Registration Dossier
Registration Dossier
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EC number: 939-066-9 | CAS number: -
Endpoint summary
Administrative data
Key value for chemical safety assessment
Genetic toxicity in vitro
Description of key information
In vitro gene mutation in bacteria: Weight of evidence: Based on experimental data on analogue substances (test method similar to OECD 471), Fatty acids, C16-18 (even numbered), ammonium salts was determined to be negative for genetic toxicity in vitro.
In vitro cytogenicity in mammalian cells: Weight of evidence: Based on experimental data on analogue substances (test method similar to OECD 473), Fatty acids, C16-18 (even numbered), ammonium salts was determined to be negative against chromosomal aberrations.
In vitro gene mutation study in mammalian cells: Key study: Experimental results on test item (OECD 476, GLP study). Fatty acids, C16-18 (even numbered), ammonium salts was considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells with and without metabolic activation.
Link to relevant study records
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 1989
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Remarks:
- A standard NTP (National Toxicology Program) protocol.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium, other: TA100, TA1535, TA97, TA98, TA104
- Metabolic activation:
- with and without
- Metabolic activation system:
- metabolic activation enzymes and cofactors from Aroclor 1254-induced male Sprague-Dawley rat and Syrian hamster liver (10 and 30%)
- Test concentrations with justification for top dose:
- Strains TA100, TA1535, TA97, TA98: 0 (control), 10, 33, 100, 333, 1000, 3333 and 10000 µg/plate
Strain TA104: 0 (control), 10, 33, 100, 333, 1000, 3333 and 10000 µg/plate - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO (Dimethyl Sulfoxide)
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- not specified
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
In the standard protocol (preincubation) for conducting the Ames assay, a test tube containing a suspension of one strain of Salmonella typhimurium plus S9 mix or plain buffer without S9, is incubated for 20 minutes at 37 ºC with the test chemical. Control cultures, with all the same ingredients except the test chemical, are also incubated. In addition, positive control cultures are prepared; these contain the particular bacterial tester strain under investigation, the various culture ingredients, and a known potent mutagen. After 20 minutes, agar is added to the cultures and the contents of the tubes are thoroughly mixed and poured onto the surface of Petri dishes containing standard bacterial culture medium. The plates are incubated, and bacterial colonies that do not require an excess of supplemental histidine appear and grow. These colonies are comprised of bacteria that have undergone reverse mutation to restore function of the histidine -manufacturing gene. The number of colonies is counted after 2 days. - Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 97
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle 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
- Vehicle 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
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium, other: TA104
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation occurred at >= 1000 µg/plate. - Conclusions:
- Stearic acid showed no mutagenicity in any tested strain, with or without metabolic activation.
- Executive summary:
A standard NTP (National Toxicology Program) protocol was performed to investigate the mutagenicity of Stearic acid. Test substance at 0 (control), 10, 33, 100, 333, 1000, 3333 and 10000 µg/plate was tested on Salmonella typhimurium TA100, TA1535, TA97, TA98 and TA104 by the preincubation assay (37°C, 20 min) with and without metabolic activation (induced male Sprague Dawley rat and male Syrian hamster livers at 10 and 30%).
Stearic acid showed no mutagenicity in any tested strain, with or without metabolic activation, under test conditions.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
- GLP compliance:
- yes
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 from rat liver, induced with phenobarbital and 5,6-benzoflavone
- Test concentrations with justification for top dose:
- 0 (control), 156, 313, 625, 1250, 2500 and 5000 µg/plate
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: DMSO
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- other: 2-(2-Furyl)-3-(5-nitro-2-furyl)acrylamide
- Remarks:
- Without metabolic activation; strains: TA100, TA98, WPr2 uvrA
- Positive controls:
- yes
- Positive control substance:
- sodium azide
- Remarks:
- Without metabolic activation; strain TA1535
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- Remarks:
- Without metabolic activation; strain TA1537
- Positive controls:
- yes
- Positive control substance:
- other: 9-Aminoanthracene
- Remarks:
- With metabolic activation, all strains
- Details on test system and experimental conditions:
- METHOD OF APPLICATION: preincubation
PLATES PER TEST: 3
NUMBER OF REPLICATIONS: 2
- Key result
- Species / strain:
- S. typhimurium TA 1535
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle 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
- Vehicle 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
- Vehicle 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
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Key result
- Species / strain:
- E. coli WP2
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Docosanoic acid was non-mutagenic in the bacterial reverse mutation test with and without metabolic activation.
- Executive summary:
A bacterial reverse mutation test was performed according to Japanese Guidelines for Screening Mutagenicity Testing of Chemicals and OECD Guidelines 471 and 472 on Docosanoic acid. Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA were exposed up to 5000 µg/plate test item with and without metabolic activation. Toxicity was not observed up to 5000 µg/plate in all strains. Docosanoic acid did not induce gene mutations with or without metabolic activation under test conditions.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 3 (not reliable)
- Rationale for reliability incl. deficiencies:
- other: Scientific Panel Review Article. Test method similar to OECD 471, basic data was given but no results on negative controls were reported. No data on GLP.
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 471 (Bacterial Reverse Mutation Assay)
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- S. typhimurium, other: TA98, TA100, TA1535, TA1537 and TA1538
- Metabolic activation:
- with and without
- Metabolic activation system:
- hepatic S9 fractions from rats induced with Aroclor 1254
- Test concentrations with justification for top dose:
- 50 mg/ml Stearic Acid suspensions in distilled water (50 µg/plate)
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- not specified
- Positive controls:
- yes
- Positive control substance:
- 9-aminoacridine
- sodium azide
- other: 2-aminoanthracene and 4-nitro-o-phenylenediamine in dimethyl sulfoxide
- 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:
- not specified
- 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:
- not specified
- 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:
- not specified
- 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:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- valid
- Conclusions:
- Stearic Acid had no mutagenic activity over background in the strains tested with and without metabolic activation.
