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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study (OECD) with acceptable restrictions - only 1000 PCE scored/animal - no data on GLP status - reduced reporting

Data source

Referenceopen allclose all

Reference Type:
publication
Title:
Micronucleus test with mice on 39 food additives and 8 miscellaneous chemical substances
Author:
Hayashi M., Kishi M., Sofuni T. and Ishidate M., Jr.
Year:
1988
Bibliographic source:
Fd. Chem. Toxic., 26, 487-500, (1988)
Reference Type:
publication
Title:
SIDS Initial Assessment Report For SIAM 17 - Ammonium Chloride (12125-02-9) - submitted on Janaury 30th 2004
Author:
OECD SIDS
Year:
2004
Bibliographic source:
OECD

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
yes
Remarks:
(reduced reporting, only 1000 PCE scored for micronucleus)
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
Details on test material:
- Name of test material (as cited in study report): Ammonium chloride
- Source: Japan Food Additive Association, Tokyo
- Analytical purity 99.7%

Test animals

Species:
mouse
Strain:
other: ddY
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Shizuoka Agricultural Cooperative Association for Laboratory Animals, Shizuoka
- Age at study initiation: 8 weeks old
- Weight at study initiation: not specified
- Assigned to test groups randomly: not specified
- Fasting period before study: not specified
- Housing: not specified
- Diet: ad libitum, food pellets CE-2 (Japan Clea, Tokyo)
- Water: ad libitum
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS: not specified

Administration / exposure

Route of administration:
intraperitoneal
Vehicle:
Vehicle: saline
Duration of treatment / exposure:
Group 1: animals were sacrificed 24 hour after single application
Group 2: 4 days
Frequency of treatment:
Group 1: single injection
Group 2: 4 injections at 24 hour intervals
Post exposure period:
no
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 62.5, 125, 250, 500 mg/kg
Basis:
nominal conc.
(group 1; single injection/dose/animal)
Remarks:
Doses / Concentrations:
0, 31.3, 62.5, 125, 250 mg/kg
Basis:
nominal conc.
(group 2; 4 injections/dose/animal)
No. of animals per sex per dose:
6 male animals/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
- Name: Mitomycin C
- Route of administration: i.p
- Doses / concentrations: 2.0 (mg/kg bw)

Examinations

Tissues and cell types examined:
Bone marrow cells taken from the femur were used
Details of tissue and slide preparation:
PRELIMINARY RANGE FINDING STUDY
- Number of animals: 2
- The maximum dose of NH4Cl was determined by pilot experiments using the multisampling at multi-dose levels method (Hayashi et al., 1984, Mutation Res. 141, 165).

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
- Group 1: single dose injection; sacrifice and sampling were performed 24 hours post injection
- Group 2: injection for 4 consecutive days at 24 hour-intervals; sacrifice and sampling were performed 24 hours after last injection

DETAILS OF SLIDE PREPARATION:
- Femoral marrow cells were flushed out with foetal bovine serum and fixed with methanol and stained with Giemsa

METHOD OF ANALYSIS:
- Conducted "blind": Yes
- No. of PCE s/mouse scored for micronucleus: 1000
- Parameters checked: Ratio of PCE to NCE (%PCE) in 1000 cells (measure of bone marrow cell toxicity) and frequency of micronucleated PCE ± standard deviation (%MNPCE)

Evaluation criteria:
- To confirm the validity of the experiments, the frequencies of MNPCEs in concurrent negative and positive control groups were compared with the historical data. Historical negative control data, indicated that the MNPCEs per mouse followed binomial distributions with P = 0.00209 and n = 1000 (laboratory 1) and P = 0.00200 and n = 1000 (laboratory 2)

A test article is considered positive in this assay if all of the following criteria are met and negative in this assay if none of the criteria are met.
- one or more treatment groups show a statistically significant increase (p < 0.01) from the spontaneous level of MNPCEs and the trend test indicate a positive dose response (p < 0.05).
Statistics:
A two-stage statistical procedure was used. In the first step of the procedure, the frequency of MNPCEs of the treatment group was compared with the distribution specified by historical control data. In the second step, dose response relationship was tested by Cochran-Armitage trend test.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Remarks:
(after single and multiple treatment)
Toxicity:
no effects
Remarks:
(%PCE comparable to controls at all doses of both treatment regimens. No mortality was observed in any dose group and treatment regimen)
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY: not reported

RESULTS OF DEFINITIVE STUDY
INDUCTION OF MICRONUCLEI (% MNPCE)
After single exposure
- control: 0.18 ± 0.18
- 62.5 mg/kg bw: 0.12 ± 0.12
- 125.0 mg/kg bw: 0.15 ± 0.14
- 250.0 mg/kg bw: 0.13 ± 0.05
- 500.0 mg/kg bw: 0.12 ± 0.08
- Mitomycin C (2.0 mg/kg bw): 4.18 ± 1.3 (p < 0.01)

After 4 injections at 24 hour intervals
- control: 0.20 ± 0.09
- 31.3.0 mg/kg bw: 0.25 ± 0.19
- 62.5.0 mg/kg bw: 0.17 ± 0.10
- 125.0 mg/kg bw: 0.20 ± 0.18
- 250.0 mg/kg bw: 0.17 ± 0.08
- Mitomycin C (2.0 mg/kg bw): 7.15 ± 3.92 (p < 0.01)

RATIO OF PCE/NCE (% PCE):
After single exposure
- control: 56.8 ± 4.7
- 62.5 mg/kg bw: 60.9 ± 4.2
- 125.0 mg/kg bw: 61.7 ± 3.8
- 250.0 mg/kg bw: 64.3 ± 2.5
- 500.0 mg/kg bw: 56.9 ± 6.1
- Mitomycin C (2.0 mg/kg bw:): 52.3 ± 4.6

After 4 injections at 24 hour intervals
- control: 59.9 ± 8.3
- 31.3 mg/kg bw: 67.2 ± 13.5
- 62.5 mg/kg bw: 63.7 ± 4.5
- 125.0 mg/kg bw: 64.0 ± 9.2
- 250 mg/kg bw: 61.6 ± 6.9
- Mitomycin C (2.0 mg/kg bw:): 32.2 ± 11.0

- Appropriateness of dose levels and route: route of exposure is prescribed by OECD TG 474. No mortality occurred 24 hours after single dose treatment or the multiple dosing regimen, even at the highest doses employed.

- Statistical evaluation: A Monte-Carlo simulation study showed that the probability of a type I error (the probability of a false positive) in the two step statistical method applied was, in general closer to the nominal significance level (P = 0.01) than that of the usual conditional binomial test (Kastenbaum and Bowman) which is commonly used to evaluate micronucleus test data. Hence this statistical method was a more powerful method of analysis than the conditional binomial test.

Any other information on results incl. tables

Table 1: Single dose injection

 

Test substance      Mortality
----------------------------------------------------
Saline                         0/6
Ammonium chloride
62.5(mg/kg)                0/6
125(mg/kg)                0/6
250(mg/kg)                0/6
500(mg/kg)                0/6

.............................................................................
Mitomycin C
2.0(mg/kg)                 0/6
----------------------------------------------------

 

Table 2: Four times injection every 24 hours (i.p.)
----------------------------------------------------
Test substance    Mortality
----------------------------------------------------
Saline                         0/6
Ammonium chloride
31.3(mg/kg)              0/6
62.5(mg/kg)              0/6
125(mg/kg)                0/6
250(mg/kg)               0/6

..............................................................................
Mitomycin C
2.0(mg/kg)                0/6
----------------------------------------------------

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative