15°C in the dark. These solutions were thawed immediately before use.

TEST SYSTEM:
Cultured peripheral human lymphocytes were used as test system. Blood was collected from healthy adult, non-smoking, male volunteers. The Average Generation Time (AGT) of the cells and the age of the donor at the time the AGT was determined to be between 12.7 and 13.5h.

CELL CULTURE
- Blood samples : Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 μg/ml respectively) and 30 U/ml heparin.
- Lymphocyte cultures: Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin was added.
- Environmental conditions: All incubations were carried out in a controlled environment in the dark, in which optimal conditions were a humid atmosphere of 80 - 100% (actual range 45 - 91%), containing 5.0 ± 0.5% CO2 in air, at a temperature of 37.0 ± 1.0°C (actual range 34.8 - 37.6°C). Temperature and humidity were continuously monitored throughout the experiment. The CO2 percentage was monitored once on each working day. Temporary deviations from the temperature (in the range of 34.8 - 36.0°C), humidity (with a maximum of 30%) and CO2 percentage (with a maximum of 1%) occurred that were caused by opening and closing of the incubator door, but the time of these deviations did not exceed 2 hours. Based on laboratory historical data these deviations are considered not to affect the study integrity.
The temporary deviations from the humidity with a minimum of 45% are explained in section "any other information on results".

METABOLIC ACTIVATION SYSTEM:
- Preparation of S9-fraction: The animals were housed at WIL Research Europe in a special room under standard laboratory conditions and allowed to acclimatise for at least 5 days. The rats were orally dosed at three consecutive days with a suspension of phenobarbital (80 mg/kg body weight) and ß-naphthoflavone (100 mg/kg body weight) in corn oil (they were denied access to food for 3 to 4 hours preceding each dosing). One day after the final exposure (24 h), the rats were sedated using oxygen/carbon dioxide and then killed by decapitation. The rats received a limited quantity of food during the night before sacrifice. The livers of the rats were removed aseptically, and washed in cold (0°C), sterile 0.1 M sodium phosphate buffer (pH 7.4) containing 0.1 mM Na2-EDTA. The livers were minced in a blender and homogenised in 3 volumes of phosphate buffer with a Potter homogeniser. The homogenate was centrifuged for 15 min at 9000 g. The supernatant (S9) was transferred into sterile ampules, which were stored in liquid nitrogen (-196°C) for a maximum of 1 year. The S9 batch was characterised in a bacterial reverse mutation assay in Salmonella typhimurium tester strain TA98 with the mutagens Benzo-(a)-pyrene (Sigma) and 2-aminoanthracene (Sigma) at concentrations of 5 μg/plate and 1 μg/plate, respectively. These mutagens require metabolic activation for exerting their mutagenic effects.
- Preparation of S9-mix: S9-mix was prepared immediately before use and kept on ice. S9-mix components contained per ml: 1.63 mg MgCl2.6H2O; 2.46 mg KCl; 1.7 mg glucose-6-phosphate; 3.4 mg NADP; 4 μmol HEPES.The solution was filter (0.22 μm)-sterilized. To 0.5 ml S9-mix components 0.5 ml S9-fraction was added (50% (v/v) S9-fraction) to complete the S9-mix.
Metabolic activation was achieved by adding 0.2 ml S9-mix to 5.3 ml of a lymphocyte culture (containing 4.8 ml culture medium, 0.4 ml blood and 0.1 ml (9 mg/ml) phytohaemagglutinin). The concentration of the S9-fraction in the exposure medium was 1.8% (v/v).

