Registration Dossier

Toxicological information

Genetic toxicity: in vivo

Currently viewing:

Administrative data

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Limited documentation

Data source

Reference
Reference Type:
publication
Title:
Micronucleus test in mice on 39 food additives and eight miscellaneous chemicals.
Author:
Hayashi M et al.
Year:
1988
Bibliographic source:
Fd Chem Toxic 26: 487-500.

Materials and methods

Principles of method if other than guideline:
The maximum doses of the test compounds were determined by pilot experiments using the multisampling at multi-dose levels method according to Hayashi M et al (1984). A pilot experiment for the micronucleus test. The multi-sampling at multi-dose levels method. Mutat Res 141:, 165.
GLP compliance:
not specified
Type of assay:
micronucleus assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Ammonium chloride
EC Number:
235-186-4
EC Name:
Ammonium chloride
Cas Number:
12125-02-9
Molecular formula:
ClH4N
IUPAC Name:
ammonium chloride

Test animals

Species:
mouse
Strain:
other: ddY
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
male ddY mice
- Source: Shizuoka Agricultural Cooperative Association for Laboratory Animals, Shizuoka
- Age at study initiation: 8 weeks
- Diet (ad libitum): food pellets CE-2 (Japan Clea, Tokyo)
- Water: ad libitum
No further data.

ENVIRONMENTAL CONDITIONS: no data

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: physiol. saline
Duration of treatment / exposure:
single dose (pilot study) or 4 doses, divided by 24 hour intervals (see table below, Remarks on results)
Post exposure period:
24 hours after dosing
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day
Remarks:
single dosing
Dose / conc.:
125 mg/kg bw/day
Remarks:
single dosing
Dose / conc.:
250 mg/kg bw/day
Remarks:
single dosing
Dose / conc.:
500 mg/kg bw/day
Remarks:
single dosing
Dose / conc.:
31.3 mg/kg bw/day
Remarks:
multiple dosing
Dose / conc.:
62.5 mg/kg bw/day
Remarks:
multiple dosing
Dose / conc.:
125 mg/kg bw/day
Remarks:
multiple dosing
Dose / conc.:
250 mg/kg bw/day
Remarks:
multiple dosing
No. of animals per sex per dose:
6 mice per group
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C, 2.0 mg/kg bw

Examinations

Tissues and cell types examined:
bone marrow (femur)
Details of tissue and slide preparation:
Mice were killed by cervical dislocation at the appropriate time after an administration. Femoral marrow cells were flushed out with fetal bovine serum and smeared on clean glass slides. Cells were fixed with methanol for 5 min, and stained with Acridine Orange for the pilot experiment and with Giemsa for the full-scale test.
The preparations were coded and analysed without any knowledge of the treatment. One thousand polychromatic erythrocytes per mouse were scored using a light microscope, with a high power objective (x 100), and the number of micronucleated polychromatic erythrocytes (MNPCEs) was recorded. The proportion of polychromatic erythrocytes (PCEs) among the total erythrocytes was also evaluated by observing 1000 erythrocytes on the same slide.
Statistics:
The dose-response relationships were tested using the Cochran-Armitage trend test. A positive dose-response was considered significant at P < 0.05.

Results and discussion

Test results
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

Any other information on results incl. tables

There was no statistically significant increase in the frequency of micronucleated polychromatic erythrocytes.

Compound

Route

No.

of

doses

Time

between

doses

(hr)

Sampling

Time

(hr)

Dose

level

(mg/kg)

NMPCE

(%)

PCE

(%)

Mortality

Single dosing

Ammonium chloride

ip

1

24

0

62.5

125

250

500

0.18 +/- 0.18

0.12 +/- 0.12

0.15 +/- 0.14

0.13 +/- 0.05

0.12 + -/ 0.08

56.8 +/- 4.7

60.9 +/- 4.2

91.7 +/- 3.8

64.3 +/- 2.5

64.3 +/- 2.5

0/6

0/6

0/6

0/6

0/6

Mitomycin

(positive control)

ip

1

24

2.0

4.18 +/- 1.30 *

52.3 +/- 4.6

0/6

Repeated dosing

Ammonium chloride

ip

4

24

24

0

31.3

62.5

125

250

0.20 +/- 0.09

0.25 +/- 0.19

0.19 +/- 0.10

0.20 +/- 0.08

0.17 +/- 0.08

59.9 +/- 8.3

67.2 +/- 13.5

63.7 +/- 4.5

64.0 +/- 9.2

61.6 +/- 6.9

0/6

0/6

0/6

0/6

0/6

Mitomycin

(positive control)

ip

1

24

2.0

7.15 +/- 3.92 *

32.2 +/- 11.0

0/6

(*) p<0.01

Applicant's summary and conclusion