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Registration Dossier
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EC number: 235-186-4 | CAS number: 12125-02-9
Genetic toxicity: in vivo
Administrative data
- Endpoint:
- in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
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:
- 1 988
- 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)
- Author:
- OECD SIDS
- Year:
- 2 004
- Bibliographic source:
- OECD- submitted on Janaury 30th 2004
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
- 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
Constituent 1
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source: Japan Food Additives Association, Tokyo, Japan
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, Japan
- Age at study initiation: 8 weeks old
- Diet: ad libitum, food pellets CE-2 (Japan Clea, Tokyo)
- Water: ad libitum
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
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Group 1; single injection/dose/animal
- Dose / conc.:
- 62.5 mg/kg bw/day (nominal)
- Remarks:
- Group 1; single injection/dose/animal
- Dose / conc.:
- 125 mg/kg bw/day (nominal)
- Remarks:
- Group 1; single injection/dose/animal
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Remarks:
- Group 1; single injection/dose/animal
- Dose / conc.:
- 500 mg/kg bw/day (nominal)
- Remarks:
- Group 1; single injection/dose/animal
- Dose / conc.:
- 0 mg/kg bw/day (nominal)
- Remarks:
- Group 2; 4 injections/dose/animal
- Dose / conc.:
- 31.3 mg/kg bw/day (nominal)
- Remarks:
- Group 2; 4 injections/dose/animal
- Dose / conc.:
- 62.5 mg/kg bw/day (nominal)
- Remarks:
- Group 2; 4 injections/dose/animal
- Dose / conc.:
- 125 mg/kg bw/day (nominal)
- Remarks:
- Group 2; 4 injections/dose/animal
- Dose / conc.:
- 250 mg/kg bw/day (nominal)
- Remarks:
- 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 PCEs/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
- Key result
- 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 (i.p.)
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
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