Ammonium bromide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ammonium bromide was negative in in vitro bacterial mutation test (Ames test). It was also negative in an in vitro TK-assay with mouse lymphoma cells. In addition all tests available with sodium bromide were negative (in vitro bacterial mutation test, in vitro mammalian cytogenetic test, in vitro Unscheduled DNA Synthesis in mammalian cells).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-07-29 to 1997-09-04

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

S. typhimurium:
TA 1535, TA 1537, TA 98, TA 100
(TA 1535 and TA 100 : base-pair substitutions, TA 1537 and TA 98 : frameshift-mutations)
E. coli:
WP2 uvr A (base-pair substitutions, base-pair reversions)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

other: All Salmonella typhimurium strains tested: - are histidine-auxotroph - contain deep rough (rfa) mutation and uvrB mutation

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor induced rat liver S9

Test concentrations with justification for top dose:

17, 50, 167, 500, 1667 and 5000 µg per plate for the toxicity test (pre-test) and for mutation tests

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: +S9: 2-Aminoanthracene (2-AAN) for all strains -S9: Sodium Azide (NaN3) for TA 1535 and TA 100 9-Aminoacridine (9-AA) for TA 1537 2-Nitrofluorene (2-NF) for TA98 N-Ethyl-N-nitro-N-nitrosoguanidin (ENNG) for WP2 uvr A

Details on test system and experimental conditions:

Two independent mutation tests were conducted using 5 bacterial strains. Triplicate plates were prepared for each bacterial stain and dose level in both the presence and absence of S9 mix. Ammonium bromide was dissolved and diluted in ultra-pure water
The first test performed used the direct plate method for which 2 ml of soft agar were dispensed into small sterile tubes. To this 0,5 ml of S9 mix or 0,05 M phosphate buffer, pH 7,4 were added followed by 0,1 ml of bacteria and 0,1 ml of solvent or test solution. The tube contains, which were continually cooling, were mixed and poured onto minimal medium. These plates contained 25 ml of 1,5 % purified agar in Vogel-Bonner Medium E with 2 % glucose. When the soft agar had set, the plates were inverted and incubated at 37°C for 2 days.
The second test used the pre-incubation method. 0,5 ml of S9 mix or 0,05 M phosphate buffer, pH 7,4 were dispensed into small sterile tubes. This was followed by 0,1 ml bacteria and, finally the solvent or test solution (0,1 ml). The tube top was then screwed tightly and the tube placed in a shaking incubator at 37°C for 20 minutes. At the end of this time, the tube top was removed and 2 ml of soft agar added to the tube. The tube contains, which were continually cooling, were mixed and poured onto minimal medium. These plates contained 25 ml of 1,5 % purified agar in Vogel-Bonner Medium E with 2 % glucose. When the soft agar had set, the plates were inverted and incubated at 37°C for 2 days.
At the end of the incubation period the colonies were counted using a Biotran III automated counter at maximum sensitivity ie colonies of 0,1 mm or more diameter were counted. The plates were also examined for precipitates and, microscopically, for microcolony growth.
Each concentration was tested in triplicate

A toxicity test using strain TA 100 only was performed in the presence and absence of S9 mix to establish suitable dose levels for the mutation tests. No toxicity was detected in any of the concentrations used.

Evaluation criteria:

A test was considered acceptable if: the bacteria demonstrated their typical response to crystal violet, ampicillin and u.v. light; at least two of the vehicle control plates were within lab-internal defined ranges; on at least two of the positive control plates there were twice (or in the case of TA 100 1,5-times) the mean vehicle control mutant numbers per plate; no toxicity or contamination was observed in at least 4 dose levels; in cases where a mutagenic response was observed, that no more than one dose level was discarded before the dose which gave the highest significant mean colony number.

Statistics:

No data

Species / strain:

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 nor precipitates, but tested up to recommended limit concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The number of revertants per plate was comparable to that observed for untreated or solvent controls; the positive controls induced the expected increased in the number of revertants per plate thereby confirming the sensitivity and reliability of the test system used

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Up to and including the maximum concentration of 5 mg/plate, there was no cytotoxic effect (i.e. clearing of background lawn or reduction in the number of spontaneous revertants) observable.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table 1: Results of thein vitroGene Mutation Assay (Plate Incorporation Test) with Ammonium Bromide

