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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Read-across from lithium fluoride: non-mutagenic in a bacterial reverse mutation assay (Ames test).

Read-across from lithium hydroxide: non-mutagenic in three in vitro tests (Ames test, Chromosome aberration test, In vitro Mammalian Cell Gene Mutation Test).

Read-across from sodium phosphate monobasic: non-mutagenic in a bacterial reverse mutation assay (Ames test) as well as in a chromosome aberration tests.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2015-07-01 to 2015-07-10
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(21st July 1997)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
(30th May 2008)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
TOXI-COOP Toxikological Research Zenter Zrt., Hungary
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 1812
- Expiration date of the lot/batch: May 2025
- Purity test date: 12. May 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature; Avoid contact with acid
- Stability under test conditions: Stable under recommended test conditions
Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA1535, TA100) and frameshift (TA1537, TA98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– → trp+ reversions. The Escherichia coli WP2 uvrA strain detects mutagens that cause other base-pair substitutions (AT to GC).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix ( Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver)
Test concentrations with justification for top dose:
Rangefinding experiment: 5, 16, 50, 160, 500, 1600, 5000 µg/plate with and without S9 mix
Main experiments: 16, 50, 160, 500, 1600, 5000 µg/plate with and without S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Test substance was suspendable in ultrapure water.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ultrapure water and DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitro-1,2-phenylene-diamine (NPD)
Remarks:
without S9 mix (TA 98)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ultrapure water and DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9 mix (TA 100, TA 1535)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ultrapure water and DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix (TA 1537)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ultrapure water and DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 mix (WP2 uvrA)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Ultrapure water and DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene (2AA)
Remarks:
with S9 mix (TA 98, TA 100, TA 1535, TA 1537, WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Experiment I: in agar (plate incorporation); Experiment II: preincubation

DURATION
- Preincubation period: 20 min at 27 °C (only Experiment II)
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduced background lawn
Evaluation criteria:
A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or;
- a reproducible biologically relevant positive response for at least one of the dose groups occurs in at least one strain with or without metabolic activation.

An increase is considered biologically relevant if:
- in strain TA 100 the number of reversions is at least twice as high as the reversion rate of the vehicle control
- in strain TA 98, TA 1535, TA 1537 and Escherichia coli WP2 uvrA the number of reversions is at least three times higher than the reversion rate of the vehicle control.

According to the guidelines, the biological relevance of the results was the criterion for the interpretation of results, a statistical evaluation of the results was not regarded as necessary.
Criteria for a Negative Response:
A test article is considered non-mutagenic in this bacterial reverse mutation assay if it produces neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups, with or without metabolic activation.
Statistics:
Not applicable.
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No

RANGE-FINDING/SCREENING STUDIES:
The toxicity of the test item was determined with strains Salmonella typhimurium TA 98 and TA 100 in a pre-experiment. 7 concentrations were tested for toxicity and mutation induction with 3 plates each. The experimental conditions in this pre-experiment were the same as described below for the main experiment I (plate incorporation test) and included non-activated and S9 activated test conditions with appropriate positive and negative controls. The test item concentrations, including the controls (untreated, vehicle and positive reference) were tested in triplicate.
In the toxicity test the concentrations examined were: 5000, 1600, 500, 160, 50, 16 and 5 μg/plate.
The obtained revertant colony numbers were slightly lower than the revertant colony numbers of the vehicle control in S. typhimurium TA 100, at the examined concentration of 5000 μg/plate, without and with addition of exogenous metabolic activation (±S9 Mix); furthermore at 500 μg/plate, without metabolic activation (-S9 Mix). In these cases the revertant colony numbers were below the corresponding historical control data ranges; however were evaluated as being within the biological variability range of the applied test system.
Slightly higher revertant colony counts were obtained in S. typhimurium TA 98 at 160 μg/plate without metabolic activation (-S9 Mix) and at 16 μg/plate, with addition of metabolic activation (+S9 Mix). These slightly higher revertant counts remained in the corresponding historical control data and biological variability range of the applied test system.
Conclusions:
Under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, it is considered non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