- Executive summary:
Stearic Acid was tested for mutagenicity using the Ames test with Salmonella typhimurium strains TA98, TA1OO, TA1535, TA1537, and TA1538. Spot tests were performed using 50 mg/ml Stearic Acid suspensions in distilled water (50 µg/plate) with and without microsomal activation from hepatic S9 fractions from rats induced with Aroclor 1254 (50 µg/plate). Positive controls were 2-aminoanthracene and &nitro-o-phenylenediamine in dimethyl sulfoxide, 9-aminoacridine in ethanol, and sodium azide in distilled water. Stearic Acid had no mutagenic activity over background in the strains tested with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- 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)
- GLP compliance:
- not specified
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- other: S. typhimurium TA 1535, TA 1537, TA1538, TA 98, TA 100 and E. coli WP2 uvr A
- Metabolic activation:
- with and without
- Metabolic activation system:
- Rat liver S9 mix (induced with polychlorinated biphenyl, KC500).
- Test concentrations with justification for top dose:
- 500, 1000, 2500, 5000, 10000 and 25000 ppm.
- Vehicle / solvent:
- Sterilized air.
- Negative solvent / vehicle controls:
- yes
- Remarks:
- (sterilized air)
- Positive controls:
- yes
- Remarks:
- (Without metabolic activation)
- Positive control substance:
- 2-nitrofluorene
- Remarks:
- (Strains TA 100, TA 98, and TA 1538)
- Positive controls:
- yes
- Remarks:
- (Without metabolic activation)
- Positive control substance:
- N-ethyl-N-nitro-N-nitrosoguanidine
- Remarks:
- (Strains TA 1535, and WP2uvrA)
- Positive controls:
- yes
- Remarks:
- (Without metabolic activation)
- Positive control substance:
- 9-aminoacridine
- Remarks:
- (Strain TA 1537)
- Positive controls:
- yes
- Remarks:
- (With metabolic activation)
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- (Strains TA 100, TA 98, TA 1537, and TA 1538)
- Positive controls:
- yes
- Remarks:
- (With metabolic activation)
- Positive control substance:
- other: 2-aminoanthracene
- Remarks:
- (Strains TA 1535, and WP2uvrA)
- Details on test system and experimental conditions:
- Tests were performed in duplicate.
The agar plates were exposed without a lid in a glass chamber.
Exposure time: 48 h at 37 ºC.
The number of revertant colonies on each plate was scored with an automated colony counter. - 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
- 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
- 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
- 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
- Positive controls validity:
- valid
- Key result
- Species / strain:
- S. typhimurium TA 1538
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle 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:
- not specified
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Ammonia was negative in an Ames test with and without metabolic activation.
- Executive summary:
A bacterial reverse mutation assay was performed with Ammonia with Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 1538, and Escherichia coli WP2uvrA. The tested concentrations were: 500, 1000, 2500, 5000, 10000, and 25000 ppm. The test method was similar to OECD guideline 471. Ammonia was negative in an Ames test with and without metabolic activation.
- Endpoint:
- in vitro gene mutation study in bacteria
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 4 (not assignable)
- Rationale for reliability incl. deficiencies:
- documentation insufficient for assessment
- Remarks:
- A paper-disk test with an initial concentration of 107 cells/plate gave a doubling of mutant frequency. This test is not considered to be a standard test. No GLP.
- Principles of method if other than guideline:
- -Principle of test: A bacterial reverse mutation assay was performed with Ammonia, aqueous solution and E. coli Sd-4-73, without metabolic activation. The method employs streptomycin dependent mutants of Escherichia coli, which cannot grow on streptomycin free nutrient agar, but will produce a zone or a ring of streptomycin-independent revertant colonies around a drop or crystal of mutagen on the agar.
- GLP compliance:
- no
- Type of assay:
- bacterial reverse mutation assay
- Species / strain / cell type:
- E. coli, other: Sd-4-73
- Additional strain / cell type characteristics:
- other: Deficiency: streptomycin
- Metabolic activation:
- without
- Untreated negative controls:
- not specified
- Negative solvent / vehicle controls:
- not specified
- True negative controls:
- not specified
- Positive controls:
- not specified
- Details on test system and experimental conditions:
- Initial cell concentration: 1E+07 cells/plate
Administration: direct plating - Key result
- Species / strain:
- E. coli, other: Sd-4-73
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not specified
- Untreated negative controls validity:
- not specified
- Positive controls validity:
- not specified
- Conclusions:
- Aqueous ammonia was found to be not mutagenic in a bacterial reverse mutation assay without metabolic activation.
- Executive summary:
A bacterial reverse mutation assay was performed with Ammonia, aqueous solution and Eschericia coli Sd-4-73, without metabolic activation. The method employs streptomycin dependent mutants of Escherichia coli, which cannot grow on streptomycin free nutrient agar, but will produce a zone or a ring of streptomycin-independent revertant colonies around a drop or crystal of mutagen on the agar. Aqueous ammonia was found to be not mutagenic in a bacterial reverse mutation assay without metabolic activation.
- Endpoint:
- in vitro cytogenicity / chromosome aberration 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 473 (In Vitro Mammalian Chromosome Aberration Test)
- GLP compliance:
- yes
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- other: Chinese hamster lung (CHL) cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9 from rat liver, induced with phenobarbital and 5,6-benzoflavone
- Test concentrations with justification for top dose:
- -S9 mix (24hr continuous exposure): 0, 350, 700, 1400, 2800 µg/mL
-S9 mix (48hr continuous exposure): 0, 288, 575, 1150, 2300 µg/mL
-S9 mix (short-term exposure): 0, 875, 1750, 3500 µg/mL
+S9 mix(short-term exposure): 0, 875, 1750, 3500 µg/mL - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: 1% Carboxymethylcellulose sodium
- Negative solvent / vehicle controls:
- yes
- Positive controls:
- yes
- Positive control substance:
- mitomycin C
- Remarks:
- Without metabolic activation
- Positive controls:
- yes
- Positive control substance:
- cyclophosphamide
- Remarks:
- With metabolic activation
- Details on test system and experimental conditions:
- For continuous treatment, cells were treated for 24 or 48 hours without S9. For short-term treatment, cells were treated for 6 hours with and without S9 and cultivated with fresh media for 18 hours.
Plates per test: 2 - Key result
- Species / strain:
- other: Chinese hamster lung (CHL) cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- cytotoxicity
- Remarks:
- (at 2800 µg/mL, 24h, -S9 and at 2300 µg/mL, 48h, -S9).