Concentration [µg per plate]	Number of mutant cells*
without metabolic activation
	TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
Solvent	8	6	24	105	2
17	8	12	12	124	3
50	7	10	12	118	6
167	5	10	11	98	6
500	11	7	14	72	5
1667	8	15	11	72	3
5000	5	13	25	77	4
NaN3	179	-	-	275	-
9-AC	-	427	-	-	-
2NF	-	-	122	-	-
ENNG	-	-	-	-	225
with metabolic activation
	TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
Solvent	8	9	12	99	2
17	10	15	15	130	5
50	8	10	18	129	6
167	9	12	12	120	6
500	8	15	14	125	4
1667	11	15	15	128	2
5000	7	11	16	125	4
2-AAN	194	68	24	22	106
*           mean of three individual plates ENNG  N-ethyl-N-nitro-N-nitrosoguanidin: 2 µg/plate withE. coli 9-AC    9-aminoacridine: 80 µg/plate with TA 1537 2AAN  2-aminoanthracene: 20 µg/plate with E. coli; 2 µg/plate with TA 1535 and Ta 1537; 0,5 µg/plate with TA 98 and TA 100 2NF      2-nitrofuorene: 1 µg/plate with TA 98 NaN3    Sodium azide: 1 µg/plate with TA 1535 and TA 100

Table 2: Results of thein vitroGene Mutation Assay (Pre-Incubation Test) with Ammonium Bromide

Concentration [µg per plate]	Number of mutant cells*
without metabolic activation
	TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
Solvent	6	5	16	63	5
17	10	8	19	62	5
50	9	7	18	65	5
167	8	5	17	62	4
500	13	7	17	58	5
1667	8	9	16	52	5
5000	10	5	19	54	7
NaN3	113	-	-	383	-
9-AC	-	441	-	-	-
2NF	-	-	211	-	-
ENNG	-	-	-	-	142
with metabolic activation
	TA 1535	TA 1537	TA 98	TA 100	WP2 uvr A
Solvent	9	5	18	74	3
17	9	5	19	71	4
50	9	6	20	74	4
167	6	7	20	66	4
500	7	8	23	84	4
1667	9	10	21	83	5
5000	7	9	20	81	3
2-AAN	134	100	105	281	374
*           mean of three individual plates ENNG  N-ethyl-N-nitro-N-nitrosoguanidin: 2 µg/plate withE. coli 9-AC    9-aminoacridine: 80 µg/plate with TA 1537 2AAN  2-aminoanthracene: 20 µg/plate with E. coli; 2 µg/plate with TA 1535 and Ta 1537; 0,5 µg/plate with TA 98 and TA 100 2NF      2-nitrofuorene: 1 µg/plate with TA 98 NaN3    Sodium azide: 1 µg/plate with TA 1535 and TA 100

Conclusions:

Interpretation of results (migrated information):
negative

Ammonium bromide was not mutagenic to Salmonella typhimurium or Escherichia coli, when tested in ultra-pure water up to a predetermined maximum limit of 5 mg/plate with and without a metabolic activation system.

Executive summary:

Purpose of the study was to test ammonium bromide for mutagenic activity in Salmonella typhimurium strains TA 1535, TA 1537, TA98 and TA 100 and Escherichia coli WP2uvrA.

Bacteria were tested at concentrations ranging from 17 to 5000 µg ammonium bromide per plate in presence and absence of S9 mix in triplicate plates each. No cytotoxicity (clearing of background lawn) or precipitation was observed up to and including the maximum concentration of 5 mg/plate.

The reliability and sensitivity of the test system was confirmed by parallel testing of positive and negative controls.

A test substance was considered mutagenic if:

- a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 1535, TA 1537 and WP2 uvrA) or 1.5-times (strains TA 100) the colony count of the corresponding vehicle control was observed

 - a related dose response where mutagens require metabolic activation was seen

- a reproducible effect in independent tests was observed

No mutagenic activity was observed in any of the 5 bacterial strains tested both in the absence and presence of S9 mix. Positive controls were shown to have significantly increased the number of revertants per plate and results obtained were within the ranges expected for each bacterial strain and activation condition.

Ammonium bromide was not mutagenic to Salmonella typhimurium or Escherichia coli, when tested in ultra-pure water up to a predetermined maximum limit of 5 mg/plate with and without a metabolic activation system.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

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

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

Target gene:

Mouse lymphoma L5178Y cells (clone 3.7.2.C)

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

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.

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'.

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

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.

Reference 3

Endpoint:

genetic toxicity in vitro

Remarks:

Type of genotoxicity: other: genotoxicity in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1994

Reliability:

2 (reliable with restrictions)

GLP compliance:

no

Species / strain / cell type:

other: primary cultures: Wistar rat thymic lymphocytes

Metabolic activation:

without

Test concentrations with justification for top dose:

0, 0.5, 1, 10, 50, 100, 150 mM

Metabolic activation:

without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

1 mM

Remarks on result:

other: other: primary cultures: Wistar rat thymic lymphocytes

Remarks:

Migrated from field 'Test system'.

Toxicity, as measured by trypan blue exclusion and LDH leakage was noted from 50mM NaBr. The MTT assay revealed a more pronounced toxic effect of NaBr with a significant cell damage detected at 1 mM.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

other: EPA FIFRA 82-2

Deviations:

yes

Remarks:

Salmonella typhimurium TA 1538 was used instead of TA 102 or Escherichia coli WP2 uvrA or WP2 uvrA (pKM101).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Salmonella typhimurium TA 1538 was used instead of TA 102 or Escherichia coli WP2 uvrA or WP2 uvrA (pKM101).

