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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1997-08-05 to 1997-10-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1198
Report date:
1998

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay

Test material

Constituent 1
Reference substance name:
Ammonium bromide
EC Number:
235-183-8
EC Name:
Ammonium bromide
Cas Number:
12124-97-9
IUPAC Name:
ammonium bromide
Details on test material:
- Name of test material (as cited in study report): Ammonium bromide
- Physical state: White powder
- Analytical purity: 98.5%
- Lot/batch No.: 970027/1
Sodium bromide is an inorganic salt that dissociates to its composite ions in aqueous solutions at environmental pH and temperature. Comparison of the available data on the various bromide salts have shown that the bromide ion is the relevant ion for determination of the toxicological profile with simple cations such as potassium, sodium or ammonium, that are ubiquitous in nature, having little or no influence on the bromide ion properties. It is therefore justified to read-across data from other inorganic bromide salts to sodium bromide.

Method

Target gene:
Mouse lymphoma L5178Y cells (clone 3.7.2.C)
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Metabolic activation system:
Arochlor induced rat liver S9
Test concentrations with justification for top dose:
Toxicity Test:
0.5/1.5/5/15/150/500/1500/5000 µg/mL
Mutagenicity Assays:
(1000)/2000/3000/4000/5000 µg/mL both in presence and absence of S9 mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: solubility
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
no
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Ethylmethanesulphonate (dissolved in dimethylsulphoxide) and Methylmethanesulphonate (dissolved in water) in the absence of S9 mix; 3-methylcholanthrene (dissolved in dimethylsulphoxide) in presence of S9 mix
Details on test system and experimental conditions:
The test substance was dissolved in water and cells exposed to 1000-5000 µg/mL final concentration both in the presence and absence of metabolic activation (S9 mix). Cell sample cultures were incubated either with test solution, vehicle or positive control for four hours at 37°C and 10 rpm on a rotating drum. After this, cells were sedimented (200 g, 5 min) and resuspended in fresh medium (20 mL). To reach a cell density of 3 x 105/mL this step was repeated. Cells were returned to the rotating drum and allowed for expression of genetic lesions for 2 days at 37°C. On Day 2 all control cultures plus the cultures from the 4 highest concentrations giving adequate survival were selected for expression of genetic damage. This was determined by performing 2 parallel cloning assays: the viability assay and the mutant selection assay. For the viability assay each culture was diluted (2 x 103 cells/ml) and three samples of 0,1 ml were added to 25 ml of cloning medium, poured into a 90 mm Petri dish, giving 200 cells per plate. For the mutant selection assay, trifluorothymidine (TFT) stock solution was added to the cloning medium to a final concentration of 3µg/ml. Duplicate samples of cells (1,5 x 106) were suspended in 2 x 25 ml cloning medium (containing TFT) and poured into Petri dishes (100 mm). The dishes were prepoured with cloning medium to make an underlay (20 ml each). This underlay compensates for the larger volume of Petri dishes and prevents the cells from making contact with the floor of the dish. All plates were gelled at room temperature until the agar had set, then incubated at 37°C in an atmosphere of 5% Co2:95% air until the colonies were fully developed (usually 14 days). The colonies were then counted using a “Domino” image analyser.
Doses in mutation experiments should extend into the toxic range, the maximum usable limit allowing cell growth and subsequent cloning efficiency giving at least 10% of the concurrent vehicle control values. Therefore a initial toxicity test was performed in the absence and presence of S9 mix. Concentrations of ammonium bromide used were in the range of 0,5 – 5000 µg/ml. Exposure of cells was similar to that of the mutation assays. Following treatment the cell population densities were recorded over two days using a haemocytometer, then the total suspension growths were expressed as percentages of the vehicle control group.
Evaluation criteria:
Results from any tube of exposed cells were rejected if mean cloning efficency was greater than 120%.
Results from any treatment were inadequate if there were less than 2 acceptable cultures. Where results were obtained from a single culture, they may have been included as supporting evidence.
Controls
Results from the vehicle cultures were rejected if mean cloning efficency was less than 60%.
Results from vehicle control cultures were required to give a mean mutant fraction less than 150 per 10E06 survivors and at least 20 per 10E06 survivors.
Results from positive control cultures were required to give mutant fractions of at least double the vehicle control value and equal to or greater than 100 per 10E06 survivors. Cloning efficiencies greater than 10% were also required.
Test materials
Individal cultures were rejected if the relative growth was less than 10% of the mean vehicle control or cloning efficiency after the expression period was less than 10%.
An experiment was liable to rejection of there were acceptable results from less than 3 dose levels.
Interpretation of toxicity
Results should be obtained from concentrations in RTG range 10- 20%
Test negative if test material response not significantly higher than negative control at pre-set limit (reduction of total relative growth to 20% or 5000 µg/mL)
Positive response: response at a single dose is significant if cloning efficiency is at least 10% of concurrent vehicle and mean mutant fraction of 2 cultures is at least 1.7 fold higher than the mean control value.
Test material positive if 2/2 positive experiments recorded with same activation condition or if in one or other activation condition; the first experiment indicated a positive but did not meet the criteria due to lack of results from a critically toxic concentration; the second experiment gave an unequivocal positive.
Statistics:
Weak positive and equivocal results can be subjected to statistical analysis by ANOVA and t-test, for confirmation of the significance of the response.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
5000 µg/mL
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
-S9: No evidence of mutagenic activity was obtained in any ammonium bromide-treated cultures. The mean relative total growth at the highest concentration (5000 µg/ml) were 57,5% and 73,5% in the absence of S9 mix. These experiment was classed as negative.
+S9: No evidence of mutagenic activity was obtained in any ammonium bromide-treated cultures. The mean relative total growth at the highest concentration (5000 µg/ml) were 58% and 71% in the presence of S9 mix. These experiment was classed as negative.
Cytotoxicity: At a concentration of 5000 µg/mL ammonium bromide caused significant reduction in relative suspension growth both in the presence and in the absence of S9 mix.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: Results of Mouse Lymphoma Mutation Test absence and presence of metabolic activation (S9 mix)