STUDY DESIGN
- Dose range finding test
Lymphocytes (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 to selected doses of Lithium cryolite for 3 h, 24 h and 48 h in the absence of S9-mix or for 3 h in the presence of S9-mix. A negative control was included at each exposure time.
The highest tested concentration was determined by the solubility of Lithium cryolite in the culture medium at the 3 h exposure time. At the 24 and 48 h exposure time, Lithium cryolite was tested beyond the limit of solubility to obtain adequate toxicity data.
After 3 h exposure to Lithium cryolite in the absence or presence of S9-mix, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and cells were rinsed with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20 - 22 h (24 h fixation time). The cells that were exposed for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately (24 h and 48 h fixation time).
Cytotoxicity of Lithium cryolite in the lymphocyte cultures was determined using the mitotic index.
- Cytogenetic assay: Lithium cryolite was tested in the absence and presence of 1.8% (v/v) S9-fraction in duplicate in two independent experiments. To be able to select appropriate dose levels for scoring of chromosome aberrations several repeat assays had to be performed (cytogenetic assays 2A to 2D).
First cytogenetic assay:
Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of Lithium cryolite for 3 h in the absence and presence of S9-mix. After 3 h exposure, the cells were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 ml HBSS. After a second centrifugation step, HBSS was removed and cells were resuspended in 5 ml culture medium and incubated for another 20 - 22 h (24 h fixation time). Appropriate negative and positive controls were included in the first cytogenetic assay.
Based on the mitotic index of the dose range finding test and the first cytogenetic assay and on the solubility of Lithium cryolite in the culture medium appropriate dose levels were selected for the second cytogenetic assay. The independent repeat was performed with the following modifications of experimental conditions.
Second cytogenetic assay:
Lymphocytes were cultured for 48 ± 2 h and thereafter exposed in duplicate to selected doses of Lithium cryolite for 24 h and 48 h in the absence of S9-mix and for 3 h in the presence of S9-mix. After 3 h exposure, the cells exposed to Lithium cryolite in the presence of S9-mix were separated from the exposure medium by centrifugation (5 min, 365 g). The supernatant was removed and the cells were rinsed once with 5 ml of HBSS and incubated in 5 ml culture medium for another 44 - 46 h (48 h fixation time). The cells that were treated for 24 h and 48 h in the absence of S9-mix were not rinsed after exposure but were fixed immediately after 24 h and 48 h (24 h and 48 h fixation time).
Appropriate negative and positive controls were included in the second cytogenetic assay.
- Chromosome preparation:
During the last 2.5 - 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 365 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).
- Preperation of the slides:
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. At least 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 Sörensenbuffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded in a 1:10 mixture of xylene/pertex and mounted with a coverslip in an automated coverslipper.
- Mitotic index/dose selection for scoring of the cytogenetic assay:
The mitotic index of each culture was determined by counting the number of metaphases from at least 1000 cells (with a maximum deviation of 5%). At least three analysable concentrations were used for scoring of the cytogenetic assay. Chromosomes of metaphase spreads were analysed from those cultures with an inhibition of the mitotic index of about 50% or above 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. If dose related cytotoxicity was observed, the highest concentration analysed at the 24 and 48 h continuous exposure times was based on toxicity irrespective of the solubility of Lithium cryolite in the culture medium. However, the extent of precipitation may not interfere with the scoring of chromosome aberrations.
- Analysis of slides for chromosome aberrations
To prevent bias, all slides were randomly coded before examination of chromosome aberrations and scored. One hundred 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 ± 2 centromeres (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, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant 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, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria are 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, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.
If P (one-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 control 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.

Key result

Species / strain:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

at 3 µg/mL: no significative effect, at 100 µg/mL: significative effect with P<0.01, at 300 µg/mL significative effect with P<0.001.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 3 µg/mL: 25% of reduction in MI, at 100 µg/mL: 31% of reduction in MI, at 300 µg/mL: 69 % of reduction in MI.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

at 200 µg/mL: no significative effect, at 370 µg/mL: significative effect with P<0.001, at 470 µg/mL significative effect with P<0.001.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 200 µg/mL: 5% of reduction in MI, at 370 µg/mL: 39% of reduction in MI, at 470 µg/mL: 58 % of reduction in MI.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: peripheral human lymphocytes

Metabolic activation:

with

Genotoxicity:

negative

Remarks:

at 300, 400 and 500 µg/mL: no significative effect.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

at 300 µg/mL: 11% of reduction in MI, at 400 µg/mL: 21% of reduction in MI, at 500 µg/mL: 4 % of reduction in MI.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

The ability of Lithium cryolite to induce chromosome aberrations in human peripheral lymphocytes was investigated in two independent experiments. The highest concentration analysed was selected based on the solubility of the test substance in the culture medium (3 h exposure period) or on toxicity, inhibition of the mitotic index of about 50% or greater (24 and 48 h exposure period).