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

other: not applicable

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9 mix: prepared from Sprague-Dawley rats after stimulation with a single intraperitoneal injection of 500 mg /kg Aroclor 1254 (200 mg/ml in Arachis oil) five days prior to sacrifice

Test concentrations with justification for top dose:

Dose range finding: 5, 50, 500, 5000 µg/plate;
Mutation test: 50, 150, 500, 1500 and 5000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

solvent (water)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: as stated under remarks

Remarks:

Positive control substances used: With metabolic activation: 2-Aminoanthracene (all strains with S9 mix) No metabolic activation: 2-Nitrofluorene (TA 1538, TA 98), 9-Aminoacridine (TA 1537), N-Ethyl-N-nitro-N-nitrosoguanidine (TA 1535, TA 100)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Preincubation period: not applicable, only a plate-incorporation test was performed
- Exposure duration: 72 hours



SELECTION AGENT (mutation assays): histidine deficient agar

NUMBER OF CELLS EVALUATED: n.a.; 2 x 10E8 bacterial cells/0.1 mL were used in the test per plate and dose level

NUMBER OF REPLICATIONS: three plates were prepared per tester strain.



DETERMINATION OF CYTOTOXICITY
- Method: relative total growth


OTHER:
A preliminary toxicity test was performed for each bacterial strain used. The highest concentration used was 5 g of sodium bromide dissolved in 1 ml of solvent. Three 10-fold serial dilutions were also tested.
Per tester strain five concentrations of sodium bromide were tested and three bottles were used at each dose level.
The mean number of revertant colonies for all treatment groups was compared with those obtained for negative and positive control groups. The effect of metabolic activation was assessed by comparing the results obtained both in the presence and absence of the liver microsomal fraction for each treatment group. A compound was deemed to provide evidence of mutagenic potential if a statistically significant dose-related increase in the number of revertant colonies of at least twice the concurrent solvent control was obtained in two separate experiments

Evaluation criteria:

The reliability and sensitivity of the test system was confirmed by parallel testing of positive and negative controls.

A compound was deemed to provide evidence of mutagenic potential if a statistically significant dose-related increase in the number of revertant colonies of at least twice the concurrent solvent control was obtained in two separate experiments.

Species / strain:

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

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:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Table A6.6.1/02-1:     Results of the in vitro Gene Mutation Assay (Plate Incorporation Test) with Sodium Bromide
Concentration [µg per plate]	Mean number of mutant cells*
without metabolic activation
Dose level [µg/plate]	TA 1535		TA 1537		TA 1538		TA 98		TA 100
	Test 1	Test 2	Test 1	Test 2	Test 1	Test 2	Test 1	Test 2	Test 1	Test 2
5000	10	12	10	10	14	12	19	25	115	121
1500	13	13	9	11	13	12	25	23	120	97
500	13	15	14	9	16	14	20	21	115	109
150	13	12	13	10	12	9	24	24	131	116
50	10	11	10	8	14	11	23	25	131	107
0	12	10	12	10	13	10	27	19	121	104
solvent	15	15	14	9	12	11	23	29	129	110
ENNG 3 - 5	161	80	-	-	-	-	-	-	338	282
9-AC 80	-	-	x	1073	-	-	-	-	-	-
NF 1 - 2	-	-	-	-	57	62	106	112	-	-
with metabolic activation
Dose level [µg/plate]	TA 1535		TA 1537		TA 1538		TA 98		TA 100
	Test 1	Test 2	Test 1	Test 2	Test 1	Test 2	Test 1	Test 2	Test 1	Test 2
5000	10	12	10	10	13	9	18	21	128	110
1500	12	14	10	9	11	10	28	24	129	91
500	8	12	11	12	12	12	17	22	133	113
150	11	12	9	8	13	10	22	23	135	130
50	9	12	13	11	10	10	23	29	133	122
0	11	13	14	10	16	11	21	21	145	110
solvent	13	17	10	9	15	11	27	23	131	122
2-AA 0.5 - 2	145	126	83	58	147	127	181	118	471	445

*:             mean from three individual plates

X:            too many colonies for counting

ENNG:   N-ethyl-N-nitro-N-nitrosoguanidin: 5 µg/plate for TA 1535, 3 µg/plate for TA 100

9-AC:      9-aminoacridine: 80 µg/plate for TA 1537

AA:         2-aminoanthracene: 2 µg/plate for TA 1535 and TA1537; 0,5 µg/plate for TA 1538 and TA 98; 1µg/plate for TA 100

NF:         2-nitrofuorene: 2 µg/plate for TA 1528, 1 µg/plate for TA 98

Conclusions:

Interpretation of results (migrated information):
negative

No evidence for mutagenic potential of sodium bromide was obtained in this bacterial test system at the dose levels used.