Lithium fluoride was tested in the bacterial reverse mutation assay according to OECD guideline 471. Five bacterial strains were used to investigate the mutagenic potential of lithium fluoride in two independent experiments, in a plate incorporation test (Experiment I, Initial Mutation Test) and in a pre-incubation test (Experiment II, Confirmatory Mutation Test). The test item was dissolved (suspended) in ultrapure water resulting in concentrations of 16, 50, 160, 500, 1600 and 5000 µg/plate. Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. All of the validity criteria, regarding the investigated strains, negative and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with Lithium fluoride, technical at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the actual historical control data ranges, were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system. The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, Lithium fluoride, technical is considered non-mutagenic in this bacterial reverse mutation assay.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1999-11-25 to 2000-01-17
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
July 1997
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: 27.05.97
- Expiration date of the lot/batch: 19 November 2000

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Stability under test conditions: Stability of aqueous solution: at least 96 hrs
Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- -> his+ reversions. The Salmonella typhimurium strains are constructed to differentiate between base pair (TA 1535, TA 100) and frameshift (TA 1537, TA 98) mutations. The Escherichia coli WP2 uvrA (trp) reversion system measures trp– -> trp+ reversions. The Escherichia coli WP2 uvrA detect mutagens that cause other base-pair substitutions (AT to GC).
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
Lithium hydroxide was tested in concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate with and without S9 mix.
Vehicle / solvent:
None
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
Without S9: TA 1535
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
Without S9: TA 1537
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: daunimycine
Remarks:
Without S9: TA 98
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Without S9: TA 100
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
Without S9: WP2uvrA
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9: TA 1537, TA 1535, TA 98, TA 100, E. coli WP2uvrA
Details on test system and experimental conditions:
The test substance was dissolved in Milli-Q-water. The test substance was ground and the stock solution was filter (0.22 µm)-sterilized. Test substance concentrations were prepared directly prior to use.

Range finding study:
Lithium Hydroxide was tested in the tester strains TA 100 and WP2uvrA with concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate in the absence and in the presence of S9 mix.

Mutation assay:
Based on the results of the dose range finding study, Lithium Hydroxide was tested up to concentrations of 5000 µg/plate in the absence and in the presence of S9-mix in two mutation experiments. The first mutation experiment was performed with the strains TA 1535, TA 1537 and TA 98 and the second mutation experiment was performed with the strains TA 1535, TA 1537, TA 98 TA 100 and WP2uvrA.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in any tester strain at any concentration is not greater than two times the solvent control value, with or without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) It induces a number of revertant colonies, dose related, greater than two-times the number of revertants induced by the solvent control in any tester strains, either with or without metabolic activation.
However, any mean plate count of less than 20 is considered to be not significant.
b) The positive response should be reproducible in at least one independently repeated experiment.
Statistics:
Not indicated
Key result
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
GENOTOXICITY:
Please refer to tables 1 and 2, which are presented under Sect. "Remarks on results including tables and figures"
- without metabolic activation: No increase in the number of revertants/plate observed
- with metabolic activation: No increase in the number of revertants/plate observed

CYTOTOXICITY:
No reduction of the bacterial background lawn was observed in all dose levels tested.

Experiment 1

Mutagenic response of lithium hydroxide in the Salmonella typhimurium reverse mutation assay and the Escherichia coli reverse mutation assay:

Dose (μg/plate)

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

 

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

Without S9-mix

positive control

219±33

435±100

371±39

466±22

172±32

solvent control

12±6

7±3

16±3

65±2

9±2

 

3

 

 

 

77±13

12±3

10

 

 

 

72±7

11±3

33

 

 

 

79±8

8±2

100

12±1

7±5

18±3

74±8

12±3

333

15±1

7±1

16±4

65±7

7±3

1000

14±3

4±2

19±3

80±8

9±2

3330

11±3

8±3

12±4

77±13

5±2

5000

12±1

6±2

12±5

77±11

4±1

With S9-mix[1]

positive control

296±23

703±22

1346±230

1199±176

237±37

solvent control

14±6

6±2

26±3

90±10

11±3

 

3

 

 

 

96±4

12±3

10

 

 

 

99±5

12±4

33

 

 

 

95±9

9±3

100

11±3

6±3

31±4

78±11

12±2

333

15±1

4±1

27±9

101±5

10±1

1000

18±7

9±1

26±6

83±12

11±3

3330

15±6

5±1

25±3

86±14

7±4

5000

14±2

4±1

22±3

65±1

4±1

Solvent control: 0.1 mL Milli-Q water

[1]The S9-mix contained 5% (v/v) S9 fraction

Experiment 2

Mutagenic response of lithium hydroxide in the Salmonella typhimurium reverse mutation assay and in the escherichia coli reverse mutation assay:

Dose (μg/plate)

Mean number of revertant colonies/3 replicate plates (±S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain

 

TA 1535

TA 1537

TA 98

TA 100

WP2uvrA

Without S9-mix

positive control

195±2

287±105

642±125

608±24

696±16

solvent control

10±1

4±2

15±4

69±10

8±1

 

100

12±4

4±2

13±2

79±13

10±1

333

8±3

4±3

16±7

68±10

8±3

1000

12±5

5±2

15±2

70±7

11±2

3330

11±4

4±2

10±4

60±11

6±1

5000

7±1

5±2

11±3

64±8

7±3

With S9-mix[1]

positive control

203±14

367±45

551±25

709±131

70±8

solvent control

9±1

3±2

23±3

67±6

11±5

 

100

10±4

3±1

23±3

84±14

12±3

333

8±2

3±3

25±4

70±8

12±2

1000

9±4

6±3

22±6

68±11

7±2

3330

11±5

3±2

14±4

49±6

7±2

5000

5±3

3±2

13±2

54±8

3±1

 

Solvent control: 0.1 mL Milli-Q water

[1]The S9-mix contained 5% (v/v) S9 fraction

Conclusions:
Based on the results of this study it is concluded that lithium hydroxide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

Lithium hydroxide was tested in the Salmonella typhimurium reverse mutation assay according to OECD Guideline 471. The test was performed with four histidine-requiring strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 and TA 98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2uvrA in two independent experiments. Lithium hydroxide was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix. Lithium hydroxide did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at all concentrations tested. Reduction in the number of revertants was observed in the tester strain TA 1535, TA 98, TA 100 and WP2uvrA. Lithium hydroxide did not induce a dose-related, two-fold, increase in the number of revertant (His+) colonies in each of the four tester strains (TA 1535, TA 1537, TA 98 and TA 100) and in the number of revertant (Trp+) colonies in the tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that lithium hydroxide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2000-04-06 to 2000-08-31
Reliability:
1 (reliable without restriction)
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
Principles of method if other than guideline:
NA
GLP compliance:
no
Type of assay:
other: In vitro mammalian chromosome aberration test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: 27.05.97
- Expiration date of the lot/batch: 19 November 2000

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature; in the dark
- Stability under test conditions: At least 96 hrs
Target gene:
Not applicable
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
Untreated negative controls:
yes
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
yes
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
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) increase 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.
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 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 our 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.

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.

No other information

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:

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 ug/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.

 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2010-01-20 to 2010-07-27
Reliability:
1 (reliable without restriction)
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
NA
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: Mammalian cell gene mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- lot/batch No.of test material: 1047

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In a closed container. Keep away from acids and water
Target gene:
Thymidine kinase (TK)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The indicator cell used for this study was the L5178Y mouse lymphoma cell line that is heterozygous at the TK locus (+/-). The particular clone (3.7.2C) used in this assay is isolated by Dr. Donald Clive (Burroughs Wellcome Company, Research Triangle Park, NC).
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
12.5, 25, 50 100 and 200 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Aqua ad iniectabilia
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
positive control for non-activation mutation studies.
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
not specified
Positive controls:
not specified
Positive control substance:
3-methylcholanthrene
Remarks:
positive control for assays performed with S9 metabolic activation.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

ASSAY WITHOUT METABOLIC ACTIVATION
The cells for the experiments were obtained from logarithmically growing laboratory stock cultures and were seeded into a series of tubes at 1 x 107 cells per tube. The cells were pelleted by centrifugation, the culture medium was removed, and the cells were resuspended in a final volume of 20.0 mL of treatment medium that contained 5% heat inactivated fetal bovine serum. The dosed tubes were closed, vortexed and placed on a roller drum at approx. 37 °C at 10 - 15 rpm for an exposure period of 3 hours. The cells were washed and resuspended in growth medium.
Cell densities were adjusted to 2 x 105/mL and the cells were plated for survival and incubated for the expression period in parallel, i.e. an aliquot of the cells was diluted to 8 cells/mL and 0.2 mL of each culture were placed in two 96 well microtiter plates (192 wells, averaging 1.6 cells/well) and incubated for 1 week at 37 ± 0.4 °C whereas the rest of the cells was incubated for 2 days at 37 ± 0.4 °C for the expression period.
The cells for the plating of survival were counted after 1 week and the number of viable clones was recorded. The cells in the expression period were maintained below 106 cells per mL and a minimum of 4 concentration levels plus positive and negative control was selected for 5-trifluoro-thymidine (TFT) resistance.
At the end of the expression period, the selected cultures were diluted to 1 x 104 cells/mL and plated for survival and TFT resistance in parallel (plating efficiency step 2). The plating for survival was similar to the above described method. For the plating for TFT resistance, 3 μg/mL TFT (final concentration) were added to the cultures and 0.2 mL of each suspensions placed into four 96-well microtiter plates (384 wells, averaging 2 x 103 cells/well). The plates were incubated for 12 days at 37 ± 0.4 °C and wells containing clones were identified microscopically and counted.
In addition, the number of large and small colonies was recorded with an automated colony counter that can detect colony diameters equal or greater than 0.2 to 0.3 mm. Large colonies are defined as >= 1/3 and small colonies < 1/3 of the well diameter of 6 mm.

