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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro

Ammonium sulfate was not mutagenic in bacteria (Ames test), yeasts, and mammalian cells (HPRT) with and without metabolic activation systems. It did not induce chromosomal aberrations in mammalian or human cell cultures.

In some cases, detailed data on purity are lacking. However, the terms "food grade" and "analytical grade" imply high purity.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Test with Salmonella typhimurium TA 102 or Escherichia coli WP2 strains not included.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S-9
Test concentrations with justification for top dose:
20, 100, 500, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: distilled water
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene, N-methyl-N'-nitro-N-nitrosoguanidine, 4-nitro-o-phenylendiamine, 9-aminoacridine chloride monohydrate
Remarks:
positive control substance depending on the tester strain and activation condition; positive control substances were dissolved in dimethylsulfoxide (DMSO)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
NUMBER OF REPLICATIONS: 3 test plates per dose or per control

DETERMINATION OF CYTOTOXICITY
- Method: reduced his- background growth
Evaluation criteria:
In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results.
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and 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
Positive controls validity:
valid

No increase in the number of his+ revertants was observed. No bacteriotoxic effect (reduced his- background growth) was observed. The test substance was completely soluble; no precipitation was observed at any concentration tested.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
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)
Remarks:
Harlan Cytotest Cell Research GmbH, In Leppsteinswiesen 19, 64380 Rossdorf, Germany
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:
- Vehicle(s)/solvent(s) used: acetone
- Justification for choice of solvent/vehicle: 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:
ethylmethanesulphonate
Remarks:
without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
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 SYSTAT11 (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.

COMPARISON WITH HISTORICAL CONTROL DATA:

ADDITIONAL INFORMATION ON CYTOTOXICITY:

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, Ammonium sulphate is considered to be non-mutagenic in this HPRT assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Limited documentation; only one dose level, no standard test system for chromosomal aberrations.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
2 prepatation times and one concentration only
GLP compliance:
not specified
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
- Type and identity of media: McCoy's 5A medium supplemented with 20% fetal calf serum, 0.06 mL phytohemagglutinin M, 100 U/mL penicillin and 0.1 mg/mL dihydrostreptomycin sulfate.
Metabolic activation:
without
Test concentrations with justification for top dose:
ca. 423 mg/ml (3.2 M)
Vehicle / solvent:
water, medium or Alu I shipping buffer (KPO4 = 20 mM ; KC1= 50 mM; EDTA = 0 .1 mM ; dithioerythritol = 10 mM; glycerin = 50% (v/v); pH = 7.5)
Negative solvent / vehicle controls:
yes
Positive controls:
no
Details on test system and experimental conditions:
Lymphocytes were isolated from whole blood in a Ficoll gradient. 1x10E6 lymphocytes were incubated in 5 mL plastic tubes in 2.5 mL McCoy's 5A medium.

METHOD OF APPLICATION: in medium

DURATION
The cultures were incubated for 50 h, including a treatment with colcemid (0.08 µg/ml) for 3 h. Treatment with Alu I, (NH4)2S04 or Alu I shipping buffer was done at culture times of 20 and 46 h.

SPINDLE INHIBITOR (cytogenetic assays): colcemid, 0.08 µg/ml

NUMBER OF REPLICATIONS: up to 7

NUMBER OF CELLS EVALUATED: 200 metaphases
Key result
Species / strain:
lymphocytes: human
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified

Effects of the restriction endonuclease Alu I on chromosomes were more pronounced when the cells were treated additionally

with the test substance. The chromosomal aberrations rates in cells treated with ammonium sulfate alone were not increased. In control cells, 5.5% of aberrant metaphases in 200 analysed cells were observed. In cells treated with ammonium sulfate (20h), 6 % of aberrant metaphases were observed. The treatment with ammonnium sulfate induced no clastogenic effect in human lymphocytes.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo:

No in vivo genotoxicity tests are available. Based on the negative results from in vitro studies and the negative results in the bone marrow micronucleus test in vivo with ammonium chloride a mutagenic activity of ammonium sulfate in vivo is unlikely.



Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
no data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Limited documentation
Principles of method if other than guideline:
The maximum doses of the test compounds were determined by pilot experiments using the multisampling at multi-dose levels method according to Hayashi M et al (1984). A pilot experiment for the micronucleus test. The multi-sampling at multi-dose levels method. Mutat Res 141:, 165.
GLP compliance:
not specified
Type of assay:
micronucleus assay
Species:
mouse
Strain:
other: ddY
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
male ddY mice
- Source: Shizuoka Agricultural Cooperative Association for Laboratory Animals, Shizuoka
- Age at study initiation: 8 weeks
- Diet (ad libitum): food pellets CE-2 (Japan Clea, Tokyo)
- Water: ad libitum
No further data.