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Conclusions:
- Docosanoic acid was non clastogenic in the in-vitro mammalian chromosome aberration test with and without metabolic activation.
- Executive summary:
An In-vitro Mammalian Chromosome Aberration Test was performed on Docosanoic acid according to the Japanese Guidelines for Screening Mutagenicity Testing of Chemicals and OECD Guideline 473. Chinese hamster lung (CHL) cells were exposed to the test item as the following:
For 24hr continuous exposure, at 0, 350, 700, 1400, 2800 µg/mL without metabolic activation;
For 48hr continuous exposure, at 0, 288, 575, 1150, 2300 µg/mL without metabolic activation;
For 6hr short-term exposure, at 0, 875, 1750, 3500 µg/mL without metabolic activation;
For 6hr short-term exposure, at 0, 875, 1750, 3500 µg/mL with metabolic activation;
Cytotoxicity was observed in the continuous exposure treatments at the highest dose. Docosanoic acid did not induce structural chromosomal aberrations with or without metabolic activation under test conditions.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Principles of method if other than guideline:
- -Principle of test: A mutagenic test with Ammonium sulfate was performed on Chinese Hamster Ovary cells. Various concentrations were tested: 0, 106, 211, and 423 mg/mL.
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Test concentrations with justification for top dose:
- 0, 0.8, 1.6, and 3.2 M
(0, 106, 211, and 423 mg/mL). - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- yes
- Remarks:
- (Alu buffer)
- True negative controls:
- no
- Positive controls:
- no
- Details on test system and experimental conditions:
- Pellets of 4 E+06 cells were made from cultures grown for 2 days and washed once with 1 mL newborn calf serum (NCS). The pellets received 24 units Alu I in a volume of 8 μL containing Alu I (3.4 or 4 μL) and NCS. After mixing, the cells were incubated for 20 minutes at 37 °C in the incubator.
Ammonium sulfate was dissolved in distilled water and 10 μL were added 5 minutes after the addition of the Alu I. The cells were mixed and reincubated for a further 15 minutes. Respective controls were set up in the same way, but instead of Alu I the cells were treated with the buffer in which the Alu I was shipped.
After the treatment the cells of each pellet were seeded in 2 6-cm petri dishes and incubated in medium containing fetal calf serum instead of NCS (10 %), and bromodeoxyuridine (BrdUrd) in a final concentration of 2 E-05 M.
Preparations were made 18 hours later including a 2-hour treatment with Colcemid. The chromosomes were differentially stained following the method of Hill and Wolff (1982) and exclusively first posttreatment metaphases were analyzed with respect to chromosomal aberrations. The number of dicentric chromosomes (DIC) per 100 metaphases were calculated from all polycentric chromosomes in such a way that from the polycentric chromosomes with more than 2 centromeres, one centromere was subtracted and the remaining number of centromeres was taken as the number of DIC.
Number of metaphases analyzed / number of independent experiments:
Alu I: 1100 / 11
Alu I + 0.8 M ammonium sulfate: 200 / 2
Alu I + 1.6 M ammonium sulfate: 200 / 2
Alu I + 3.2 M ammonium sulfate: 600 / 6
Alu buffer (control): 1100 / 11
Alu buffer + 3.2M ammonium sulfate: 200 / 2 - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Additional information on results:
- 3.2 M Ammonium sulfate did not induce chromosomal aberrations in Chinese hamster ovary (CHO) cells. However, high concentrations of ammonium sulfate increased the frequency of chromosome-type aberrations in CHO cells induced by the restriction endonuclease Alu I.
The results for cultures treated with Alu I, Alu I + ammonium sulfate and control cultures were as follows:
Treatment / Percent aberrant metaphases / DIC*
Alu I / 40.7 / 34.3
Alu I + 0.8 M (NH4)2SO4 / 59.0 / 22.0
Alu I + 1.6 M (NH4)2SO4 / 76.5 / 42.5
Alu I + 3.2 M (NH4)2SO4 / 98.2 / 240.3
control (Alu buffer) / 16.1 / 4.1
Alu buffer + 3.2 M (NH4)SO4 / 22.5 / 6.0
*DIC: Percent aberrant metaphases, including achromatic lesions and dicentric chromosomes per 100 metaphases as calculated from all polycentric chromosomes induced in CHO cells by 24 units Alu I in the presence or absence of salts. - Conclusions:
- Treatment of Chinese Hamster Ovary (CHO) cells with 3.2 M (423 mg/mL) ammonium sulfate in the absence of a metabolic activation system, did not result in chromosomal aberrations.
- Executive summary:
A mutagenic test with Ammonium sulfate was performed on Chinese Hamster Ovary cells. Various concentrations were tested: 0, 106, 211, and 423 mg/mL. Treatment of Chinese Hamster Ovary (CHO) cells with 3.2 M (423 mg/mL) ammonium sulfate in the absence of a metabolic activation system, did not result in chromosomal aberrations. However, ammonium sulfate enhanced the frequency of chromosome type aberrations, which had been induced by the restriction endonuclease Alu I, but this is not indicative of a mutagenic effect of ammonium sulfate.
- Endpoint:
- in vitro cytogenicity / chromosome aberration study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Principles of method if other than guideline:
- -Principle of test: The frequencies of chromosomal aberrations induced by the restriction endonuclease Alu I (recognition site AG/CT) can be elevated to a similar extent by additional treatments with a single-strand-specific endonuclease from Neurospora crassa (EC 3.1.30.1), or with ammonium sulfate in which the Neurospora endonuclease is suspended.
- GLP compliance:
- not specified
- Type of assay:
- in vitro mammalian chromosome aberration test
- Species / strain / cell type:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Test concentrations with justification for top dose:
- 3.2 M (423 mg/mL)
- Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: distilled water
- Untreated negative controls:
- no
- Negative solvent / vehicle controls:
- no
- True negative controls:
- no
- Positive controls:
- no
- Details on test system and experimental conditions:
- Chinese hamster ovary cells (CHO) were grown in McCoy's 5A medium, supplemented with 10% fetal calf serum, 100 units penicillin and 0.128 mg/mL dihydrostreptomycin sulfate. 4E +06 cells from exponentially growing cultures were centrifuged and repelleted in 1 mL newborn calf serum (NCS).
