Registration Dossier
Registration Dossier
Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.
The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.
Use of this information is subject to copyright laws and may require the permission of the owner of the information, as described in the ECHA Legal Notice.
EC number: 235-186-4 | CAS number: 12125-02-9
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
- In vitro bacterial gene mutation (OECD
471): negative (with and without metabolic activation)
- In vitro mammalian gene mutation (OECD 476): negative (with and
without metabolic activation) (read across to CAS 7783-20-2 and CAS
1111-78-0)
- In vitro cytogenicity (equivatent OECD 473): negative (positive
without metabolic activation)
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- data from handbook or collection of data
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- not specified
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Target gene:
- Histidine operon
- Species / strain / cell type:
- other: Salmonella typhimurium TA98, TA100, TA1535, TA1537 and TA1538
- Species / strain / cell type:
- E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- rat liver S-9 fraction mixed with a series of cofactors (MgCl2, KCl, Glu-6-p, NADP, phosphate buffer (NaH2PO4, Na2HPO2.2H2O)
- Test concentrations with justification for top dose:
- 0, 4, 20, 100, 500, 2500, 5000 µg/plate
- Vehicle / solvent:
- - Vehicle: water
- Untreated negative controls:
- yes
- Remarks:
- sterility controls
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- without S9 mix; TA100, TA1535
- Untreated negative controls:
- yes
- Remarks:
- sterility control
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- without S9 mix; TA1537
- Untreated negative controls:
- yes
- Remarks:
- sterility control
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- without S9 mix; TA98, TA1538
- Untreated negative controls:
- yes
- Remarks:
- sterility control
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- other: N-methyl, N-nitro, N-nitrosoguanidine
- Remarks:
- without S9 mix; E.coli WP2 uvrA
- Untreated negative controls:
- yes
- Remarks:
- sterility control
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- benzo(a)pyrene
- other: 2-aminoanthracene
- Remarks:
- with S9 mix; all tested strains
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period: no data
- Exposure duration: 48 - 72 hours at 37 °C
NUMBER OF REPLICATIONS: 2 experiments; triplicate cultures/experiment
DETERMINATION OF CYTOTOXICITY
- Method: a clearing of the bacterial background lawn - Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- 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
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
- Deviations:
- not specified
- Qualifier:
- according to guideline
- Guideline:
- other: Ishidate, M. et al, Mutation Research 48, 337
- Principles of method if other than guideline:
- According to the method Ishidate, M.,Jr. und Odashima, S.: Mutation Research 48, 337
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian chromosome aberration test
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source: Japan Food Additives Association, Tokyo, Japan - Target gene:
- not applicable
- Species / strain / cell type:
- other: Chinese hamster lung fibroblasts (CHL)
- Details on mammalian cell type (if applicable):
- - Origins: The cell line was originally established from the lung of a newborn female at the Cancer Research Institute, Tokyo
- Modal number: 25
- Doubling time: 15 hours - Metabolic activation:
- without
- Test concentrations with justification for top dose:
- The authors reported that three doses were tested. However, results for only two doses were report
- 0.3 and 0.4 mg/mL. The third dose tested could not be identified - Vehicle / solvent:
- - Vehicle: physiol. saline
- Untreated negative controls:
- yes
- Remarks:
- (untreated cells)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Remarks:
- In the study a total of 242 substance were tested. Of these substances, 54 were positive for causing chromosomal aberration, of which some have been shown to be also positive for carcinogenicity in rodents.
- Positive control substance:
- other: example of positively tested substance (chromosomal aberration + carcinogenicity): potassium bromate
- Remarks:
- Based on arguments cited above, the test system is considered to be adequatedly sensitive in distinguishing between substance capable of causing chromosomal aberration and substances not causing chromosomal aberration.
- Details on test system and experimental conditions:
- DURATION
- Exposure duration: 24 and 48 hours
SPINDLE INHIBITOR (cytogenetic assays):
- Name: Colcemid
- Concentration: 0.2 µg/mL
- Time schedule for addition: 2 hours before harvesting
STAIN: Giemsa solution (1.5%, at pH 6.8; E. Merck) for 12-15 mins
NUMBER OF REPLICATIONS: In case no dose dependency was found in the first experiment, a repeat experiment was performed at similar dose levels
NUMBER OF CELLS EVALUATED: 100 well spread metaphases
The incidence of polyploid cells as well as cells with structural chromosomal aberrations such as chromatid or chromosomal gaps, breaks, exchanges, ring formations, fragmentations, and others, was recorded for each culture plate. - Evaluation criteria:
- The result was considered to be
- negative if the incidence of aberration is less than 4.9 %.