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The number of polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. 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.

In the first cytogenetic assay, 8% and 6% of reduction in the mitotic index was observed at the highest tested concentration (i.e. 300 µg/mL) in the absence and presence of S9-mix respectively. The doses of 75, 100 and 300 μg/mL were selected for scoring of chromosome aberrations. Lithium cryolite did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix.

To obtain more information about the possible clastogenicity of Lithium cryolite, a second cytogenetic assay was performed in which human lymphocytes were continuously exposed to Lithium cryolite in the absence of S9-mix for 24 or 48 hours. In the presence of S9-mix, cells were fixed after 48 hours following a 3 hour exposure to Lithium cryolite. The following dose levels were selected for the second cytogenetic assay:
Without S9-mix : 3, 10, 30, 75, 100, 200 and 300 μg/ml culture medium (24 and 48 h exposure time, 24 and 48 h fixation time).
With S9-mix : 3, 10, 30, 75, 100, 200 and 300 μg/ml culture medium (3 h exposure time, 48 h fixation time).

In the absence of S9-mix (24h exposure time), 59% of reduction in the mitotic index was observed at the concentration of 200 µg/mL. In the absence of S9-mix (48 h exposure time) no appropriate dose levels could be selected for scoring of chromosome aberrations since at the concentration of 200 μg/ml not enough cytotoxicity was observed (36%), whereas the next higher concentration of 300 μg/ml was too toxic for scoring (75%). Since no precipitate was observed at the highest dose level of 300 μg/ml and no severe toxicity was observed, no concentrations could be selected for the scoring of chromosome aberrations for the 3 hour exposure period. The experiment was repeated in cytogenetic assay 2A:
Without S9-mix : 10, 100, 200, 225, 250, 275 and 300 μg/ml culture medium (48 h exposure time, 48 h fixation time).
With S9-mix : 10, 100, 200, 300, 400 and 5001 μg/ml culture medium (3 h exposure time, 48 h fixation time).

In the presence of S9-mix (3h exposure time), 11%, 21% and 4% of reduction in the mitotic index was observed at the three highest tested concentrations (i.e. 300, 400 and 500 µg/mL respectively).
In the absence of S9-mix (48 h exposure time) no appropriate dose levels could be selected for scoring of chromosome aberrations since at the highest concentration of 300 μg/ml not enough cytotoxicity was observed (33%). The experiment was repeated in cytogenetic assay 2B:
Without S9-mix : 10, 200, 225, 250, 275, 300, 325, 350, 375 and 4001 μg/ml culture medium (48 h exposure time, 48 h fixation time).

No appropriate dose levels could be selected for scoring of chromosome aberrations since at the highest concentration of 400 μg/ml not enough cytotoxicity was observed (13%). The experiment was repeated in cytogenetic assay 2C:
Without S9-mix : 10, 225, 300, 450, 500, 600, 700, 800, 900 and 1000 μg/ml culture medium (48 h exposure time, 48 h fixation time).

No precipitate was observed up to 500 μg/ml.
No appropriate dose levels could be selected for scoring of chromosome aberrations since at the concentration of 300 μg/ml not enough cytotoxicity was observed (28%), whereas the next higher concentration of 450 μg/ml was too toxic for scoring (79%). The experiment was repeated in cytogenetic assay 2D:
Without S9-mix: 10, 200, 235, 270, 300, 335, 3701, 4001, 4351, 4701, 5001, 5351, 5701 and 6001 μg/ml culture medium (48 h exposure time, 48 h fixation time). 58% of reduction in the mitotic index was observed at the concentrations of 435 and 470 µg/mL.