ASSAY WITH METABOLIC ACTIVATION
The activation assay is often run concurrently with the non-activation assay; however, it is an independent assay performed with its own set of solvent and positive controls. In this assay, the above-described activation system was added to the cells together with test item.
Evaluation criteria:
The minimum criterion considered necessary to demonstrate mutagenesis for any given treatment was a mutant frequency that was >= 2 times the concurrent background mutant frequency. The observation of a mutant frequency that meets the minimum criterion for a single treated culture within a range of assayed concentrations was not sufficient evidence to evaluate a test item as a mutagen.
A concentration-related or toxicity-related increase in mutant frequency should be observed.
The ratio of small: large colonies will be calculated from the results of the determination of small to large colonies.
If the test item is positive, the ratio of small to large colonies for the test item will be compared with the corresponding ratios of the positive and negative controls. Based on this comparison, the type of the mutagenic properties (i.e. basepair substitutions, deletions or large genetic changes frequently visible as chromosomal aberrations) of the test item will be discussed.
A test item is evaluated as non-mutagenic in a single assay only if the minimum increase in mutant frequency is not observed for a range of applied concentrations that extends to toxicity causing 10% to 20% relative growth or a range of applied concentrations extending to at least twice the solubility limit in culture media.
Statistics:
No data
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the main study, cytotoxicity (decreased survival) was noted immediately after treatment (plating efficiency step 1) and in the following plating for 5-trifluoro-thymidine (TFT) resistance (plating efficiency step 2) in the presence and absence of metabolic activation at the top concentration of 200 µg/mL.
Cytotoxicity is defined as a reduction in the number of colonies by more than 50% compared with the negative control. Exposure to the test item at the concentration of 200 µg/mL in the absence of metabolic activation resulted in relative survival of 28% and 34% (plating efficiency step 1) and 20% and 33% (plating efficiency step 2), and in the presence of metabolic activation of 28% and 26% (plating efficiency step 1) and 23% and 17% (plating efficiency step 2). Therefore, the test item was considered cytotoxic at the top concentration of 200 ug/mL.

No relevant change in pH and osmolality was noted.

No other information

Conclusions:
Under the present test conditions, lithium hydroxide monohydrate, tested up to a pronounced cytotoxic concentration in the absence and presence of metabolic activation in two independent experiments, was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions positive controls exerted potent mutagenic effects.
In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, lithium hydroxide monohydrate also did not exhibit clastogenic potential at the concentration-range investigated.
According to the evaluation criteria for this assay, these findings indicate that lithium hydroxide monohydrate, tested up to a cytotoxic concentration in the absence and presence of metabolic activation did neither induce mutations nor had any chromosomal aberration potential.
Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK +/- cells to test the potential of lithium hydroxide to cause gene mutation and/or chromosome damage according to OECD Guideline 476 and the EU method B.17. Lithium hydroxide monohydrate was assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK +/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing 2 exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; this experiment with S9 mix was carried out twice. The test item was dissolved in aqua ad iniectabilia. A correction factor of 1.73 was used. The dose-levels and concentrations given in the text and tables refer to lithium hydroxide monohydrate. The limit of solubility was about 34 mg/mL. In the preliminary experiment without and with metabolic activation, concentrations tested were 0.25, 1, 2.5, 10, 25, 100 and 200 µg/mL. Cytotoxicity (decreased survival) was noted at the top concentration of 200 μg/mL. Hence, in the experiments without or with metabolic activation the concentrations of 12.5, 25, 50 100 and 200 µg/mL were used. In the main study, cytotoxicity (decreased survival) was noted immediately after treatment (plating efficiency step 1) and in the following plating for 5-trifluoro-thymidine (TFT) resistance (plating efficiency step 2) in the presence and absence of metabolic activation at the top concentration of 200 μg/mL. Methylmethanesulfonate was employed as positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene in the presence of exogenous metabolic activation. The mean values of mutation frequencies of the negative controls ranged from 61.61 to 98.34 per 106 clonable cells in the experiments without metabolic activation, and from 68.23 to 82.61 per 106 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data-range. The mutation frequencies of the cultures treated with lithium hydroxide monohydrate ranged from 64.74 to 92.63 per 106 clonable cells (3 hours exposure) and 50.42 to 92.34 per 106 clonable cells (24 hours exposure) in the experiments without metabolic activation and 75.88 to 105.59 per 106 clonable cells (3 hours exposure, first assay) and 45.04 to 99.10 per 106 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