Chemical

Concentration

[µg/ml]

Without S9 mix

With S9 mix

assay 1

assay 3

assay 2

assay 4

Mutant fraction*

RTG

%

Mutant fraction*

RTG

%

Mutant fraction*

RTG

%

Mutant fraction*

RTG

%

Water

(100 µl added)

24

87

35

93

26

109

38

98

47

82

39

87

32

103

38

100

19

119

49

106

20

97

37

87

33

112

52

114

36

91

37

116

EMS

250

235

38

332

68

-

-

-

-

244

41

301

79

-

-

-

-

MMS

15

266

19

206

16

-

-

-

-

294

16

267

18

-

-

-

-

3-MC

2,5

-

-

-

-

200

32

250

61

-

-

-

-

199

29

244

54

Ammonium Bromide

2000

30

103

47

84

28

100

36

93

25

67

44

87

25

95

34

87

3000

45

63

34

83

19

86

32

87

30

80

32

80

26

86

42

83

4000

20

70

45

62

29

68

39

60

28

70

43

54

19

71

39

64

5000

17

59

34

68

15

62

33

72

24

56

45

79

29

54

35

70

EMS       Ethylmethanesulphonate

MMS     Methylmethanesulphonate

3-MC     3-methylcholanthrene

*             [x 106] 200 (total mutant count) / (total viable count)

RTG       relative total growth

 

 

Table 2: Results of Mouse Lymphoma Toxicity Test (Pre-Test) in the absence and presence of metabolic activation (S9 mix)

Chemical

Concentration (µg/ml)