ENVIRONMENTAL CONDITIONS: no data
Route of administration:
intraperitoneal
Vehicle:
- Vehicle(s)/solvent(s) used: physiol. saline
Duration of treatment / exposure:
single dose (pilot study) or 4 doses, divided by 24 hour intervals (see table below, Remarks on results)
Post exposure period:
24 hours after dosing
Dose / conc.:
62.5 mg/kg bw/day
Remarks:
single dosing
Dose / conc.:
125 mg/kg bw/day
Remarks:
single dosing
Dose / conc.:
250 mg/kg bw/day
Remarks:
single dosing
Dose / conc.:
500 mg/kg bw/day
Remarks:
single dosing
Dose / conc.:
31.3 mg/kg bw/day
Remarks:
multiple dosing
Dose / conc.:
62.5 mg/kg bw/day
Remarks:
multiple dosing
Dose / conc.:
125 mg/kg bw/day
Remarks:
multiple dosing
Dose / conc.:
250 mg/kg bw/day
Remarks:
multiple dosing
No. of animals per sex per dose:
6 mice per group
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C, 2.0 mg/kg bw
Tissues and cell types examined:
bone marrow (femur)
Details of tissue and slide preparation:
Mice were killed by cervical dislocation at the appropriate time after an administration. Femoral marrow cells were flushed out with fetal bovine serum and smeared on clean glass slides. Cells were fixed with methanol for 5 min, and stained with Acridine Orange for the pilot experiment and with Giemsa for the full-scale test.
The preparations were coded and analysed without any knowledge of the treatment. One thousand polychromatic erythrocytes per mouse were scored using a light microscope, with a high power objective (x 100), and the number of micronucleated polychromatic erythrocytes (MNPCEs) was recorded. The proportion of polychromatic erythrocytes (PCEs) among the total erythrocytes was also evaluated by observing 1000 erythrocytes on the same slide.
Statistics:
The dose-response relationships were tested using the Cochran-Armitage trend test. A positive dose-response was considered significant at P < 0.05.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Positive controls validity:
valid

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

Compound

Route

No.

of

doses

Time

between

doses

(hr)

Sampling

Time

(hr)

Dose

level

(mg/kg)

NMPCE

(%)

PCE

(%)

Mortality

Single dosing

Ammonium chloride

ip

1

24

0

62.5

125

250

500

0.18 +/- 0.18

0.12 +/- 0.12

0.15 +/- 0.14

0.13 +/- 0.05

0.12 + -/ 0.08

56.8 +/- 4.7

60.9 +/- 4.2

91.7 +/- 3.8

64.3 +/- 2.5

64.3 +/- 2.5

0/6

0/6

0/6

0/6

0/6

Mitomycin

(positive control)

ip

1

24

2.0

4.18 +/- 1.30 *

52.3 +/- 4.6

0/6

Repeated dosing

Ammonium chloride

ip

4

24

24

0

31.3

62.5

125

250

0.20 +/- 0.09

0.25 +/- 0.19

0.19 +/- 0.10

0.20 +/- 0.08

0.17 +/- 0.08

59.9 +/- 8.3

67.2 +/- 13.5

63.7 +/- 4.5

64.0 +/- 9.2

61.6 +/- 6.9

0/6

0/6

0/6

0/6

0/6

Mitomycin

(positive control)

ip

1

24

2.0

7.15 +/- 3.92 *

32.2 +/- 11.0

0/6

(*) p<0.01

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro Studies

Ammonium sulfate (purity 99.5%) was not mutagenic in the standard plate and pre-incubation Ames test performed in 4 strains of Salmonella typhimurium (TA1535, TA100, TA1537, TA98) with and without a metabolic activation system up to and including the maximum tested concentration of 5000 µg/plate. No cytotoxic effects were observed (BASF, 1989).

Ammonium sulfate (food grade; no further data on purity) was also not mutagenic in Salmonella typhimurium strains TA1535, TA1537 and TA1538, and in Saccharomyces cerevisiae D4 with and without metabolic activation systems. Again, no cytotoxic effects were observed up to the highest tested concentration of 50000 ppm (Litton Bionetics, 1975).

Treatment of Chinese Hamster Ovary (CHO) (Tuschy and Obe, 1988) cells and treatment of human lymphocytes (Obe et al., 1986) with 3.2 M (423 mg/ml) ammonium sulfate (analytical grade; no further data on purity) 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 1. A similar effect was observed with other salts (magnesium chloride, calcium chloride and sodium chloride), and is not indicative of a mutagenic effect of ammonium sulfate (Obe et al., 1986; Tuschy and Obe, 1988).

A study was performed to investigate the potential of Ammonium sulphate to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster (BASF, 2010). The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a treatment time of 4 hours with and 24 hours without metabolic activation. The highest concentration of 1320 µg/mL was equal to a molar concentration of about 10 mM. No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments. Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

In vivo Studies

There are no in vivo studies available with ammonium sulfate. Ammonium chloride (99.7% pure) gave negative results in a micronucleus test in mice (Hayashi et al., 1988). This micronucleus test was conducted with bone marrow in ddY mice. Animals had received a single and 4 times i.p. injection. A single dose test and 4 times dose test were dosed with 62.5 - 500 mg/kg and 31.3 - 250 mg/kg as MTD (maximum tolerated dose), respectively, with mitomycin C as positive control. No increase of erythrocytes with micronuclei was observed at any group. The potential of ammonium sulfate to induce mutagenic effects in vivo is considered to be negligible, because there is no evidence of a mutagenic effect from in vitro studies.


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 the available data, the substance is not considered to be classified for genetic toxicity under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EU) No 2016/1179.