Under the present test conditions, lithium hydroxide monohydrate, tested up to a pronounced cytotoxic concentration in the absence and presence of metabolic activation in two independent experiments, was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Under these conditions positive controls exerted potent mutagenic effects. In addition, no change was noted in the ratio of small to large mutant colonies. Therefore, lithium hydroxide monohydrate also did not exhibit clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay, these findings indicate that Lithium hydroxide monohydrate, tested up to a cytotoxic concentration in the absence and presence of metabolic activation did neither induce mutations nor had any chromosomal aberration potential.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102 have not been tested in this publication. Test was only performed with metabolic activation.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
The Salmonella typhimurium histidine (his) reversion system measures his- → his+ reversions.
Species / strain / cell type:
S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94, TA98
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with
Metabolic activation system:
S9 mix (polychlorinated biphenyl induce rat liver)
Test concentrations with justification for top dose:
Six different concentrations up to a maximum of 100 mg/plate (the highest non-cytotoxic dose)
Vehicle / solvent:
- Vehicle used: phosphate buffer
- Justification for choice of vehicle: Solubility of the test substance was adequate in the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
no
Details on test system and experimental conditions:
METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: Tests were performed in duplicate.

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
If the number of colonies was twice as high as the control colonies that test was regarded as positive.
Key result
Species / strain:
S. typhimurium, other: TA92, TA1535, TA100, TA1537, TA94, TA98
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Conclusions:
Under the described test conditions, sodium phosphate monobasic is regarded as not mutagenic in a bacterial reverse mutation test with metabolic activation.
Executive summary:

In a reverse mutation assay in bacteria equivalent or similar to OECD guideline 471, strains of S. typhimurium (TA92, TA1535, TA100, TA1537, TA94, TA98) were exposed to sodium phosphate monobasic at six different concentrations up to 100 mg/plate in the presence of mammalian metabolic activation (pre-incubation). Sodium phosphate monobasic, an overnight culture of Salmonella and 3.75 mL of S9 mix were incubated for 20 min at 37 °C. After pre-incubation top agar was added, mixed and poured into petri dishes. After 48 hours at 37 °C histidine independent colonies were counted. There was no evidence or a concentration related positive response of induced colonies over background. Sodium phosphate monobasic is regarded as non-mutagenic.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
no
Type of assay:
other: in vitro mammalian chromosome aberration test
Species / strain / cell type:
mammalian cell line, other: Chinese hamster lung fibroblast cell line (CHL)
Details on mammalian cell type (if applicable):
- Type and identity of media: Minimum Essential Medium (MEM;GIBCO) supplemented with 10 % calf serum
- Properly maintained: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
without
Test concentrations with justification for top dose:
Three concentrations up to a maximum of 1 mg/mL (the highest non cytotoxic dose used in this experiment)
Vehicle / solvent:
- Vehicle used: physiol. saline
- Justification for choice of vehicle: Solubility of the test substance was adequat in this vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
no
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 and 48 hours

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 µg/mL) was added to the cultures two hours befor harvesting.
STAIN (for cytogenetic assays): with Giemsa solution (1.5 %; at pH 6.8)

NUMBER OF REPLICATIONS: 1

NUMBER OF CELLS EVALUATED: 100 well spread metaphases were analysed.