- ambiguous if the incidence of aberration is between 5.0 - 9.9 % and
- positive if the incidence of aberration is above 10 % - Key result
- Species / strain:
- other: Chinese hamster lung fibroblasts (CHL) cells
- Metabolic activation:
- without
- Genotoxicity:
- positive
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- RANGE-FINDING/SCREENING STUDIES:
- The maximum dose was chosen based on the cytotoxicity and osmolarity. The maximum dose chosen for the main study was the concentration needed for 50 % cell growth inhibition, estimated using a cell dosimeter (Monocellater, Olympus Co. Ltd.). - Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- weight of evidence
- Justification for type of information:
- REPORTING FORMAT FOR THE ANALOGUE APPROACH
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
In aqueous solution, ammonium salts are completely dissociated into NH4+ and a corresponding anion. This equilibrium depends on temperature, pH and ionic strength of the water in the environment. Un-ionized NH3 species exists in the aquatic environments and the fraction (NH3/(NH3 +NH4+)) steeply increases with elevated pH value or temperature. It is well known that toxicity to aquatic organisms has been attributed to un-ionized ammonia (NH3) species, and NH4 + species is considered to be non- or significantly less-toxic (Emerson et al., 1975). However, recent developments in assessing ammonia toxicity clearly show that in contrast to earlier assumptions where un-ionized ammonia was considered to be the toxic component, both the uncharged and charged molecule are toxic. Therefore, a joint toxicity model has been proposed, with ammonia causing most toxicity at high pH values and ammonium ion also contributing to toxicity at lower pH values (U.S. EPA 1999, OECD 2007).
2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
Source substance: diammonium sulfate, CAS 7783-20-2
Target substance: ammonium chloride, CAS 12125-02-9
3. ANALOGUE APPROACH JUSTIFICATION
It is generally accepted, that the principal toxic component of ammonium salts such as ammonium chloride or -sulphate is ammonia, rather than the corresponding anion (see also: OECD 2004, SIDS ammonium chloride or OECD 2007 ammonium sulphate). Therefore toxicity values for ammonium salts (such as: ammonium -sulphates, phosphates, carbonates, chlorides or nitrates), where the major toxic component is ammonia, can be considered as equivalent. Consequently, this hazard assessment comprises the total topic of ammonia toxicity. - Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- 10 mM
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Remarks on result:
- other:
- Remarks:
- (BASF SE, 2010)
- Endpoint:
- in vitro gene mutation study in bacteria
- Remarks:
- Type of genotoxicity: gene mutation
- Type of information:
- experimental study
- Adequacy of study:
- supporting study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Deviations:
- yes
- Remarks:
- (no E. coli or TA 102 tested, incomprehensive reporting of results and methods)
- Principles of method if other than guideline:
- According to Ames, McCann and Yamasaki (1975) Mutation Research 31, 347
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source: Japan Food Additives Association, Tokyo, Japan - Target gene:
- Histidine operon
- Species / strain / cell type:
- other: Salmonella typhimurium TA 92, TA 1535; TA 100; TA 1537; TA 94; TA 98
- Metabolic activation:
- with and without
- Metabolic activation system:
- Kanechlor KC-400 induced rat liver S-9 fraction mixed with a series of cofactors (MgCl2, KCl, Glu-6-p, NADP, phosphate buffer (NaH2PO4, Na2HPO2.2H2O)
- Test concentrations with justification for top dose:
- 6 concentrations were tested with the highest concentration tested being 10 mg/plate
- Vehicle / solvent:
- - Vehicle: phosphate buffer
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Remarks:
- In the study, a total of 200 substance were tested. Of these substances, 14 were positive for causing point mutations the salmonella typhimurium strains tested.
- Positive control substance:
- other: calcium hypochlorite (CAS No. 7778-54-3), Cinnamic aldehyde (CAS No. 104-55-2), Erythorbic acid (CAS No. 89-65-6).
- Remarks:
- Based on arguments cited above, the test system is considered to be adequatedly sensitive in distinguishing between substances that cause point mutations from those that do not cause point mutations.
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
DURATION
- Preincubation period (Exp. 2): 20 min
- Exposure duration: 48 hours at 37 °C
NUMBER OF REPLICATIONS: duplicate culture were sowed per concentration (repeat experiments were performed (with different doses) only if no dose dependent reponse was noticed. - Evaluation criteria:
- - A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice the colony count of the corresponding solvent control is observed.
