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Genetic toxicity in vitro

Description of key information

No mutagenic potential was observed in a bacterial reverse mutation assay conducted with ammonia. A mouse lymphoma assay with the source substance LiOH*H2O was also negative. The target substance can therefore regarded as non mutagenic.

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Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010-01-20 to 2010-07-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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:
mammalian cell gene mutation assay
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 ug/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:
without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
with S9-mix
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 degree 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 degree C whereas the rest of the cells was incubated for 2 days at 37 ± 0.4 degree 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 degree 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 ug/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 ug/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.
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 ug/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 ug/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:
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 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Bacteria strains were only tested in duplicate.
GLP compliance:
not specified
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
Additional strain / cell type characteristics:
not specified
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 mix (rat liver induced with polychlorinated biphenyl, KC500)
Test concentrations with justification for top dose:
500, 1000, 2500, 5000, 10000, 25000 ppm
Vehicle / solvent:
- Vehicle used: sterilised air
- Justification for choice of vehicle: substance is volatile.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2NF
Positive control substance:
2-nitrofluorene
Remarks:
without S9 mix (TA100, TA98, TA1538)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
ENNG
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
without S9 mix (TA1535, WP2 uvrA)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
9AC
Positive control substance:
9-aminoacridine
Remarks:
without S9 mix (TA1537)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
B(a)P
Positive control substance:
benzo(a)pyrene
Remarks:
with S9 mix (TA100, TA98, TA1537, TA1538)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
2AA
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9 mix (TA1535, WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: via air, agar plates were placed upside down without a lid in a glass chamber

DURATION
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS: 2

DETERMINATION OF CYTOTOXICITY
- Method: background bacterial lawn check
Rationale for test conditions:
The test substance is a gas at room temperature.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Table 1: results

Dose (µg/plate)

Mean number of revertant colonies/2 replicate plates with different strains of Salmonella typhimurium and E.coli

 

TA1535

TA1537

TA98

TA100

TA1538

WP2 uvrA

 

 

 

Results with S9

Solvent control

27.8±8.4

10.3±3.8

60.7±14.1

190.6±49.3

58.3±14.2

28.8±6.1

500

31

9

63

229

37

31

1000

40

7

76

220

59

33

2500

35

9

66

181

53

31

5000

21

6

53

195

55

28

10000

34

13

60

185

52

30

25000

31

10

68

220

40

20

positive control

138.5±24.8

96.8±24.6

546.0±29.8

956.4±212.4

510.5±183.8

132.0±50.7

 

 

 

Results without S9

Solvent control

31.3±7.0

11.4±3.0

48.3±7.7

191.8±39.5

31.8±8.6

32.0±5.0

500

25

11

64

243

13

36

1000

23

13

56

251

27

32

2500

29

8

43

198

23

28

5000

20

10

43

207

34

33

10000

23

7

69

197

32

33

25000

19

13

57

227

25

19

positive control

1001.4±168.5

745.0±59.1

503.2±121.9

700.0±52.8

295.6±46.5

136.8±39.8

 

 

 

 

 

 

 

Conclusions:
Under the present experimental conditions, ammonia did not show any genotoxic effects in the tested bacteria strains.
Executive summary:

The mutagenicity of ammonia was assessed in an Ames assay equivalent or similar to OECD guideline 471. In a reverse mutation assay in bacteria, strains (TA98, TA100, TA1535, TA1537, TA1538, WP2 uvrA) of S. typhimurium and E.coli were exposed to ammonia gas at concentrations of 500, 1000, 2500, 5000, 10000 and 25000 ppm in the presence and absence of mammalian metabolic activation.

The test chemical was tested up to a limit concentration. The prepared minimal glucose agar plate was placed upside down without a lid inside a glass chamber. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence or a concentration related positive response of induced mutant colonies over background.

Based on the above results the substance is not regarded as mutagenic in a bacterial reverse mutation assay.

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

Additional information

Bacterial reverse mutation assay (Ames test)

The mutagenicity of ammonia was assessed in an Ames assay equivalent or similar to OECD guideline 471. In a reverse mutation assay in bacteria, strains (TA98, TA100, TA1535, TA1537, TA1538, WP2 uvrA) of S. typhimurium and E.coli were exposed to ammonia gas at concentrations of 500, 1000, 2500, 5000, 10000 and 25000 ppm in the presence and absence of mammalian metabolic activation. The test chemical was tested up to a limit concentration. The prepared minimal glucose agar plate was placed upside down without a lid inside a glass chamber. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence or a concentration related positive response of induced mutant colonies over background. Based on the above results the substance is not regarded as mutagenic in a bacterial reverse mutation assay.

Mammalian cell gene mutation assay

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 ug/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 ug/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 10^6 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.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result the target substance is not considered to be classified and labelled as mutagenic under Regulation (EC) No 1272/2008.