Executive summary:

Materials and Methods

The toxicity potential of sodium bromide (98 %) was examined in Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538 (EPA FIFRA 84-2).

Tester strains were treated with 50, 150, 500, 1500 and 5000 µg/plate sodium bromide in the presence and absence of a metabolic activation system (S9 mix) in triplicate each and revertant colonies were counted after 72-hour incubation time.

No cytotoxicity (clearing of background lawn) or precipitation was observed up to and including the maxium concentration of 5 mg/plate.

The reliability and sensitivity of the test system was confirmed by parallel testing of positive and negative controls.

A compound was deemed to provide evidence of mutagenic potential if a statistically significant dose-related increase in the number of revertant colonies of at least twice the concurrent solvent control was obtained in two separate experiments

Results and Diskussion

Sodium bromide was not toxic towards all tester strains in the dose range finding study up to and including a concentration of 5000 µg/plate. Therefore, 5000 µg/plate was chosen as the top dose level in the mutation test.

No substantial increase in revertant colony numbers of any of the five tester strains when compared with revertants in solvent controls were observed following treatment with sodium bromide at any dose level, either in the presence or in the absence of metabolic activation (S9 mix).

Positive controls were shown to have significantly increased the number of revertants per plate and results obtained were within the ranges expected for each bacterial strain and activation condition.

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

other: not applicable

Species / strain / cell type:

mammalian cell line, other: cultured human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix: prepared from male Sprague-Dawley rats. Mixed-function oxidase system in the liver was stimulated following a single intraperitoneal injection of 500 mg/kg Aroclor 1254 (200 mg/ml in Arachis oil) five days prior to sacrifice.

Test concentrations with justification for top dose:

Preliminary toxicity test:
9.77; 19.53; 39.06; 78.13; 156.25; 312.5; 625; 1250; 2500; 5000 µg/mL
Metaphase analysis without metabolic activation:
500, 2500 and 5000 µg/mL
Metaphase analysis with metabolic activation:
500.2; 2501 and 5002 µg/mL

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

solvent (water)

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

cyclophosphamide dissolved in distilled water at a final concentration of 20 µg/mL

Migrated to IUCLID6: and ethylmethane sulphonate dissolved in dimethylsulphoxide (1000 µg/mL)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: Human lymphocyte cell suspensions of 1 mL were prepared and incubated for a period of 48-hours in the presence of phytohaemagglutinin.
- Exposure duration: 48 hours without metabolic activation. With metabolic activation: two hours after dosing cultures treated with S9 mix were centrifuged, the cell pellet resuspended in fresh medium and incubated for a further 20 hours
- Expression time (cells in growth medium): 2 days
- Fixation time (start of exposure up to fixation or harvest of cells): cells were arrested in metaphase by addition of colchicine for a further 2 hours.



SPINDLE INHIBITOR (cytogenetic assays): colchicine


NUMBER OF REPLICATIONS: Each concentration was examined in duplicate. Four cultures were used for solvent controls (50 µL of distilled water) and two cultures for the positive control


NUMBER OF CELLS EVALUATED: Five slides were prepared from each culture and a total of 100 metaphase spreads were examined from each culture (giving 200 metaphases per concentration).


DETERMINATION OF CYTOTOXICITY
- Method: other: no data

Species / strain:

mammalian cell line, other: cultured human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES:
A preliminary toxicity test indicated that with and without metabolic activation, no decrease of the mitotic index below 50% of the solvent control was observed at any dose level. The highest dose level was the maximum achievable concentration of 5002 µg/mL was therefore chosen as the highest dose level for the metaphase analysis in addition of lower dose levels of 2501 and 500.2 µg/mL.

Table A6.6.2/01-1    Summary of results after treatment of human lymphocytes with Sodium Bromide:Number of aberratinonwith and without metabolic activation
Treatment	Metaphases scored	Without metabolic activation (no. of aberrations per 100 cells)		With metabolic activation (no. of aberrations per 100 cells)
		Exc. Gaps	Inc. gaps	Exc. Gap		Inc. gaps
Solvent Control (sterile distilled water)	100	0	0		0		0
	100	0	0		0		0
	100	0	0		0		0
	100	0	0		0		0
500,2 µg/ml Sodium Bromide	100	0	0		0		0
	100	1	1		0		0
2501 µg/ml Sodium Bromide	100	0	0		0		0
	100	1	1		0		0
5002 µg/ml Sodium Bromide	100	1	1		0		0
	100	0	0		0		0
1000 µg/ml Ethylmethane sulphonate	100	34	37		-		-
	100	30	30		-		-
20 µg/ml Cyclophosphamide	91	-	-		48.4		52.7
	100	-	-		42		48