Based on these observations the following doses were selected for scoring of chromosome aberrations:
Without S9-mix : 3, 100 and 300 μg/ml culture medium (24 h exposure time, 24 h fixation time).
200, 370 and 470 μg/ml culture medium (48 h exposure time, 48 h fixation time).
With S9-mix : 300, 400 and 500 μg/ml culture medium (3 h exposure time, 48 h fixation time).

In this second cytogenetic assay, at the 3 h exposure period, Lithium cryolite did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the presence of S9-mix.
In the absence of S9-mix, both at the 24 and 48 h prolonged exposure periods, Lithium cryolite induced a statistically significant, dose dependent increase in the number of cells with chromosome aberrations both when gaps were included and excluded. Since the type of aberrations included exchanges (48 h exposure period), observed when gaps were included and excluded and moreover the number of cells with chromosome aberrations was higher than the historical control data range, the increases were considered biologically relevant.

No effects of Lithium cryolite on the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Lithium cryolite does not induce numerical chromosome aberrations. It was observed that only at the prolonged exposure period of 48 h there was an increase in the number of polyploid cells. This might indicate that Lithium cryolite does disturb mitotic processes and cell cycle progression under the experimental conditions described.

Remarks on result:

other: other: 24h exposure time, 24h fixation time

Remarks:

Migrated from field 'Test system'.

List of protocol deviations:

1. In the second cytogenetic assay and cytogenetic assays 2B and 2C the humidity was several times below the range as mentioned in the protocol (80-100%) with a minimum of 45%, for a maximum of 2.5 hours.

Evaluation: These deviations were only for a short time and were caused by opening and closing of the incubator door during handling of the cell cultures. The number of cells with chromosome aberrations found in the negative control cultures of the second cytogenetic assay was within the laboratory historical negative control data range. Cytogenetic assays 2B and 2C were not used for the scoring of chromosomal aberrations. Therefore, these deviations of the humidity have no effect on the results of the study.

2. The experimental end date was 26 February 2014, which is not within the period mentioned in the time schedule of the protocol.

Evaluation: Due to repeat experiments the experimental end date was not met. Changing the experimental end date has no influence on the study integrity.

The study integrity was not adversely affected by the deviations.

List of standard operating procedures deviations:

Any deviations from standard operating procedures were evaluated and filed in the study file. There were no deviations from standard operating procedures that affected the integrity of the study.

Conclusions:

Lithium cryolite is clastogenic in human lymphocytes with prolonged incubations of 24 and 48 hours.

Executive summary:

The ability of Lithium cryolite to induce chromosome aberrations in cultured peripheral human lymphocytes (with repeat experiment) was investigated in a study performed according to the OECD Testing Guideline 473 and under GLP.

In the first cytogenetic assay, Lithium cryolite was tested up to 300 μg/ml for a 3 h exposure time with a 24 h fixation time in the absence and presence of 1.8% (v/v) S9-fraction. Lithium cryolite precipitated in the culture medium at this dose level.

In the second cytogenetic assay, Lithium cryolite was tested up to 300 μg/ml for a 24 h continuous exposure time with a 24 h fixation time and up to 470 μg/ml for a 48 h continuous exposure time with a 48 h fixation time in the absence of S9-mix. Appropriate toxicity was reached at these dose levels. In the presence of S9-mix Lithium cryolite was tested up to 500 μg/ml for a 3 h exposure time with a 48 h fixation time. Lithium cryolite precipitated in the culture medium at this dose level.

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. 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.

In the first cytogenetic assay, Lithium cryolite did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix.

In the second cytogenetic assay, at the 3 h exposure period, Lithium cryolite did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the presence of S9-mix.

In the absence of S9-mix, both at the 24 and 48 h prolonged exposure periods, Lithium cryolite induced a statistically significant, dose dependent increase in the number of cells with chromosome aberrations both when gaps were included and excluded. Since the type of aberrations included exchanges (48 h exposure period), observed when gaps were included and excluded and moreover the number of cells with chromosome aberrations was high above the historical control data range, the increases were considered biologically relevant.

No effects of Lithium cryolite on the number of cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that Lithium cryolite does not induce numerical chromosome aberrations. It was observed that only at the prolonged exposure period of 48 h there was an increase in the number of polyploid cells. This might indicate that Lithium cryolite does disturb mitotic processes and cell cycle progression under the experimental conditions described.