Without S9 mix

With S9 mix

Daily Suspension Count x 105/ml

Total Suspension Growth

RSG

%

Daily Suspension Count x 105/ml

Total Suspension Growth

RSG

%

Day 1

Day 2

Day 1

Day 2

Water

100 µl added

11.4

15.2

19.3

100

11

15.2

18.6

100

Ammonium

Bromide

0,5

12.6

15.2

21.3

110

11.6

15

19.3

104

1,5

11.4

14

17.7

92

12

14.2

18.9

102

5

14

14.6

22.7

118

11.2

16.6

20.7

111

15

13.6

13.4

20.2

105

13.2

11

16.1

87

50

15

13.8

23

119

11.2

11

13.7

74

150

12.4

15.2

20.9

108

11.6

11.2

14.4

77

500

14.8

12.4

20.4

106

11.2

11

13.7

74

1500

14.4

13.6

21.8

113

10.4

12.6

14.6

78

5000

7

14

10.9

56

4.7

12.6

6.6

35

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Ammonium bromide is not mutagenic in mouse lymphoma L5178Y cells when tested to the pre-set maximum concentration of 5000 µg/ml both in the presence and absence of a metabolic activation system
Executive summary:

The objective of the study was to determine the potential of ammonium bromide to induce mutations at the thymidine kinase locus: tk+tk-to tk-tk-of mouse lymphoma L5178Y cells.

The test substance was dissolved in water and cells exposed to 1000-5000 µg/mL final concentration both in the presence and absence of metabolic activation (S9 mix). Cell sample cultures were incubated either with test solution, vehicle or positive control for four hours at 37°C and 10 rpm on a rotating drum. After this, cells were sedimented (200 g, 5 min) and resuspended in fresh medium (20 mL). To reach a cell density of 3 x 105/mL this step was repeated. Cells were returned to the rotating drum and allowed for expression of genetic lesions for 2 days at 37°C. On Day 2 all control cultures plus the cultures from the 4 highest concentrations giving adequate survival were selected for expression of genetic damage. This was determined by performing 2 parallel cloning assays: the viability assay and the mutant selection assay. For the viability assay each culture was diluted (2 x 103cells/ml) and three samples of 0,1 ml were added to 25 ml of cloning medium, poured into a 90 mm Petri dish, giving 200 cells per plate. For the mutant selection assay, trifluorothymidine (TFT) stock solution was added to the cloning medium to a final concentration of 3µg/ml. Duplicate samples of cells (1,5 x 106) were suspended in 2 x 25 ml cloning medium (containing TFT) and poured into Petri dishes (100 mm). The dishes were prepoured with cloning medium to make an underlay (20 ml each). This underlay compensates for the larger volume of Petri dishes and prevents the cells from making contact with the floor of the dish. All plates were gelled at room temperature until the agar had set, then incubated at 37°C in an atmosphere of 5% Co2:95% air until the colonies were fully developed (usually 14 days). The colonies were then counted using a “Domino” image analyser.

Doses in mutation experiments should extend into the toxic range, the maximum usable limit allowing cell growth and subsequent cloning efficiency giving at least 10% of the concurrent vehicle control values. Therefore a initial toxicity test was performed in the absence and presence of S9 mix. Concentrations of ammonium bromide used were in the range of 0,5 – 5000 µg/ml. Exposure of cells was similar to that of the mutation assays. Following treatment the cell population densities were recorded over two days using a haemocytometer, then the total suspension growths were expressed as percentages of the vehicle control group.

The preliminary toxicity test showed that ammonium bromide caused a significant reduction in the relative suspension growth only at the pre-set maximum concentration of 5000 µg/ml (56 % and 35 % in the absence and presence of s9 mix respectively).

No evidence of mutagenic activity was obtained from cultures treated with ammonium bromide in any of the 4 assays with and without metabolic activation. The solvent control values were within the normal ranges experienced in the performing laboratory and reported in the literature with the L5178Y cell line. The high mutant fractions obtained with EMS, MMS and 3-MC were within the normal ranges. 3-MC (which is not mutagenic in the absence of S9 mix) proved the efficacy of the S9.

Ammonium bromide is not mutagenic in mouse lymphoma L5178Y cells when tested to the pre-set maximum concentration of 5000 µg/ml both in the presence and absence of a metabolic activation system.