Table A6.6.2/01-2    Summary of results after treatment of human lymphocytes with Sodium Bromide:, Mean number of aberrant cell (with and without metabolic activation)
Treatment	Without metabolic activation		With metabolic activation (no. of aberrations per 100 cells)
	Exc. Gaps (%)	Inc. gaps (%)	Exc. Gap (%)	Inc. gaps (%)
Solvent Control (sterile distilled water)	0.0	0.0	0.0	0.0
500,2 µg/ml Sodium Bromide	0.5	0.5	0.0	0.0
2501 µg/ml Sodium Bromide	0.5	0.5	0.0	0.0
5002 µg/ml Sodium Bromide	0.5	0.5	0.0	0.5
1000 µg/ml Ethylmethane sulphonate	19	19.5	-	-
20 µg/ml Cyclophosphamide	-	-	27.7	30.4

Conclusions:

Interpretation of results (migrated information):
negative

Sodium bromide, does not reveal any evidence of a clastogenic potential under in vitro test conditions.

Executive summary:

Materials and Methods

Sodium bromide, technical grade was tested for its ability to induce chromosomal aberrations in human lymphocytes cultured in vitro. Cultured human lymphocytes were exposed to the test substance both in the presence and in the absence of a metabolic activation system (S9 mix). Cell division was arrested using colchicine, chromosomes were prepared and examined for structural and numerical aberrations.

Results and Diskussion

A preliminary toxicity test indicated that with and without metabolic activation, no decrease of the mitotic index below 50% of the solvent control was observed at any dose level. The highest dose level was the maximum achievable concentration of 5002 µg/mL was therefore chosen as the highest dose level for the metaphase analysis in addition of lower dose levels of 2501 and 500.2 µg/mL. In both the presence and absence of metabolic activation, sodium bromide caused no statistically significant increase in the proportion of metaphase figures containing chromosomal aberrations when compared with the concurrent control. The respective positive controls caused statistically significant increases in the number of chromosomal aberrations thereby proving the sensitivity of the test system.

Reference 6

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1988

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA TCSA 560/6-83-001

Deviations:

no

GLP compliance:

yes

Type of assay:

DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro

Target gene:

other: not applicable

Species / strain / cell type:

mammalian cell line, other: HeLa S3 epitheloid cells, human cervical carcinoma

Details on mammalian cell type (if applicable):

- Type and identity of media: arginine deficient medium

Metabolic activation:

with and without

Metabolic activation system:

S9 mix: obtained from Sprague-Dawley rats after injection of Aroclor 1254 (200 mg/ml in Arachis oil) at a dosage of 500 mg/kg bw to induce microsomal enzyme activity five days prior to sacrifice.

Test concentrations with justification for top dose:

12.5; 25; 50; 100; 200; 400; 800; 1600; 3200; 6400; 12800 and 25600 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: sterile water

Untreated negative controls:

yes

Remarks:

vehicle

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-1-oxide (absence of S9 mix), 2-aminoanthracene (presence of S9 mix)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 72 hours
- Exposure duration: 180 minutes
- Expression time (cells in growth medium): 180 minutes

NUMBER OF REPLICATIONS: 4


NUMBER OF CELLS EVALUATED: One-hundred (100) non-S-phase nuclei were examined from each culture and the number of silver grains overlying these nuclei and a corresponding adjacent area of cytoplasm was counted.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (S-phase replicative synthesis of DNA)

Evaluation criteria:

100 non-S-phase nuclei were examined from each culture and the number of silver grains overlying these nuclei and a corresponding adjacent area of cytoplasm was counted. A record was kept of the number of non-S-phase nuclei with more than 3 grains net ie nuclear grain count minus cytoplasmic count (% labelled nuclei). A positive response was recorded if there was a reproducible, statistically significant increase in the number of grains per 100 nuclei of non-S-phase compound-treated cells compared with the number deposited over nuclei of solvent-treated cells.

Species / strain:

mammalian cell line, other: HeLa S3 epitheloid cells; human cervical carcinoma

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

At the highest dose level used (25600 µg/mL) normal S-phase replicative synthesis of DNA was inhibited in some of the cultures. For those cells with severe inhibition, no grain count analysis was performed.

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Other confounding effects:
Genotoxicity with metabolic activation: No. In the first test performed in the presence of metabolic activation, no significant increases in either the net or gross nuclear grain count were obtained at any concentration used. In the second test, a small but statistically significant increase (P<0.05) in the gross nuclear grain count was obtained at a single concentration of sodium bromide (1600 µg/ml) but there was no increase in the net nuclear grain count, i.e. when the corresponding cytoplasmic labelling levels were taken into account. In addition, no dose-response relationship could be observed and the increase observed is, therefore, not considered to be toxicologically significant.



ADDITIONAL INFORMATION ON CYTOTOXICITY:
At the highest dose level used (25600 µg/mL) normal S-phase replicative synthesis of DNA was inhibited in some of the cultures. For those cells with severe inhibition, no grain count analysis was performed.