Finally, it is concluded that this test is valid and that Lithium cryolite is clastogenic in human lymphocytes under the experimental conditions described in this report. The clastogenic activity is confined to prolonged incubations of 24 and 48 hours.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Lithium cryolite does not induce gene mutations in a bacterial in vitro system. An in vitro test on induction of chromosomal aberrations (human lymphocytes) is positive. As indicated in the REACH Regulation (Annex VIII), an in vivo mutagenicity test shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII. In addition, in the REACH guidance (Chapter R.7A – Endpoint Specific Guidance) it is stated that an in vivo test should be initiated as soon as possible following a positive result in an in vitro mammalian cell mutagenicity test. Based on the positive results in the in-vitro chromosome aberration test, it was decided to perform the in vivo micronucleus test with lithium cryolite.

Lithium cryolite was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect in developing erythrocytes (polychromatic erythrocytes) in the bone marrow. The test was performed according to the OECD Testing Guideline 474 and under the GLP.

Based on the results of the dose range finding study, in the main study male animals were dosed once via oral gavage with vehicle or with 2000, 1000 and 500 mg/kg body weight. A positive control group was dosed once via oral gavage with 40 mg cyclophosphamide (CP) per kg body weight. In total 6 treatment groups were used, each consisting of 5 animals.

Clinical signs of toxicity were limited to the high dose group and included a hunched posture (3 animals).

Bone marrow of the groups treated with Lithium cryolite was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with Lithium cryolite compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met.

The groups that were treated with Lithium cryolite showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

It is concluded that Lithium cryolite is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 2000 mg/kg under the experimental conditions described in this report. 

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 30 July 2014 (Study Plan completion) to 19 September 2014 (Quality Assurance statement)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: The animals were provided by Charles River, Sulzfeld, Germany.
- Age at study initiation: Young adult animals were selected (6-8 weeks old at the start of treatment).
- Number of animals : The total number of animals used in the dose range finding study was 8 and in the main study 33. In the micronucleus main study 5 male mice were treated per sampling time in each treatment group.
- Weight at study initiation: The body weights of the mice at the start of the treatment were within 20% of the sex mean. The mean body weights were 36.2 ± 2.0 g and the range was 32 – 40 g.
- Assigned to test groups randomly: yes
- Fasting period before study: Feed was withheld 3-4 h prior to dosing until administration of Lithium cryolite
- Housing: The animals were group housed (maximum 5 animals per sex per cage) in labelled Macrolon cages (type MIII height: 18 cm) containing sterilised sawdust as bedding material (Lignocel S 8-15). A shelter (disposable paper corner home) and paper bedding (Enviro-dri) was provided as cage-enrichment.
- Diet : The animals had free access to pelleted rodent diet
- Water : The animals had free access to tap-water.
- Acclimation period: at least 5 days before the start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
A controlled environment was maintained in the room with optimal conditions of approximately 10 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 21.7 - 22.5°C), a relative humidity of 40 - 70% (actual range: 38 - 64%) and a 12 hour light/12 hour dark cycle. Due to e.g. cleaning procedures, temporary deviations from the minimum level for humidity (with max. 2%) occurred. Based on laboratory historical data these deviations are considered not to affect the study integrity.

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: propylene glycol
- Justification for choice of solvent/vehicle: The vehicle was selected based on trial formulations performed at WIL Research Europe and on test substance data supplied by the sponsor.
- Concentration of test material in vehicle: no data
- Amount of vehicle : no data
- Type and concentration of dispersant aid (if powder): none

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Lithium cryolite was suspended in propylene glycol. Lithium cryolite concentrations were treated with ultra-sonic waves to obtain a homogeneous suspension. Lithium cryolite concentrations were dosed within 4 hours after preparation.

The mice received an oral intubation of a maximum tolerated (high), an intermediate and a low dose of Lithium cryolite. The route of administration was selected taking into account the possible route of human exposure during manufacture, handling and use. Feed was withheld 3-4 h prior to dosing until administration of Lithium cryolite.
The dosing volume was 5 ml/kg body weight.