Alu I was prepared by mixing stock solution with NCS to give a final volume of 8 μL which was added to the pellet.
Neurospora endonuclease or 3.2 M ammonium sulfate solution in distilled water were added to the Alu I-treated cells at different times. The treatments were finished 20 minutes after addition to Alu I by washing the cells.
Controls included cells treated with medium + newborn calf serum (8 μL) and 5 minutes later with 10 μL 3.2 M ammonium sulfate or neurospora endonuclease, added for 15 minutes. Cells were fixed and stained 22 hours after treatment, including a treatment with colcemid (0.08 μg/mL) for 2 hrs.
The following aberrations were analysed: achromatic lesions, chromatid breaks, isochromatid/chromosome breaks, chromatid intrachanges, chromatid interchanges, triradials, polycentric chromosomes, ring chromosomes and minutes. - Key result
- Species / strain:
- Chinese hamster Ovary (CHO)
- Metabolic activation:
- without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- not specified
- Vehicle controls validity:
- not examined
- Untreated negative controls validity:
- not examined
- Positive controls validity:
- not examined
- Additional information on results:
- Effects of the restriction endonuclease Alu I on chromosomes were more pronounced when the cells were treated additionally with ammonium sulfate. The chromosomal aberration rates in cells treated with ammonium sulfate alone were not increased: % ABERRANT METAPHASES (200 metaphases analyzed, 22 hr after the treatment with 3.2 M ammonium sulfate for 15 min; including achromatic lesions): 15.5% (control with Neurospora crassa
endonuclease instead of ammonium sulfate: 20%).
The authors suggest that ammonium sulfate may induce conformational changes in the chromatin which make more recognition sites available for the Alu I enzyme. The frequencies of chromosomal aberrations induced by the restriction endonuclease Alu I (recognition site AG/CT) could be elevated to a similar extent by additional treatments with a single-strand-specific endonuclease from Neurospora crassa, or with ammonium sulfate in which the Neurospora endonuclease was suspended. - Conclusions:
- No chromosomal aberrations were found upon exposure of Chinese hamster ovary cells to ammonium sulfate.
- Executive summary:
The frequencies of chromosomal aberrations induced by the restriction endonuclease Alu I (recognition site AG/CT) can be elevated to a similar extent by additional treatments with a single-strand-specific endonuclease from Neurospora crassa (EC 3.1.30.1), or with ammonium sulfate in which the Neurospora endonuclease is suspended. No chromosomal aberrations were found upon exposure of Chinese hamster ovary cells to ammonium sulfate.
- Endpoint:
- in vitro gene mutation study in mammalian cells
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- August 10, 2012 - November 5, 2012
- 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)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Type of assay:
- in vitro mammalian cell gene mutation tests using the thymidine kinase gene
- Target gene:
- Thymidine kinase, TK +/-
- Species / strain / cell type:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Metabolic activation system:
- S9-mix
- Test concentrations with justification for top dose:
- Experiment I:
With metabolic activation: 1.0, 2.0, 5.0, 10.0, 25.0, 50.0, 100.0 and 200.0 µg/mL (4h exposure period)
Without metabolic activation: 1.0, 2.0, 5.0, 10.0, 20.0, 40.0, 50.0 and 80.0 µg/mL. (4h exposure period)
Experiment II:
With metabolic activation: 3.0, 7.0, 15.0, 30.0, 60.0, 90.0, 150.0 and 180.0 µg/mL (4h exposure period)
Without metabolic activation: 0.2, 0.5, 1.0, 3.0, 8.0, 10.0, 15.0 and 20.0 µg/mL (24h exposure period) - Vehicle / solvent:
- - Vehicle(s)/solvent(s) used: RPMI cell culture medium (RPMI + 5% HS)
- Justification for choice of solvent/vehicle: Based on the results of the solubility test. - Negative solvent / vehicle controls:
- yes
- Remarks:
- RPMI cell culture medium (RPMI + 5% HS)
- Positive controls:
- yes
- Remarks:
- Without metabolic activation
- Positive control substance:
- ethylmethanesulphonate
- Remarks:
- 300 µg/mL in medium
- Positive controls:
- yes
- Remarks:
- Without metabolic activation
- Positive control substance:
- methylmethanesulfonate
- Remarks:
- 8 and 10 µg/mL in 0.9% NaCl
- Positive controls:
- yes
- Remarks:
- With metabolic activation
- Positive control substance:
- benzo(a)pyrene
- Remarks:
- 1.5 and 2.5 µg/Ml in DMSO
- Details on test system and experimental conditions:
- METHOD OF APPLICATION:
Experiment I: Short-term exposure (4h): 1x10E+07 cells were suspended in 11 mL RPMI medium with 5% horse serum (25 cm2 flasks) and exposed to designated concentrations of the test item either in the presence of absence of metabolic activation. After exposure, test item was removed by centrifugation (200 x g, 10 min) and the cells were washed twice with PBS. Subsequently the cells were suspended in 30 mL complete culture medium and incubated for an expression and growth period. The cell density was determined each day and adjusted to 3x10E+05 cells/mL in a total culture volume of 20 mL, if necessary.
Experiment II: Long-term exposure (24h): 5x10E+06 cells were suspended in 25 mL RPMI medium with 7.5% horse serum (75 cm2 flasks) and exposed to designated concentrations of the test item in the absence of metabolic activation. After exposure, test item was removed by centrifugation (200 x g, 10 min) and the cells were washed twice with PBS. Subsequently 3x10E+05 cells were suspended in 14 mL complete culture medium and incubated for an expression and growth period. The cell density was determined each day and adjusted to 3x10E+05 cells/mL in a total culture volume of 20 mL, if necessary.
DURATION
- Exposure duration:
Experiment 1: 4 hours (with and without metabolic activation)
Experiment II: 4 hours (with metabolic activation) and 24 hours (without metabolic activation)
- Expression time (cells in growth medium): 2 days, at 37 ºC in 5% Co2/95% humidified air.
- Selection time (if incubation with a selection agent): ~ 14 days at 37 ºC in 5% CO2/95% humidified air
SELECTION AGENT (mutation assays): Cells from each experimental group were seeded in four 96-well plates at a density of approximately 2000 cells/well in 200 µL selective medium with TFT.
NUMBER OF REPLICATIONS: 1 replicate per dose (negative control, 2 replicates).
NUMBER OF CELLS EVALUATED: 2000 cells/well. The mutant frequency was calculated by dividing the number of TFT resistant colonies by the number of cells plated for selection, corrected for the plating efficiency of cells from the same culture grown in the absence of TFT.
DETERMINATION OF CYTOTOXICITY:
The toxicity of the test item was determined in pre-experiments up to a maximum concentration of 5 mg/mL. For experiment I (4h exposure) six concentrations (50, 150, 500, 1000, 2000 and 5000 µg/ml) were tested with and without metabolic activation. For the 24h long-term exposure assay (Experiment II, without metabolic activation) six concentrations (0.1, 1.0, 5.0, 10.0, 40.0 and 100.0 µg/mL) were tested. The number of cells at 4h, 24h and 48h after treatment were measured and the relative suspension growths were calculated.
In the main test the following parameters were calculated:
- Mutant frequency (MF)
- Cloning efficiency (CE) and relative cloning efficiency (RCe)
- Suspension growth (SG) and relative suspension growth (RSG)
OTHER EXAMINATIONS:
Colony sizing was performed for the highest concentrations of the test item and for the negative and positive controls. Small colonies were defined by slow growth and/or morphological alteration of the cell close. - Evaluation criteria:
- The test item is considered mutagenic if the following criteria are met:
- The induced mutant frequency meets or exceeds the Global Evaluation Factor (GEF) of 126 mutants per 1E+06 cells.
- A dose-dependent increase in mutant frequency is detected.
Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (>= 40$ of total colonies) is an indication for potential clastogenic effect and/or chromosomal aberrations. - Statistics:
- The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls.
- Key result
- Species / strain:
- mouse lymphoma L5178Y cells
- Metabolic activation:
- with and without
- Genotoxicity:
- negative
- Cytotoxicity / choice of top concentrations:
- no cytotoxicity
- Vehicle controls validity:
- valid
- Positive controls validity:
- valid
- Additional information on results:
- TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH-value of the test item suspension was adjusted with HCl to the physiological range.
- Precipitation: Precipitation of the test item was noted in the pre-experiment for experiment I with metabolic activation at concentrations of 500 µg/mL and higher and without metabolic activation at concentrations of 150 µg/mL and higher. No precipitations were noted in experiment I and II with and without metabolic activation and in pre-experiment II without metabolic activation.
RANGE-FINDING/SCREENING STUDIES: The selection of the concentrations used in the main experiments was based on data from the pre-experiments. In experiment I 200.0 µg/mL (with metabolic activation) and 80 µg/mL (without metabolic activation) were selected as the highest concentrations. In experimental II 180.0 µg/mL (with metabolic activation) and 20.0 µg/mL (without metabolic activation) were selected as the highest concentrations.
TOXICITY: Growth inhibition was observed in experiment I and II with and without metabolic activation.
MUTAGENICITY: In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF) was exceed by the induced mutant frequency at only one concentration (100 µg/mL) in experiment I with metabolic activation. The exceeded threshold of the GEF could not be verified in the second independent experiment with metabolic activation. Therefore, this effect was considered as not biologically relevant.
CLASTOGENICITY: In experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation).
CONTROLS: EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects. - Conclusions:
- Test item Fatty acids, C16-18 (even numbered), ammonium salts was considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells, under test conditions (with and without metabolic activation.
- Executive summary:
An in vitro gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells was performed on test item Fatty acids, C16 -18 (even numbered), ammonium salts in accordance with OECD Guideline 476. The selection of the concentrations used in the main experiment was based on the data from the pre-experiments. In experiment I 200.0 µg/mL (with metabolic activation, 4h short-term exposure) and 80.0 µg/mL (without metabolic activation, 4h short-term exposure) were selected as the highest concentrations. In experiment II 180.0 µg/mL (with metabolic activation, 4h short-term exposure) and 20.0 µg/mL (without metabolic activation, 24h long-term exposure) were selected as the highest concentrations. No precipitation was noted in the main experiment. Growth inhibition was observed in experiment I and II with and without metabolic activation. In experiment I and II no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). The Global Evaluation Factor (GEF) was exceed by the induced mutant frequency at only one concentration (100 µg/mL) in experiment I with metabolic activation. The exceeded threshold of the GEF could not be verified in the second independent experiment with metabolic activation. Therefore, this effect was considered as not biologically relevant. Additionally, in experiment I and II colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (with and without metabolic activation). EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects. Based on these results, test item Fatty acids, C16-18 (even numbered), ammonium salts was considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells, under test conditions (with and without metabolic activation.
Referenceopen allclose all
TA100
Dose |
No Activation |
No Activation |
10% HLI |
30% HLI |
10% RLI |
30% RLI |
(Negative) |
(Negative) |
(Negative) |
(Negative) |
(Negative) |
(Negative) |
|
Protocol |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Concentrations: 0 (control), 10, 33, 100, 333, 1000, 3333 and 10000 µg/plate and positive control.
Precipitation at 1000, 3333 and 10000 µg/plate.
TA1535
Dose |
No Activation |
No Activation |
No Activation |
10% HLI |
30% HLI |
10% RLI |
30% RLI |
(Equivocal) |
(Negative) |
(Negative) |
(Negative) |
(Negative) |
(Negative) |
(Negative) |
|
Protocol |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Concentrations: 0 (control), 10, 33, 100, 333, 1000, 3333 and 10000 µg/plate and positive control.
Precipitation at 1000, 3333 and 10000 µg/plate.
TA97
Dose |
No Activation |
No Activation |
No Activation |
10% HLI |
30% HLI |
10% RLI |
30% RLI |
30% RLI |
30% RLI |
(Negative) |
(Negative) |
(Negative) |
(Negative) |
(Negative) |
(Negative) |
(Not valid) |
(Equivocal) |
(Negative) |
|
Protocol |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Concentrations: 0 (control), 10, 33, 100, 333, 1000, 3333 and 10000 µg/plate and positive control.
Precipitation at 1000, 3333 and 10000 µg/plate.