Table A6.6.3/02-1               Results of UDS assay by Autoradiography: Mean net number of grains per 100 nuclei

Experiment No./S9 mix added	H2O	DMSO	4NQO	2AA	Dose of Sodium Bromide, technical grade (µg/ml)*
					^12.5	^25	^50	^100	^200	^400	^800	^1600	^3200	^6400	^12800	^25600
1/no	26	-6	1481/ 3065	-	44	21	28	-33	52	47	5	37	-5	-61	-11	S
2/no	36	29	1820/ 3663	-	38	11	2	11	18	33	21	19	-33	-51	-35	-35
3/yes	-57	-30	-	37/ 769	-49	-10	-39	-48	-53	-37	-100	-65	-16	-95	-84	-64
4/yes	-36	-22	-	135/ 16	-36	-15	-19	-32	-64	35	-32	-70	-4	-28	-19	-25

DMSO:                          Dimethylsulfoxide

4NQO:                           4-Nitroquinoline-1-oxide (result of lowest/highest concentration used, is given (0.02 and 0.32 µg/ml))

2AA:                             2-Aminoanthracene (result of lowest/highest concentration used, is given (2.5 and 40 µg/ml))

S:vere inhibition of S-phase incorporation of ³HTdR

Table A6.6.3/02-2              Results of UDS assay by Autoradiography: % nuclei with > 3 net grains

Experiment No./S9 mix added	H2O	DMSO	4NQO	2AA	Dose of Sodium Bromide, technical grade (µg/ml)
					^12.5	^25	^50	^100	^200	^400	^800	^1600	^3200	^6400	^12800	^25600
1/no	1.9	1.3	100/ 100	-	2.5	2.5	0.5	2.5	3.5	3.5	1	3	0	1.5	2	-
2/no	2.6	1.5	100/ 100	-	2.5	2	0	0.5	2	3	1.5	2	0.5	0.5	0.5	1
3/yes	2.1	1.4	-	5.5/ 79	2	0	1.5	4.5	1.5	1	0	1.5	3.5	4.5	1	1.5
4/yes	1.5	1.8	-	9.5/ 3.5	0.5	2	0.5	1.5	1.5	1	1.5	3	0.5	0.5	1.5	1

DMSO:                          Dimethylsulfoxide

4NQO:                           4-Nitroquinoline-1-oxide (result of lowest/highest concentration used, is given (0.02 and 0.32 µg/ml))

2AA:                             2-Aminoanthracene (result of lowest/highest concentration used, is given (2.5 and 40 µg/ml))

Conclusions:

Interpretation of results (migrated information):
negative

Sodium bromide has shown no evidence of DNA damaging activity in this in vitro UDS test for mutagenic potential using cultured HeLa S3 cells .

Executive summary:

Materials and Methods

The study was designed to test sodium bromide for mutagenic potential by measuring its ability to induce DNA repair in cultured human epitheloid cells (HeLa S3 cells). HeLa S3 cells were plated at a density of 105 cells/well of multi-well tissue culture dishes and incubated at 37°C for 96 hours before culture medium was replaced by arginine deficient medium (ADM). After a further 24 hours of incubation this medium was replaced by fresh ADM and cells incubated for another 48 hours before treatment. To 2 mL of cell culture 20 µL of radioactive DNA precursor (63H)-thymidine was added to give a final activity of 5 µCi/mL. S9 mix (100 µL/culture) was then added to those cultures requiring metabolic activation. Finally a 100 µL aliquot of sodium bromide solution was added at twelve concentrations ranging from 12.5 to 25600 µg/mL. 4-nitroquinoline-1-oxide and 2-aminoanthracene, both dissolved in dimethylsulfoxide, served as control compounds in absence and presence of S9 mix respectively. After incubation for 180 minutes the cultures were harvested, washed, fixed, stained and processed for autoradiography. One-hundred (100) non-S-phase nuclei were examined from each culture and the number of silver grains overlying these nuclei and a corresponding adjacent area of cytoplasm was counted.

Results and Diskussion

The highest concentration of sodium bromide used (25600 µg/mL) was toxic to the cells. The test substance did not show any statistically significant increase in the nuclear grain count in either test in the absence of S9 mix. In the first test in the presence of S9 mix Sodium Bromide did not cause statistically significant increases in nuclear grain count. In the second test in the presence of S9 mix a small but statistically significant increase (P less than 0.05) in the gross nuclear grain count was obtained at a single concentration of sodium bromide (1600 µg/mL). No increase in the grain count was apparent, however, when the corresponding cytoplasmic labelling levels were taken into account, i.e. there was no increase in the net nuclear grain count. There was also a small increase in the net nuclear grain count at 400 µg/ml of test substance, but no increase in the gross nuclear grain count at the same concentration. These two increases are thought to be due to chance variation rather than a treatment-related effect since the two increases were small, not reproducible between tests and unrelated to dose.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Ammonium bromide was negative in an in vivo mouse bone marrow micronucleus test.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: US EPA Pesticide Assessment Guidelines Subdivision F series 84-2

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese MHW Guidelines on Toxicity studies

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Olac Limited, Shaw´s Farm, Blackthorn, Bicester, Oxfordshire, United Kingdom
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 21-35 g

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: water
- Concentration of test material in vehicle: Test material was dissolved in water to give the required dosing concentrations and treat animals with a constant 10 mL/kg bw.
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Duration of treatment / exposure:

animals were treated twice with the same dosage (400, 800 and 1600 mg/kg bw/day), interval between applications was 24 hours.

Frequency of treatment:

2

Post exposure period:

48 h after first treatment

Remarks:

Doses / Concentrations:
400, 800 or 1600 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

vehicle control: 5 male + 5 female
low and mid dose group: 5 male
high dose group: 10 male + 10 female (5 males and 5 females assessed)
Positive control group: 5 male

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide

- Route of administration: oral/gavage
- Doses / concentrations: 50 mg/kg bw given at 10 mL/kg bw

Tissues and cell types examined:

Tissue: bone marrow
Number of cells: 2000 per animal
Type of cells: Poly- and normochromatic erythrocytes in bone marrow
Parameters: frequency of micronucleated cells

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Prior to the conduct of the main study, a dose-range-finding study was performed:
In a first test, one male and female mouse each was treated at 5 dose levels of 50, 125, 350, 800 and 2000 mg/kg bw. Since no toxicity was observed, the test material was further investigated in a limited test consisting of one group of 3 male and 3 female mice dose with 2000 mg/kg bw at 0 and 24 h. In both trials, mice were regularly observed for clinical signs or mortality following dosing on the first day of dosing and twice daily until the end of the observation period. Surviving animals were killed on day 4. No animal died following dosing. Clinical signs of subdued behaviour, hunched appearance, piloerection and rolling gait were observed. This apparent non-toxicity was further investigated in a limit test. Three male and three female mice were given 2 daily doses of 2000 mg ammonium bromide/kg/day. One female died following dosing. Clinical signs consisted of subdued behaviour, hunched appearance, ploerection, rolling gait, tremours, unwilling to move, unable to use hind limbs properly, cold, discharge (eyes), eyes half closed, pale as well as wet and stained perigenital region and ventral surfaces.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Bone marrow cells were examined 48 h after first treatment.

METHOD OF ANALYSIS:
Two thousand (2000) polychromatic erythrocytes (PCE) per animal were scored for micronuclei and the frequency of micronucleated cells determined. Miconucleated normochromatic erythrocytes (MN-NCE) in mature red blood corpuscles were also recorded and the PCE/NCE ratio determined by counting a minimum of 1000 erythrocytes (PCE + NCE) per marrow preparation.

Evaluation criteria:

Two thousand PCEs per animal were scored for micronuclei and the frequency of micronucleated PCEs determined. The PCE/NCE ratio as a measure of systemic toxicity was determined by using a minimum of 1000 erythrocytes (PCE + NCE) per marrow preparation.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

not specified

Remarks:

maximum tolerable dose was determined to be 1600 mg/kg/day

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 50 to 2000 mg/kg/day and 2000 mg/kg/day for the limit test (2 daily doses)
- Clinical signs of toxicity in test animals: subdued behaviour, hunched appearance, piloerection and rolling gait (dose-range finding) and subdued behaviour, hunched appearance, ploerection, rolling gait, tremours, unwilling to move, unable to use hind limbs properly, cold, discharge (eyes), eyes half closed, pale as well as wet and stained perigenital region and ventral surfaces (limit test).



RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): There was no indication that ammonium bromide induced bone marrow micronuclei in the treated mice.
- Ratio of PCE/NCE (for Micronucleus assay):
PCE/NCE (water): 1.04
PCE/NCE (400 mg AmBr): 0.84
PCE/NCE (800 mg AmBr): 0.76
PCE/NCE (1600 mg AmBr): 0.9
PCE/NCE (50 mg cyclophosphamide): 0.58