Lithium cryolite concentrations were prepared on the day of administration.

Duration of treatment / exposure:

Range finding study: single dose, the observation period after dosing was one to 4 days. During this period mortality and physical condition were recorded at least once a day.

Main study : single dose. Three dose levels were used at the first sampling time. At the second sampling time only the highest dose was used. The first sampling time was 24 h after treatment and the second sampling time was 48 h after treatment.

Frequency of treatment:

Single dose.

Remarks:

Doses / Concentrations:
Dose range finding study : 1500 and 2000 mg/kg
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
Main study : 500, 1000 and 2000 mg/kg
Basis:
nominal conc.

No. of animals per sex per dose:

Range finding study :
at 1500 mg/kg : 1 male and 1 female
at 2000 mg/kg: 3 males and 3 females

Main study:
Based on the results of the dose range finding test a full study with one sex was performed. Since there were no substantial differences in toxicity between sexes, only male animals were used in the main study.
At least five male mice were used per sampling time in each treatment group. The animals were dosed once.

Control animals:

yes, concurrent vehicle

Positive control(s):

The positive control used in the micronucleus test was cyclophosphamide (CP; CAS no. 50-18-0) dissolved in physiological saline dosed as a single oral intubation of 40 mg/kg body weight.
The route and frequency of administration of the positive control were the same as those of the test substance.

Details of tissue and slide preparation:

ISOLATION OF BONE MARROW:
Bone marrow of the groups treated with Lithium cryolite was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative control group was isolated 24 hours after dosing and bone marrow of the positive control group was isolated 48 hours after dosing. The animals were
sacrificed by cervical dislocation. Both femurs were removed and freed of blood and muscles. Both ends of the bone were shortened until a small opening to the marrow canal became visible. The bone was flushed with approximately 2 ml of fetal calf serum. The cell suspension was collected and centrifuged at 216 g for 5 min.

PREPARATION OF BONE MARROW SMEARS
The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the remaining serum. A drop of the cell suspension was placed on the end of a clean slide, which was previously immersed 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 the animal number. The drop was spread by moving a clean slide with round-whetted sides at an angle of approximately 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.

STAINING OF THE BONE MARROW SMEARS
The slides were automatically stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer. This staining is based on Giemsa. The dry slides were automatically embedded in a 1:10 mixture of xylene /pertex and mounted with a coverslip in an automated coverslipper.

ANALYSIS OF THE BONE MARROW SMEARS FOR MICRONUCLEI
To prevent bias, all slides were randomly coded before examination. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000 x. The number of
micronucleated polychromatic erythrocytes was counted in at least 2000 polychromatic erythrocytes (with a maximum deviation of 5%). The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating at least the first 1000 erythrocytes at the same time.
Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated. In the group of the animals treated with the positive control substance only four animals were used for the scoring of micronucleated polychromatic erythrocytes.

Evaluation criteria:

Equivocal results should be clarified by further testing using modification of experimental conditions.

A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.
A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.

The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1500 and 2000 mg/kg bw
- Clinical signs of toxicity in test animals:
One male and one female animal were dosed with 1500 mg/kg bw. Within 1 hour after dosing both animals had a hunched posture.
In total three male and three female animals were dosed with 2000 mg/kg bw. No signs of toxicity were observed in one male and one female animal within 2 days after dosing. Within 2 hours after dosing 2 female and 2 male animals had a hunched posture. One male and female animal had recovered from the treatment within 21 hours after dosing and one female animal had recovered from the treatment within 47 hours. Within 71 hours after dosing one of the recovered female animals was lethargic, had a rough coat and a hunched posture. One male animal died within 47 hours after dosing.