TA98
Dose |
No Activation |
No Activation |
10% HLI |
30% HLI |
10% RLI |
30% RLI |
(Negative) |
(Negative) |
(Negative) |
(Negative) |
(Negative) |
(Negative) |
|
Protocol |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Concentrations: 0 (control), 10, 33, 100, 333, 1000, 3333 and 10000 µg/plate and positive control.
Precipitation at 1000, 3333 and 10000 µg/plate.
TA104
Dose |
No Activation |
No Activation |
10% HLI |
30% HLI |
10% RLI |
30% RLI |
(Equivocal) |
(Negative) |
(Negative) |
(Negative) |
(Negative) |
(Negative) |
|
Protocol |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Preincubation |
Concentrations: 0 (control), 10, 33, 100, 333 and 1000 µg/plate and positive control.
Precipitation at 1000 µg/plate.
Results of the bacterial reversión test of docosanoic acid (1st trial) (direct method: -S9):
Compound |
Dose (µg/plate) |
Revertant colonies per plate (Mean ± SD) |
||||
TA100 |
TA1535 |
WP2 uvrA |
TA98 |
TA1537 |
||
Test substance |
0 |
95±12 |
15±1 |
25±2 |
23±3 |
9±2 |
156 |
94±3 |
11±1 |
27±3 |
21±2 |
8±3 |
|
313 |
94±4 |
12±4 |
24±1 |
21±4 |
10±2 |
|
625 |
89±2 |
14±3 |
26±1 |
18±2 |
8±1 |
|
1250 |
82±4 |
13±1 |
25±3 |
21±3 |
8±3 |
|
2500 |
80±2 |
11±1 |
26±1 |
17±3 |
10±4 |
|
5000 |
78±2 |
9±2 |
28±1 |
14±4 |
10±3 |
|
Positive control |
|
687±12 |
422±7 |
173±10 |
460±27 |
791±28 |
+: Visible precipitation was occurred at the end of exposure period
Positive controls: TA100: TA98: AF-2, 0.01 µg/plate; TA1535: NaN3: 0.5 µg/plate; WP2 uvrA: AF-2, 0.01 µg/plate: TA98: AF-2, 0.1 µg/plate; TA1537 ACR, 80 µg/plate.
Results of the bacterial reversión test of docosanoic acid (1st trial) (direct method: +S9):
Compound |
Dose (µg/plate) |
Revertant colonies per plate (Mean ± SD) |
||||
TA100 |
TA1535 |
WP2 uvrA |
TA98 |
TA1537 |
||
Test substance |
0 |
112±4 |
13±1 |
31±2 |
33±2 |
7±1 |
156 |
103±4 |
12±3 |
27±3 |
30±3 |
10±3 |
|
313 |
106±10 |
10±4 |
26±1 |
34±4 |
11±3 |
|
625 |
97±3 |
12±2 |
27±1 |
29±4 |
10±3 |
|
1250 |
97±5 |
12±4 |
28±5 |
30±2 |
10±1 |
|
2500 |
98±3 |
12±3 |
27±3 |
30±2 |
10±3 |
|
5000 |
99±9 |
9±2 |
28±2 |
29±1 |
9±1 |
|
Positive control |
|
723±12 |
383±5 |
516±5 |
366±19 |
172±8 |
+: Visible precipitation was occurred at the end of exposure period
Positive controls: TA100: 2-AA, 1 µg/plate; TA1535: 2-AA: 2 µg/plate; WP2 uvrA: 2-AA, 10 µg/plate; TA98: 2-AA, 0.5 µg/plate; TA1537: 2-AA: 2 µg/plate.
Results of the bacterial reversión test of docosanoic acid (2nd trial) (direct method: -S9):
Compound |
Dose (µg/plate) |
Revertant colonies per plate (Mean ± SD) |
||||
TA100 |
TA1535 |
WP2 uvrA |
TA98 |
TA1537 |
||
Test substance |
0 |
95±2 |
16±2 |
26±3 |
23±2 |
5±1 |
156 |
94±3 |
14±1 |
25±1 |
23±3 |
6±2 |
|
313 |
95±3 |
14±1 |
26±2 |
23±4 |
6±2 |
|
625 |
90±0 |
17±1 |
24±3 |
22±2 |
7±0 |
|
1250 |
89±5 |
15±1 |
25±3 |
21±2 |
5±2 |
|
2500 |
85±5 |
15±2 |
23±1 |
20±1 |
7±1 |
|
5000 |
88±3 |
15±3 |
22±2 |
20±1 |
4±1 |
|
Positive control |
|
734±14 |
433±3 |
128±6 |
439±7 |
503±6 |
+: Visible precipitation was occurred at the end of exposure period
Positive controls: TA100: TA98: AF-2, 0.01 µg/plate; TA1535: NaN3: 0.5 µg/plate; WP2 uvrA: AF-2, 0.01 µg/plate: TA98: AF-2, 0.1 µg/plate; TA1537 ACR, 80 µg/plate.
Results of the bacterial reversión test of docosanoic acid (2nd trial) (activation method: +S9):
Compound |
Dose (µg/plate) |
Revertant colonies per plate (Mean ± SD) |
||||
TA100 |
TA1535 |
WP2 uvrA |
TA98 |
TA1537 |
||
Test substance |
0 |
107±3 |
15±2 |
29±2 |
26±2 |
14±1 |
156 |
100±2 |
15±1 |
28±1 |
29±2 |
14±2 |
|
313 |
99±4 |
13±1 |
29±3 |
30±4 |
14±1 |
|
625 |
103±3 |
14±2 |
27±2 |
30±4 |
15±2 |
|
1250 |
101±4 |
12±2 |
28±5 |
28±2 |
13±1 |
|
2500 |
98±2 |
13±2 |
26±2 |
26±1 |
11±1 |
|
5000 |
96±5 |
10±1 |
26±2 |
24±1 |
12±2 |
|
Positive control |
|
678±17 |
393±5 |
521±22 |
423±31 |
173±13 |
+: Visible precipitation was occurred at the end of exposure period
Positive controls: TA100: 2-AA, 1 µg/plate; TA1535: 2-AA: 2 µg/plate; WP2 uvrA: 2-AA, 10 µg/plate; TA98: 2-AA, 0.5 µg/plate; TA1537: 2-AA: 2 µg/plate.
Stearic Acid had no mutagenic activity over background in the strains tested with and without metabolic activation.