OTHER EXAMINATIONS:
- Determination of polyploidy: Yes
Evaluation criteria:
The results were considered to be negative if the incidence was less than 4.9 %, equivocal if it was between 5.0 and 9.9 %, and positive if it was more than 10.0 %.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
not examined
Conclusions:
Under the described test conditions, sodium phosphate monobasic is regarded as not clastogenic.
Executive summary:

In a chromosome aberration test equivalent or similar to OECD guideline 473, a Chinese hamster fibroblast cell was exposed to sodium phosphate monobasic (three different doses for 24 h and 48 h). The maximum dose of each sample was choosen via a preliminary test (dose needed for 50 % cell-growth inhibition). Untreated cells and solvent-treated cells were used as negative controls when the incidence of aberrations was less than 3 %. The results were considered as negative if the incidence was <= 4.9 % and positive if it was > 10 %. The test results for sodium phosphate monobasic were negative.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Genetic toxicity in vitro:

Genetic toxicity tests with lithium phosphate are not available. Consequently, read-across was applied using study results obtained from lithium hydroxide, lithium fluoride and sodium phosphate monobasic.

In vitro assay with bacteria

Read-across with lithium fluoride (TOXI-COOP, 803-471-0787, 2015)

Lithium fluoride was tested in the bacterial reverse mutation assay according to OECD guideline 471. Five bacterial strains were used to investigate the mutagenic potential of lithium fluoride in two independent experiments, in a plate incorporation test (Experiment I, Initial Mutation Test) and in a pre-incubation test (Experiment II, Confirmatory Mutation Test). The test item was dissolved (suspended) in ultrapure water resulting in concentrations of 16, 50, 160, 500, 1600 and 5000 µg/plate. Each assay was conducted with and without metabolic activation (S9 Mix). The concentrations, including the controls, were tested in triplicate. In the performed experiments positive and negative (vehicle) controls were run concurrently. All of the validity criteria, regarding the investigated strains, negative and positive controls, S9 activity and number of investigated analyzable concentration levels were fulfilled. No substantial increases were observed in revertant colony numbers of any of the five test strains following treatment with lithium fluoride, technical at any concentration level, either in the presence or absence of metabolic activation (S9 mix) in the performed experiments. Sporadic increases in revertant colony numbers compared to the vehicle control values mostly within the actual historical control data ranges, were observed in both independently performed main experiments. However, there was no tendency of higher mutation rates with increasing concentrations beyond the generally acknowledged border of biological relevance in the performed experiments. The test item did not show inhibitory, cytotoxic effects in the performed experiments. The colony and background lawn development was not affected in any case; the obtained revertant colony number decreases (compared to the revertant colony numbers of the vehicle control) remained within the biological variability range of the applied test system. The reported data of this mutagenicity assay shows, that under the experimental conditions reported, the test item did not induce gene mutations by frameshift or base-pair substitution in the genome of the tester strains used. Therefore, Lithium fluoride, technical is considered non-mutagenic in this bacterial reverse mutation assay.

Read-across with lithium hydroxide (NOTOX B.V, 279067, 1999)

Lithium hydroxide was tested in the Salmonella typhimurium reverse mutation assay according to OECD Guideline 471. The test was performed with four histidine-requiring strains of Salmonella typhimurium (TA 1535, TA 1537, TA 100 and TA 98) and in the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli WP2 uvr A in two independent experiments. Lithium hydroxide was tested up to concentrations of 5000 µg/plate in the absence and presence of S9-mix. Lithium hydroxide did not precipitate on the plates at this dose level. The bacterial background lawn was not reduced at all concentrations tested. Reduction in the number of revertants was observed in the tester strain TA 1535, TA 98, TA 100 and WP2 uvr A at the limit concentration of 5000 µg/plate, indicating some bacterial toxicity. Lithium hydroxide did not induce a dose-related, two-fold, increase in the number of revertant (His+) colonies in each of the four tester strains (TA 1535, TA 1537, TA 98 and TA 100) and in the number of revertant (Trp+) colonies in the tester strain WP2 uvr A both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment. Based on the results of this study it is concluded that lithium hydroxide is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Read-across with sodium phosphate monobasic (Ishidate et al. 1984)

In a reverse mutation assay in bacteria equivalent or similar to OECD guideline 471, strains of S. typhimurium (TA92, TA1535, TA100, TA1537, TA94, TA98) were exposed to sodium phosphate monobasic at six different concentrations up to 100 mg/plate in the presence of mammalian metabolic activation (pre-incubation). Sodium phosphate monobasic, an overnight culture of Salmonella and 3.75 mL of S9 mix were incubated for 20 min at 37 °C. After pre-incubation top agar was added, mixed and poured into petri dishes. After 48 hours at 37 °C histidine independent colonies were counted. There was no evidence or a concentration related positive response of induced colonies over background. Sodium phosphate monobasic is regarded as non-mutagenic.