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1537
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 98
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 100
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium, other: 92
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium, other: 94
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- GLP compliance:
- yes
- Type of assay:
- mammalian cell gene mutation assay
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Batch No.of test material: PBG 10002877
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Refrigerator, protected against moisture - Target gene:
- hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 (rat liver; phenobarbital and β-naphthoflavone induced)
- Test concentrations with justification for top dose:
- 1st experiment:
- without S9 mix (4-hour exposure period): 0; 100; 200; 400; 800 µg/mL.
- with S9 mix (4-hour exposure period): 0; 100; 200; 400; 800 µg/mL.
2nd experiment:
- without S9 mix (24-hour exposure period): 0; 100; 200; 400; 800 µg/mL.
- with S9 mix (4-hour exposure period): 0; 200; 400; 600; 800 µg/mL. - Vehicle / solvent:
- culture medium (Ham's F12)
- Untreated negative controls:
- yes
- Remarks:
- culture medium without test substance was used (with and without S9)
- Negative solvent / vehicle controls:
- yes
- Remarks:
- culture medium
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- 3-methylcholanthrene
- ethylmethanesulphonate
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 or 24 hours
- Expression time (cells in growth medium): 7 - 9 days
- Selection time (if incubation with a selection agent): 6 - 7 days
SELECTION AGENT (mutation assays): 6-thioguanine
NUMBER OF REPLICATIONS: two independant experiments
DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; survival - Evaluation criteria:
- Assessment criteria
A finding is assessed as positive if the following criteria are met:
• Increase of the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range.
• Evidence of reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship. Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10E^6 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity. The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range.
Acceptance criteria
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
• The background mutant frequency in the negative/vehicle controls should fall within our historical negative control data range of 0 – 15 mutants per 10E^6 clonable cells.
• The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies (historical positive control data.
• At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM. - Statistics:
- Due to the clearly negative findings with mutation frequencies within the range of our laboratory's historical negative control data, a statistical evaluation was not carried out.
- Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
- Remarks:
- 10 mM
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- MUTANT FREQUENCY
In this study, no relevant increase in the number of mutant colonies was observed either without S9 mix or after the addition of a metabolising system. In both experiments after 4 and 24 hours treatment with the test substance the values for the corrected mutation frequencies (without S9 mix: MFcorr.: 0.76 – 13.80 per 10E^6 cells; with S9 mix: MFcorr.: 1.27 – 5.86 per 10E^6 cells) were close to the respective vehicle control values (MFcorr.: 0.00 – 8.15 per 10E^6 cells) and clearly within the range of our historical negative control data (without S9 mix: MFcorr.: 0.00 – 15.95 per 10E^6 cells; with S9 mix: MFcorr.: 0.00 – 12.62 per 10E^6 cells)
The positive control substances EMS (without S9 mix; 300 μg/mL) and MCA (with S9 mix; 20 μg/mL) induced clearly increased mutant frequencies as expected. The values of the corrected mutant frequencies (MFcorr.: 116.00 – 394.20 per 10E^6 cells) were clearly within our historical positive control data range (without S9 mix: MFcorr.: 48.83 – 999.46 per 10E^6 cells; with S9 mix: MFcorr.: 26.29 – 413.54 per 10E^6 cells)
CYTOTOXICITY
In both experiments in the absence and presence of S9 mix, no cytotoxicity indicated by reduced relative cloning efficiency of below 20 % relative survival was observed up to the highest required test substance concentration.
CELL MORPHOLOGY
There were no adverse observations on cell morphology.
TREATMENT CONDITIONS
The pH value of the treatment medium was adjusted by adding small amounts of HCl. Osmolarity and pH values were not influenced by test substance treatment. In this study, in the absence and presence of S9 mix no precipitation in culture medium was observed up to the highest required test substance concentration. - Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 2010
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
- Version / remarks:
- adopted July 21, 1997
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- mammalian cell gene mutation assay
- Target gene:
- hypoxanthine-guanine phosphoribosyl transferase (HPRT)
- Species / strain / cell type:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Metabolic activation system:
- Phenobarbital/b-naphthoflavone induced rat liver S9
- Test concentrations with justification for top dose:
- 82.5, 165.0, 330.0, 660.0 and 1320.0 µg/mL
- Vehicle / solvent:
- - Solvent used: acetone
- Justification for choice of solvent: The solvent was chosen according to its solubility properties and its relative nontoxicity to the cells. - Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- not specified
- Remarks:
- without metabolic activation
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- True negative controls:
- no
- Positive controls:
- yes
- Positive control substance:
- not specified
- Remarks:
- with metabolic activation
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: in medium
DURATION
- Exposure duration: 4 hour with and without metabolic activation, or 24 hours without metabolic activation.