RESULTS OF DEFINITIVE STUDY
- Doses: 500, 1500 and 2000 mg/kg bw
- Mortality and toxic signs:
The animals of the groups treated with 500 and 1000 mg /kg bw and the animals of the negative and positive control groups showed no treatment related clinical signs of toxicity or mortality.
Three animals dosed with 2000 mg/kg bw had a hunched posture within 2 hours after dosing. Within 20 hours after dosing all animals were recovered or showed no reaction to treatment.
- Micronucleated polychromatic erythrocytes:
The mean number of micronucleated polychromatic erythrocytes scored in Lithium cryolite treated groups were compared with the corresponding vehicle control group.
No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of Lithium cryolite treated animals compared to the vehicle treated animals.
The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range.
Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, the acceptability criteria of the test were met.
- Ratio polychromatic to normochromatic erythrocytes:
The animals of the groups, which were treated with Lithium cryolite showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects of this test substance on the erythropoiesis. The animals of the groups treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Table 1: Mean number of micronucleated polychromatic erythrocytes and ratio of polychromatic/normochromatic erythrocytes

Group	Treatment	Dose (mg/kg bw)	Sampling time (h)	Number of micronucleated polychromatic erythrocytes (mean +/- SD)^(1,3)	Ratio polychromatic/ normochromatic erythrocytes (mean +/- SD)^(1,4)
A	Vehicle control	0	24	2.0 ± 1.0	0.94 ± 0.06
B	Lithium cryolite	2000	24	2.6 ± 1.1	0.83 ± 0.09
C	Lithium cryolite	2000	48	2.4 ± 1.1	0.92 ± 0.04
D	Lithium cryolite	1000	24	2.0 ± 0.7	0.90 ± 0.05
E	Lithium cryolite	500	24	2.6 ± 0.9	0.83 ± 0.13
F	CP	40	48	19.5 ± 2.5^(2,5)	0.59 ± 0.08^(2)
Vehicle control =propylene glycol
CP = Cyclophosphamide.
(1)	Five animals per treatment group.
(2)	Four animals.
(3)	At least 2000 polychromatic erythrocytes were evaluated with a maximum deviation of 5%.
(4)	The ratio was determined from at least the first 1000 erythrocytes counted.
(5)	Significantly different from corresponding control group (Wilcoxon Rank Sum Test, P = 0.01).

Table 2: INDIVIDUAL DATA

Individual data (males) (group A       : oral intubation of the vehicle) (group B & C: oral intubation of Lithium cryolite at 2000 mg/kg body weight) (group D       : oral intubation of Lithium cryolite at 1000 mg/kg body weight) (group E       : oral intubation of Lithium cryolite at 500 mg/kg body weight) (group F        : oral intubation of cyclophosphamide at 40 mg/kg body weight)
	Group	Animal number	Number of micronucleated polychromatic erythrocytes	Number of polychromatic erythrocytes scored for micronuclei	Number of poly-chromatic erythrocytes^(1)	Number of normo-chromatic erythrocytes^(1)	Ratio polychromatic/ normochromatic erythrocytes^(1)
	A	1	3	2013	498	503	0.99
	A	2	3	2005	469	549	0.85
	A	3	2	2009	516	558	0.92
	A	4	1	2004	515	511	1.01
	A	5	1	2021	510	555	0.92
	B	6	4	2079	428	607	0.71
	B	7	1	2052	459	559	0.82
	B	8	3	2021	557	581	0.96
	B	9	3	2001	459	547	0.84
	B	10	2	2059	471	563	0.84
	C	11	2	2001	474	541	0.88
	C	12	2	2029	502	530	0.95
	C	13	3	2009	492	511	0.96
	C	14	4	2017	475	537	0.88
	C	15	1	2013	506	546	0.93
	D	16	3	2007	468	547	0.86
	D	17	1	2093	459	547	0.84
	D	18	2	2003	487	520	0.94
	D	19	2	2017	519	561	0.93
	D	20	2	2015	490	527	0.93
	E	21	4	2043	507	542	0.94
	E	22	3	2005	472	532	0.89
	E	23	2	2019	492	559	0.88
	E	24	2	2051	469	571	0.82
	E	25	2	2035	383	633	0.61
	F	26	23	2011	390	631	0.62
	F	27	19	2009	407	598	0.68
	F	28	19	2007	382	668	0.57
	F	29	17	2001	332	677	0.49
	F	30^(2)

(1) The ratio was determined from the first 1000 erythrocytes counted.

