Sodium bromide

Genetic toxicity: in vitro

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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Ammonium bromide

EC Number:

235-183-8

EC Name:

Ammonium bromide

Cas Number:

12124-97-9

Molecular formula:

BrH4N

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 10^5/ml		Total Suspension Growth	RSG %	Daily Suspension Count x 10^5/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.