Ammonia was negative in an Ames test with and without metabolic activation.
Aqueous ammonia was found to be not mutagenic in a bacterial reverse mutation assay without metabolic activation.
Cytotoxicity: 50% inhibition of cell proliferation was observed at 2703 µg/mL for 24 hours continous treatment and at 2242 µg/mL for 48 hours continuous treatment, respectively. Cell proliferation inhibition was not observed in short-term treatment with or without metabolic activation.
Structural chromosomal aberrations and polyploidy were not induced up to a maximum concentration of test substance under conditions of continuous treatment, and short-term treatment with and without metabolic activation.
Treatment of Chinese Hamster Ovary (CHO) cells with 3.2 M (423 mg/mL) ammonium sulfate in the absence of a metabolic activation system, did not result in chromosomal aberrations.
No chromosomal aberrations were found upon exposure of Chinese hamster ovary cells to ammonium sulfate.
Summary: Experiment I and II with metabolic activation:
|
Test groups |
Conc [µg/ml] |
RCE [µg/ml] |
RTG [%] |
MF [mutants/106cells] |
IMF [mutants/106cells] |
GEF exceded |
Statistical Significance |
Precipitate |
Exp I |
C1 |
0 |
100.0 |
100.0 |
129.1 |
/ |
/ |
- |
- |
C2 |
/ |
/ |
- |
- |
|||||
1 |
1.0 |
94.8 |
97.9 |
114.7 |
-14.4 |
- |
- |
- |
|
2 |
2.0 |
79.9 |
86.2 |
116.1 |
-13.0 |
- |
- |
- |
|
3 |
5.0 |
90.4 |
78.8 |
148.6 |
19.5 |
- |
- |
- |
|
4 |
10.0 |
87.6 |
72.7 |
122.3 |
-6.8 |
- |
- |
- |
|
5 |
25.0 |
81.1 |
70.6 |
147.0 |
17.9 |
- |
- |
- |
|
6 |
50.0 |
78.7 |
51.2 |
155.2 |
26.1 |
- |
- |
- |
|
7 |
100.0 |
74.2 |
21.8 |
289.1 |
160.0 |
+ |
+ |
- |
|
10 |
200.0 |
72.1 |
12.7 |
225.7 |
96.9 |
- |
+ |
- |
|
B[a]P |
2.5 |
60.7 |
25.4 |
848.6 |
719.5 |
+ |
+ |
- |
|
Exp II |
C1 |
0 |
100.0 |
100.0 |
86.6 |
/ |
/ |
/ |
- |
C2 |
/ |
/ |
/ |
- |
|||||
3 |
3.0 |
99.7 |
91.5 |
86.6 |
4.9 |
- |
- |
- |
|
4 |
7.0 |
109.7 |
98.1 |
91.4 |
-14.0 |
- |
- |
- |
|
5 |
15.0 |
82.1 |
74.6 |
72.5 |
16.1 |
- |
- |
- |
|
6 |
30.0 |
88.3 |
81.9 |
102.7 |
1.4 |
- |
- |
- |
|
7 |
60.0 |
89.6 |
48.7 |
101.4 |
14.9 |
- |
- |
- |
|
8 |
90.0 |
84.5 |
33.0 |
119.1 |
32.5 |
- |
+ |
- |
|
10 |
150.0 |
77.5 |
24.3 |
110.6 |
24.0 |
- |
+ |
- |
|
11 |
180.0 |
64.4 |
11.9 |
151.2 |
64.7 |
- |
+ |
- |
|
B[a]P |
1.5 |
67.2 |
37.8 |
690.7 |
604.1 |
+ |
+ |
- |
Summary: Experiment I and II without metabolic activation:
|
Test groups |
Conc [µg/ml] |
RCE [µg/ml] |
RTG [%] |
MF [mutants/106cells] |
IMF [mutants/106cells] |
GEF exceded |
Statistical Significance |
Precipitate |
Exp I |
C1 |
0 |
100.0 |
100.0 |
121.4 |
/ |
/ |
- |
- |
C2 |
/ |
/ |
- |
- |
|||||
3 |
1.0 |
116.3 |
114.4 |
111.2 |
-10.3 |
- |
- |
- |
|
4 |
2.0 |
100.7 |
98.6 |
123.1 |
1.7 |
- |
- |
- |
|
5 |
5.0 |
88.4 |
81.6 |
180.2 |
58.8 |
- |
- |
- |
|
6 |
10.0 |
104.2 |
98.0 |
128.9 |
7.4 |
- |
- |
- |
|
7 |
20.0 |
84.4 |
42.4 |
216.3 |
94.8 |
- |
+ |
- |
|
8 |
40.0 |
100.7 |
9.3 |
145.2 |
23.8 |
- |
- |
- |
|
9 |
50.0 |
78.3 |
15.1 |
180.2 |
58.8 |
- |
+ |
- |
|
12 |
80.0 |
57.1 |
10.1 |
232.9 |
111.5 |
- |
+ |
- |
|
EMS |
300 |
77.1 |
64.9 |
687.4 |
566.0 |
+ |
+ |
- |
|
MMS |
10 |
64.8 |
45.2 |
690.6 |
569.2 |
+ |
+ |
- |
|
Exp II |
C1 |
0 |
100.0 |
100.0 |
60.7 |
/ |
/ |
/ |
- |
C2 |
/ |
/ |
/ |
- |
|||||
2 |
0.2 |
105.0 |
95.4 |
67.2 |
6.6 |
- |
- |
- |
|
3 |
0.5 |
110.1 |
100.8 |
60.0 |
-0.7 |
- |
- |
- |
|
4 |
1.0 |
94.4 |
91.2 |
68.3 |
7.6 |
- |
- |
- |
|
5 |
3.0 |
85.1 |
76.6 |
83.2 |
22.5 |
- |
- |
- |
|
7 |
8.0 |
108.4 |
70.6 |
89.1 |
28.4 |
- |
- |
- |
|
8 |
10.1 |
95.8 |
52.2 |
67.2 |
6.5 |
- |
- |
- |
|
9 |
15.0 |
91.6 |
22.4 |
82.4 |
21.7 |
- |
+ |
- |
|
10 |
20.0 |
80.4 |
7.7 |
83.9 |
23.2 |
- |
- |
- |
|
EMS |
200 |
61.4 |
24.2 |
1617.7 |
1557.0 |
+ |
+ |
- |
|
MMS |
8 |
65.0 |
17.0 |
725.8 |
665.1 |
+ |
+ |
- |
C: Negative controls
RCE: Relative Cloning Efficiency
RTG: Relative Total Growth
MF: Mutant Frequency
IMF: Induced Mutant Frequency
GEF: Global Evaluation Factor
Statistical Significance: Mann Whitney test, p<0.05
B[a]P: Benzo[a]pyrene
EMS: Ethylmethanesulfonate
MMS: Methylmethanesulfonate
Endpoint conclusion
- Endpoint conclusion:
- no adverse effect observed (negative)
Genetic toxicity in vivo
Endpoint conclusion
- Endpoint conclusion:
- no study available
Additional information
IN-VITRO GENE MUTATION IN BACTERIA:
Weight of Evidence Approach (see rationale attached in IUCLID5 Section 13):
A standard NTP (National Toxicology Program) protocol was performed to investigate the mutagenicity of Stearic acid. Test substance was tested up to 10000 µg/plate on Salmonella typhimurium TA100, TA1535, TA97, TA98 and TA104 with and without metabolic activation. Stearic acid showed no mutagenicity in any tested strain, with or without metabolic activation, under test conditions.