In vitro assay with cultured Peripheral Human Lymphocytes

Read-across with lithium hydroxide (NOTOX B.V, 291228, 2000)

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 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 both experiments 1A and 1C, lithium hydroxide did not induce a statistically or biologically significant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix.

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 (both without S9-mix). 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 with S9-mix. 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. 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. Positive control chemicals mitomycin C and cyclophosphamide indicated that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Read-across with sodium phosphate monobasic (Ishidate et al. 1984)

In a chromosome aberration test equivalent or similar to OECD guideline 473, a Chinese hamster fibroblast cell was exposed to sodium phosphate monobasic (three different doses for 24 h and 48 h). The maximum dose of each sample was choosen via a preliminary test (dose needed for 50 % cell-growth inhibition). Untreated cells and solvent-treated cells were used as negative controls when the incidence of aberrations was less than 3 %. The results were considered as negative if the incidence was <= 4.9 % and positive if it was > 10 %. The test results for sodium phosphate monobasic were negative.

In vitro assay with mammalian cells

Read-across with lithium hydroxide monohydrate ( LTP, 25361, 2010)

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK +/- cells to test the potential of lithium hydroxide to cause gene mutation and/or chromosome damage according to OECD Guideline 476 and the EU method B.17. Lithium hydroxide monohydrate was assayed in a gene mutation assay in cultured mammalian cells (L5178Y TK +/-) both in the presence and absence of metabolic activation by a liver post-mitochondrial fraction (S9 mix) from Aroclor 1254-induced rats. The test was carried out employing 2 exposure times without S9 mix: 3 and 24 hours, and one exposure time with S9 mix: 3 hours; this experiment with S9 mix was carried out twice. In the preliminary experiment without and with metabolic activation, concentrations tested were 0.25, 1, 2.5, 10, 25, 100 and 200 µg/mL. Cytotoxicity (decreased survival) was noted at the top concentration of 200 μg/mL. Hence, in the experiments without or with metabolic activation the concentrations of 12.5, 25, 50 100 and 200 µg/mL were used. In the main study, cytotoxicity (decreased survival) was noted immediately after treatment (plating efficiency step 1) and in the following plating for 5-trifluoro-thymidine (TFT) resistance (plating efficiency step 2) in the presence and absence of metabolic activation at the top concentration of 200 μg/mL. The mean values of mutation frequencies of the negative controls ranged from 61.61 to 98.34 per 106 clonable cells in the experiments without metabolic activation, and from 68.23 to 82.61 per 106 clonable cells in the experiments with metabolic activation and, hence, were well within the historical data-range. The mutation frequencies of the cultures treated with lithium hydroxide monohydrate ranged from 64.74 to 92.63 per 106 clonable cells (3 hours exposure) and 50.42 to 92.34 per 106 clonable cells (24 hours exposure) in the experiments without metabolic activation and 75.88 to 105.59 per 106 clonable cells (3 hours exposure, first assay) and 45.04 to 99.10 per 106 clonable cells (3 hours exposure, second assay) in the experiments with metabolic activation. These results were within the range of the negative control values and, hence, no mutagenicity was observed according to the criteria for assay evaluation.

Methylmethanesulfonate was employed as positive control in the absence of exogenous metabolic activation and 3-Methylcholanthrene in the presence of exogenous metabolic activation and indicated that the test conditions were adequate and that the metabolic activation system functioned properly.

Lithium hydroxide monohydrate, tested up to a pronounced cytotoxic concentration in the absence and presence of metabolic activation in two independent experiments, was negative with respect to the mutant frequency in the L5178Y TK +/- mammalian cell mutagenicity test. Therefore, lithium hydroxide monohydrate also did not exhibit clastogenic potential at the concentration-range investigated. According to the evaluation criteria for this assay, these findings indicate that lithium hydroxide monohydrate, tested up to a cytotoxic concentration in the absence and presence of metabolic activation did neither induce mutations nor had any chromosomal aberration potential.

These negative findings for lithium hydroxide are supported by experience with long-term administration of e.g. lithium carbonate in humans for therapy of bipolar disorder.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available read-across data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on genetic toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.