Ammonium bromide

Genetic toxicity: in vitro

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

Test guideline 4

Qualifier:

according to guideline

Guideline:

EPA OPP 84-2

GLP compliance:

yes

Type of assay:

bacterial reverse 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:

Bromide activated chloramine (BAC) generated from ammonium bromide and sodium hypochlorite

Details on test material:

- Name of test material (as cited in study report): Ammonium bromide
- Physical state: White powder
- Analytical purity: 98.5%
- Storage condition of test material: in the dark at ambient temperature

Method

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

Species / strain 1

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 2

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

Controls

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

Results and discussion

Test results

Test results 1

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

Test results 2

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'.

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.