A bacterial reverse mutation test was performed by the Ministry of Health, Labour and Welfare (Japan, 1998) according to Japanese Guidelines for Screening Mutagenicity Testing of Chemicals and OECD Guidelines 471 and 472 on Docosanoic acid. Salmonella typhimurium TA100, TA1535, TA98, TA1537 and Escherichia coli WP2 uvrA were exposed up to 5000 µg/plate test item with and without metabolic activation. Docosanoic acid did not induce gene mutations with or without metabolic activation under test conditions.
As reported by Cosmetic Ingredient Review Panel (1987), Stearic Acid was tested for mutagenicity using the Ames test with Salmonella typhimurium strains TA98, TA1OO, TA1535, TA1537, and TA1538. Spot tests were performed using 50 mg/ml test item suspensions in distilled water (50 µg/plate) with and without microsomal activation. Stearic Acid had no mutagenic activity over background in the strains tested with and without metabolic activation.
A bacterial reverse mutation assay (test method similar to OECD 471) was performed by Shimizu H et al. (1985) with Ammonia up to 25000 ppm in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 1538, and Escherichia coli WP2uvrA. Ammonia was negative with and without metabolic activation.
A bacterial reverse mutation assay was performed by Szybalski W (1958) with Ammonia, aqueous solution in Escherichia coli Sd-4-73, without metabolic activation. The method employs streptomycin dependent mutants of Escherichia coli, which cannot grow on streptomycin free nutrient agar, but will produce a zone or a ring of streptomycin-independent revertant colonies around a drop or crystal of mutagen on the agar. Aqueous ammonia was found to be not mutagenic without metabolic activation.
According to the available data, the weight of evidence approach was applied and the substance Fatty acids, C16 -18 (even numbered), ammonium salts was determined to be negative for in vitro gene mutation in bacteria.
IN-VITRO CYTOGENICITY IN MAMMALIAN CELLS:
Weight of Evidence Approach (see rationale attached in IUCLID5 Section 13):
An in-vitro Mammalian Chromosome Aberration Test was performed by the Ministry of Health, Labour and Welfare (Japan, 1998) on Docosanoic acid according to the Japanese Guidelines for Screening Mutagenicity Testing of Chemicals and OECD Guideline 473. Chinese hamster lung (CHL) cells were exposed to the test item as the following up to 3500 µg/mL and 2800 µg/mL with and without metabolic activation respectively. Cytotoxicity was observed in the continuous exposure treatments at the highest dose. Docosanoic acid did not induce structural chromosomal aberrations with or without metabolic activation under test conditions.
As reported by Obe G et al. (1986), a mutagenic test with Ammonium sulfate was performed on Chinese Hamster Ovary cells up to 3.2 M (423 mg/mL). Ammonium sulfate in the absence of a metabolic activation system did not result in chromosomal aberrations. However, ammonium sulfate enhanced the frequency of chromosome type aberrations, which had been induced by the restriction endonuclease Alu I, but this is not indicative of a mutagenic effect of ammonium sulfate.
In accordance with Obe G et al. (1986) the frequencies of chromosomal aberrations induced by the restriction endonuclease Alu I (recognition site AG/CT) can be elevated to a similar extent by additional treatments with a single-strand-specific endonuclease from Neurospora crassa (EC 3.1.30.1), or with ammonium sulfate in which the Neurospora endonuclease is suspended. No chromosomal aberrations were found upon exposure of Chinese hamster ovary cells to ammonium sulfate.
According to the available data, the weight of evidence approach was applied and the substance Fatty acids, C16 -18 (even numbered), ammonium salts was determined to be negative for in vitro cytogenicity in mammalian cells.
IN VITRO GENE MUTATION IN MAMMALIAN CELLS:
Key study: Experimental results on Fatty acids, C16 -18 (even numbered), ammonium salts:
An in vitro gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells was performed on test item Fatty acids, C16 -18 (even numbered), ammonium salts in accordance with OECD Guideline 476 (GLP study). Based on the results from the preliminary study, the substance was tested up to 200.0 µg/mL and 80 µg/mL with and without metabolic activation respectively. Growth inhibition was observed but no biologically relevant increase of mutants was found after treatment with the test item. The Global Evaluation Factor (GEF) was exceeded by the induced mutant frequency at only one concentration (100 µg/mL) in experiment I with metabolic activation. Nevertheless, this effect was considered as not biologically relevant. Additionally, colony sizing showed no clastogenic effects induced by the test item. Test item Fatty acids, C16-18 (even numbered), ammonium salts was considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells with and without metabolic activation.
Justification for selection of genetic toxicity endpoint
No study was selected, since the studies were negative in a weight of evidence approach.
Justification for classification or non-classification
Based on the available information, Fatty acids, C16 -18 (even numbered), ammonium salts is considered to be negative for genetic toxicity, and therefore the substance is not classified according to CLP Regulation (EC) No 1272/2008.
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