Table A6.6.4/01-1:     Results for Micronucleus Test In Vivo following Administration of Ammonium Bromide to CD-1 Mice by Gavage
Treatment	sex	PCE examined	No. of MN-NCE	No. of MN-PCE	% MN-PCE	PCE/NCE
10 ml H20/kg/day	M	2000	2	1	0,05	0,95
		2000	1	0	0	0,84
		2000	0	0	0	1,03
		2000	1	4	0,2	0,65
		2000	0	2	0,1	0,87
	F	2000	0	0	0	1,58
		2000	1	2	0,1	1,45
		2000	0	2	0,1	1,27
		2000	0	0	0	0,91
		2000	2	5	0,25	0,89
	total/mean	20000	7	16	0,08	1,04 (± 0,29)
400 mg NH4Br/kg/day	M	2000	0	2	0,1	0,99
		2000	0	0	0	1,25
		2000	1	3	0,15	0,86
		2000	0	1	0,05	0,71
		2000	0	0	0	0,41
	total/mean	10000	1	6	0,06	0,84 (± 0,31)
800 mg NH4Br/kg/day	M	2000	0	1	0,05	0,82
		2000	0	3	0,15	0,89
		2000	0	2	0,1	1,09
		2000	4	2	0,1	0,69
		2000	3	3	0,15	0,32
	total/mean	10000	7	11	0,11	0,76 (± 0,29)

1600 mg NH4Br/kg/day	M	2000	2	3	0,15	0,52
		2000	0	2	0,1	0,75
		2000	0	3	0,15	0,79
		2000	0	2	0,1	0,61
		2000	0	2	0,1	1,26
	F	2000	0	0	0	0,8
		2000	1	0	0	1,14
		2000	0	3	0,15	1,63
		2000	1	2	0,1	0,64
		2000	2	3	0,15	0,87
	total/mean	20000	6	20	0,1	0,9 (± 0,34)
50 mg CPH/kg/day	M	2000	8	49	2,45	0,44
		2000	3	37	1,85	0,7
		2000	0	6	0,3	0,52
		2000	7	48	2,4	0,61
		2000	3	9	0,45	0,64
	total/mean	10000	21	149	1,49	0,58 (± 0,1)

PCE                        Polychromatic erythrocytes

NCE                        Normochromatic erythrocytes

MN                         Micronucleated

CPH                        Cyclophosphamide, positive control

total/mean               for PCE examined, MN-PCE and MN-NCE numbers total events are denoted,

for % MN-PCE and PCE/NCE (± SD) mean numbers per treatment group are given

Conclusions:

Interpretation of results (migrated information): negative
Ammonium bromide did not induce micronuclei in bone marrow cells when tested to the maximum tolerated dose of 1600 mg/kg/day in male and female mice using a 0h + 24h oral dosing and 48h sampling regimen.

Executive summary:

Materials and methods:

The study was designed to evaluate the genotoxic potential of ammonium Bromide in a micronucleus test in bone marrow erythrocytes in male and female CD-1 mice .

CD-1 mice were orally exposed at concentrations of 400, 800 and 1600 mg/kg/day of test substance at 0 and 24 hours. Bone marrow samples were taken 48 hours after the initial dose. Suitable dose levels for the main test were selected in a dose range finding and limit toxicity test. A group of 5 male mice received the positive control cyclophosphamide at 0 and 24 hours at 50 mg/kg bw. Two thousand PCEs per animal were scored for micronuclei and the frequency of micronucleated PCEs determined. The PCE/NCE ratio as a measure of systemic toxicity was determined by using a minimum of 1000 erythrocytes (PCE + NCE) per marrow preparation.

Results and diskussion:

During the dose range finding study with oral doses of Ammonium Bromide ranging from 50 to 2000 mg/kg/day clinical signs of subdued behaviour, hunched appearance, piloerection and rolling gait were observed. But no animal deaths occurred following dosing.

The non-toxicity in the dose range finding study was further investigated in a limit test using 2000 mg Ammonium Bromide/kg/day as oral doses for three male and three female mice as 2 daily doses. One female died following dosing. Clinical signs were subdued behaviour, hunched appearance, piloerection, rolling gait, tremors, unwilling to move, unable to use hindlimbs properly, cold, discharge, eyes half closed and pale, wet and stained perigenital region and ventral surfaces. Based on these investigations, the maximum tolerated dose of ammonium bromide was judged to be in the region of 1600 mg/kg/day. Dose levels of 400 and 800 mg/kg bw were selected as the low and mid dose levels, respectively.

No micronucleus induction was detected in bone marrow erythrocytes of mice dosed with ammonium bromide concentrations of 400, 800 and 1600 mg/kg/day and no effect on the PCE/NCE was noted. The positive control cyclphosphamide induced a significant increase in the number of micronucleated polychromatic erythrocytes and a suppression of the PCENCE ratio indicative for bone marrow toxicity was observed as well.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ammonium bromide was negative in in vitro bacterial mutation test (Ames test). It was also negative in an in vitro TK-assay with mouse lymphoma cells and an in vivo mouse bone marrow micronucleus test. In addition all tests available with sodium bromide were negative (in vitro bacterial mutation test, in vitro mammalian cytogenetic test, in vitro Unscheduled DNA Synthesis in mammalian cells). Based on all these results, ammonium bromide does not need to be classified with regard to mutagenicity.

Justification for classification or non-classification

Ammonium bromide is not mutagenic based on in vitro and in vivo tests performed using the substance itself and sodium bromide.