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days
- Fixation time (start of exposure up to fixation or harvest of cells): 7 to 8 days
SELECTION AGENT (mutation assays): 6-thioguanine
NUMBER OF CELLS EVALUATED: 50 colonies
DETERMINATION OF CYTOTOXICITY
- Method: relative total growth - Evaluation criteria:
- A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate in the range normally found (3.0 – 33.2 mutants per 1000000 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration. - Statistics:
- A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
- Key result
- Species / strain:
- Chinese hamster lung fibroblasts (V79)
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
The test medium was checked for precipitation or phase separation at the end of each treatment period (4 or 24 hours) before the test item was removed. No precipitation was observed up to the maximum concentration with and without metabolic activation. There was no relevant change of the pH value and the osmolarity even at the maximum concentration of the test item.
RANGE-FINDING/SCREENING STUDIES: The highest concentration in the pre-experiment was 1320 µg/mL equal to approximately 10 mM.
Referenceopen allclose all
The incidence of aberration in the untreated and solvent control was about 3.0 %. The incidence of cells with structural chromosomal aberrations at 0.4 mg/mL after 48 hr treatment was 47.0 %. The incidences of structural aberrations at 0.3 mg/mL after 24 hours and 48 hours of exposure were 11.0 % or 30 %, respectively.
Table 1: Summary of results
Exp. |
Exposure period |
Test groups |
S9 mix |
Prec.* |
Genotoxicity** MFcorr. [per 108 cells] |
Cytotoxicity |
|
CE1 [%] |
CE2 [%] |
||||||
1 |
4 hrs |
Negative control |
− |
− |
0.38 |
100.0 |
100.0 |
100 µg/mL |
− |
− |
0.76 |
100.4 |
93.6 |
||
200 µg/mL |
− |
− |
4.35 |
89.3 |
99.7 |
||
400 µg/mL |
− |
− |
1.33 |
92.7 |
103.3 |
||
800 µg/mL |
− |
− |
1.89 |
93.4 |
89.3 |
||
Positivecontrol1 |
− |
− |
299.10 |
88.4 |
74.9 |
||
2 |
24 hrs |
Negative control |
− |
− |
3.11 |
100.0 |
100.0 |
100 µg/mL |
− |
− |
5.35 |
99.7 |
106.7 |
||
200 µg/mL |
− |
− |
1.70 |
93.8 |
96.6 |
||
400 µg/mL |
− |
− |
13.80 |
95.1 |
93.4 |
||
800 µg/mL |
− |
− |
2.07 |
100.1 |
115.2 |
||
Positive control1 |
− |
− |
394.2 |
77.0 |
90.9 |
||
1 |
4 hrs |
Negative control |
+ |
− |
0.00 |
100.0 |
100.0 |
100 µg/mL |
+ |
− |
1.48 |
103.9 |
94.2 |
||
200 µg/mL |
+ |
− |
1.27 |
104.8 |
92.3 |
||
400 µg/mL |
+ |
− |
1.95 |
98.8 |
86.0 |
||
800 µg/mL |
+ |
− |
5.86 |
107.3 |
101.2 |
||
Positive control2 |
+ |
− |
116.00 |
108.8 |
89.8 |
||
2 |
4 hrs |
Negative control |
+ |
− |
8.15 |
100.0 |
100.0 |
200 µg/mL |
+ |
− |
4.39 |
102.0 |
89.6 |
||
400 µg/mL |
+ |
− |
3.20 |
103.8 |
94.9 |
||
600 µg/mL |
+ |
− |
1.90 |
105.7 |
86.9 |
||
800 µg/mL |
+ |
− |
5.48 |
109.2 |
92.6 |
||
Positive control2 |
+ |
− |
154.58 |
92.7 |
89.1 |
||
* Precipitation in culture medium at the end of exposure period |
|||||||
** Mutant frequency MFcorr.: number of mutant colonies per 10E^6 cells corrected with the CE2 value |
|||||||
*** Cloning efficiency related to the respective negative control |
|||||||
1 EMS 300 µg/mL 2 MCA 20 µg/mL |
|||||||
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Description of key information
- In vivo cytogenetics (OECD 474): negative in mouse micronucleus assay
Link to relevant study records
- 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
- 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
- Specific details on test material used for the study:
- SOURCE OF TEST MATERIAL
- Source: Japan Food Additives Association, Tokyo, Japan - 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 - 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
- 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) - 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.
- 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.