(2) Slide was not judged, hardly any cells present

Table 3 : statistics

Wilcoxon Rank Sum Test.
Number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes; treatment/control comparison.
Group	Treatment	Dose	Sex	P-value	Decision at 95%
		mg/kg bw		(one-sided)	confidence level
F	cyclophosphamide	40	males	0.01	significant

Validity criteria

A micronucleus test is considered acceptable if it meets the following criteria:

1) The incidence of micronucleated polychromatic erythrocytes in the positive control animals should be above the historical control data range. => fulfilled, see table 1 2) The positive control substance induced a statistically significant (Wilcoxon Rank Sum, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes => fulfilled, see table 3 3) The incidence of micronucleated polychromatic erythrocytes in the control animals should reasonably be within the laboratory historical control data range => fulfilled, 2 +/- 1 in the control, 1.1 +/-1.2 in the historical control. List of protocol deviations : In the positive control group, the slides of only four animals were used for the determination of the number of micronucleated polychromatic erythrocytes instead of five animals as specified in the protocol. Evaluation: The quality of the slides of one animal was poor. Hardly any cells were present. Since four analysable slides could be used for this positive control group and the incidence of micronucleated polychromatic erythrocytes was above the historical control data range (10-fold increase), this deviation had no effect on the results of the study. The study integrity was not adversely affected by the deviation. List of standard operating procedures deviations :There were no deviations from standard operating procedures that affected the integrity of the study.

Conclusions:

Lithium cryolite is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a
dose of 2000 mg/kg under the experimental conditions described in this report.

Executive summary:

Lithium cryolite was tested in the Micronucleus Test in mice, to evaluate its genotoxic effect in developing erythrocytes (polychromatic erythrocytes) in the bone marrow. The test was performed according to the OECD Testing Guideline 474 and under the GLP.

In the dose range finding study 1 male and 1 female were dosed once via oral gavage with 1500 mg Lithium cryolite per kg body weight. Since the animals showed only a hunched posture directly after treatment, an additional 3 males and 3 females were dosed once via oral gavage with 2000 mg Lithium cryolite per kg body weight. Within 2 hours after dosing 2 female and 2 male animals had a hunched posture. One male and female animal had recovered from the treatment within 21 hours after dosing and one female animal had recovered from the treatment within 47 hours. Within 71 hours after dosing one of the recovered female animals was lethargic, had a rough coat and a hunched posture. One male animal died within 47 hours after dosing.

In the main study male animals were dosed once via oral gavage with vehicle or with 2000, 1000 and 500 mg/kg body weight. A positive control group was dosed once via oral gavage with 40 mg cyclophosphamide (CP) per kg body weight. In total 6 treatment groups were used, each consisting of 5 animals.

Clinical signs of toxicity were limited to the high dose group and included a hunched posture (3 animals).

Bone marrow of the groups treated with Lithium cryolite was sampled 24 or 48 (highest dose only) hours after dosing. Bone marrow of the negative and positive control groups was harvested 24 and 48 hours after dosing, respectively.

No increase in the mean frequency of micronucleated polychromatic erythrocytes was observed in the bone marrow of animals treated with Lithium cryolite compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical vehicle control data range.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, both criteria for an acceptable assay were met.

The groups that were treated with Lithium cryolite showed no decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the concurrent vehicle control group, indicating a lack of toxic effects of this test substance on erythropoiesis. The group that was treated with cyclophosphamide showed an expected decrease in the ratio of polychromatic to normochromatic erythrocytes compared to the vehicle control, demonstrating toxic effects on erythropoiesis.

It is concluded that Lithium cryolite is not clastogenic or aneugenic in the bone marrow micronucleus test when sampled at 24 and 48 hours post dosing of male mice up to a dose of 2000 mg/kg under the experimental conditions described in this report. 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

According to the results obtained in the tests performed according to the OECD test guidelines 471, 473 and 474, Lithium cryolite does not have to be classified for mutagenicity according to the CLP and the UN GHS.