Registration Dossier

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1998
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1998

Materials and methods

Test guidelineopen allclose all
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

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
- Description: white crystalline solid
- Analytical purity: 98.5%
- Lot/batch No.: 970027/1
- Stability under test conditions: not applicable
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.

Test animals

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


Administration / exposure

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
Doses / concentrations
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

Examinations

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.

Results and discussion

Test results
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

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.