Reference
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
----------------------------------------------------
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Additional information
In vitro Gene Mutation in Bacteria Cells
In a guideline and GLP study, bacteria strains of S. typhimurium TA 98, TA 1535, TA 1537, TA 100, TA 1538 and Escherichia Coli WP2 uvrA were exposed to ammonium chloride (99.1 % pure) in water at concentrations of 0, 4, 20, 100, 500, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation using the preincubation method. No cytotoxity was observed up to the limit concentration. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background (Hoechst, 1987, summarised in OECD SIDS, 2004). The study was performed according to GLP and is acceptable for assessment.
In a second Ames test, bacteria strains of S. typhimurium TA 92, TA 94, TA 98, TA 1535, TA 1537 and TA 100 were exposed to ammonium chloride (99.7 % pure) in phosphate buffer at 6 concentrations up to 10000 µg/plate in the presence and absence of mammalian metabolic activation. The bateria cells were incuabted using the preincubation method. Results on cytotoxity were not given. The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background (Ishidate et al., 1984).
In vitro Cytogenicity in Mammalian Cells
In a mammalian cell cytogenetics assay (chromosome aberration), CHL cell cultures were exposed to ammonium chloride (99.7 % pure), in 0.9 % NaCl at 0.3, or 0.4 mg/mL without metabolic activation. A third unidentifiable concentration was also tested. There was evidence of chromosome aberration induced over background. The incidence of aberration in the untreated and solvent control was about 3.0 %. The incidence of cells with structural chromosomal aberrations at 0.4 mg/mL after 48 h treatment was 47.0 %. The incidences of structural aberrations at 0.3 mg/mL after 24 hours and 48 hours of exposure were 11.0 % and 30 %, respectively. Positive controls induced the appropriate response (Ishidate et al. 1984). This study is equivalent to OECD Guideline 473 (In vitro Mammalian Chromosome Aberration Test) with acceptable restrictions which were mainly in results of the reporting style.
In vitro Gene Mutation in Mammalian Cells
There are no data available for gene mutation toxicity in mammalian cells for ammonium chloride. Reliable studies performed with ammonium ion containing compounds ammonium sulfate (CAS 7783-20-2) or ammonium carbamate (1111-78-0) have been used to cover this endpoint.
In the study with ammonium sulfate, an HPRT test was conducted according to OECD 476 and GLP requirements (BASF SE/ Harlan CCR, 2010). CHL V79 cells were treated with the test substance at concentrations of 82.5, 165.0, 330.0, 660.0 and 1320.0 µg/mL (10 mM) with or without metabolic activation. The vehicle and positive controls induced the appropriate responses. There was no evidence of induced mutant colonies over background.
In the study with ammonium carbamate, an HPRT test was conducted according to OECD 476 and GLP requirements (BASF SE, 2010). CHO cells were treated with the test substance at concentrations from 100 to 800 µg/mL with or without metabolic activation. The vehicle and positive controls induced the appropriate responses. There was no evidence of induced mutant colonies over background.
In vivo Cytogenicity in Mammalian Cells
In a ddY mouse bone marrow micronucleus assay, 6 males per dose were treated by intraperitoneal (ip) injection with ammonium chloride (99.7% pure). Ammonium chloride was tested at an adequate dose based on a preliminary dose finding experiment. Administration was performed as a single injection at doses of 0, 62.5, 125, 250, 500 mg/kg bw and as multiple injections (4 times) at 24 hour intervals at doses of 0, 31.3, 62.5, 125, 250 mg/kg bw. Bone marrow cells were harvested 24 hours after the last injection. Mytomycin C, administered at 2.0 mg/kg bw via i.p. injection served as the positive control. 1000 PCE were scored for micronucleus. The vehicle was sodium chloride. No mortality occurred 24 hours after single dose treatment or the multiple dosing regimen. Percentage PCE/NCE was comparable in the bone marrow of treated and control animals after single and multiple exposure. The positive control induced the appropriate response. There was no significant increase in the frequency of micronucleated polychromatic erythrocytes in bone marrow after any treatment time (Hayashi, 1988). This study is acceptable for assessment. The study is equivalent to OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test) with acceptable restrictions (only 1000 PCE scored, incomprehensive reporting style).
Justification for classification or non-classification
Classification, Labelling, and Packaging
Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for
classification purposes under Regulation (EC) No 1272/2008. Based on
available data on genetic toxicity, the test item is not classified
according to Regulation (EC) No 1272/2008 (CLP), as amended for the
tenth time in Regulation (EU) No 2017/776.
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
Reproduction or further distribution of this information may be subject to copyright protection. Use of the information without obtaining the permission from the owner(s) of the respective information might violate the rights of the owner.
