Ammonium nitrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

A chromosome aberration study with ammonium nitrate was performed according to OECD test guideline 473 with and without metabolic activation. No effects were found up to the highest tested concentration of 10 mM.

Three in vitro studies were performed with nitrates from which the results can be read across to ammonium nitrate: in an in vitro Ames test performed according to OECD test guideline 471, 4 strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and an E. coli bacteria (WP2 uvr A) were exposed to nitric acid, ammonium calcium salt and did not show mutagenicity with or without metabolic activation up to the limit concentration. In a second in vitro study also with nitric acid, ammonium calcium salt human lymphocytes with or without metabolic activation according to OECD test guideline 473 were observed for chromosome abberrations. No effects were found up with either precipitating concentrations or toxic concentrations depending on the exposure time. A third in vitro study which was performed with potassium nitrate in an in vitro TK assay in L5178Y mouse lymphoma cells performed according to OECD 476, showing no genotoxicity.

Based on the results of these four studies, it is concluded that ammonium nitrate is not considered genotoxic. The read across rationale can be found in the target study records.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 05-18, 2006

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was performed with a substance analogue and the data are read across.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine gene in S. typhimurium
Tryptophan gene in E. coli

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver S9 mix

Test concentrations with justification for top dose:

dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
first and second test: 100, 333, 1000, 3330, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- Justification for choice of solvent/vehicle: at concentrations of 33.3 mg/ml and lower the test substance was dissolved in the vehicle.

Negative solvent / vehicle controls:

yes

Remarks:

Dimethyl sulfoxide

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix Migrated to IUCLID6: 5 μg/plate in saline for TA1535

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix Migrated to IUCLID6: 60 µg/plate in mili-Q water for TA1537

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9-mix Migrated to IUCLID6: 10 µg/plate in DMSO for TA98

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9-mix Migrated to IUCLID6: 650 µg/plate in DMSO for TA100

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene in DMSO (5 and/or 10%) for all tester strains

Remarks:

with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)


DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: doses of the test substance were tested in triplicates in each strain. Two independent experiments were conducted.

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the relevant colonies


OTHER EXAMINATIONS:
- Other: precipitation of the test substance

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent
control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater
than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is greater than two (2) times the concurrent control, or
the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3)
times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one
independently repeated experiment.

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

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

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: The test substance did not precipitate at the start or at the end of the incubation period


RANGE-FINDING/SCREENING STUDIES:
No toxicity and mutagenicity was observed up to concentrations of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
The negative and strain-specific positive control values were within the laboratory historical control data
ranges indicating that the test conditions were adequate and that the metabolic activation system functioned
properly.

Conclusions:

All bacterial strains showed negative responses over the entire dose range, i.e. no significant dose-related increase in the number of revertants in two independently repeated experiments.
The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metobolic activation system functioned properly.
Based on the results of this study it is concluded that CN-Nitcal is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

09-Apr-2010 to 30-May-2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was performed with a substance analogue and the data are read across.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human peripheral

Details on mammalian cell type (if applicable):

- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin (Vacuette, Greiner Bio-One, Alphen aan den Rijn, The Netherlands). Immediately after blood collection lymphocyte cultures were started.

Culture medium
Culture medium consisted of RPMI 1640 medium (Invitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (Invitrogen Corporation), L-glutamine (2 mM) (Invitrogen Corporation), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively) (Invitrogen Corporation) and 30 U/ml heparin (Sigma, Zwijndrecht, The Netherlands).

Lymphocyte cultures
Whole blood (0.4 ml) treated with heparin was added to 5 ml or 4.8 ml culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin (Remel, Europe Ltd., United Kingdom) was added.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3hr exposure; 24 hr fixation: 33, 100, 333, 1000 and 3330 µg/ml
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 33, 100, 333, 1000, 3330 and 5000 µg/ml
First cytogenetic test:
Without and with S9-mix, 3 h exposure time, 24 h fixation time: 333, 1000 and 3330 µg/ml
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 500, 1000 and 3500 µg/ml
Without S9-mix, 48 hr exposure; 48 hr fixation: 500, 1000 and 3000 µg/ml
With S9-mix, 3 hr exposure; 48 hr fixation: 500, 1000 and 3500 µg/ml

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide
- Justification for choice of solvent/vehicle:Test compound was soluble in dimethyl sulfoxide and dimethyl sulfoxide has been accepted and approved by authorities and international guidelines.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9; in Hank's Balanced Salt Solution: 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9; in Hank's Balanced Salt Solution: 10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Key result

Species / strain:

lymphocytes: human peripheral

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: No toxicity was observed at the 3 hr treatment/24 hr fixation, but tested up to precipitating concentrations. Appropriate toxicity was observed at the continous treatment of 24 and 48 hr.

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 3330 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest precipitating tested dose in the absence and presence of S9, 3 hr treatment/24 hr fixation. Toxicity was observed at dose levels of 3330 µg/ml and above in the absence of S9 for the continuous treatment of 24 and 48 hr.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity was observed up to and including the highest precipitating tested dose in the absence and presence of S9, 3 hr treatment/24 hr fixation.
- Appropriate toxicity was reached at the dose levels selected for scoring for the continuous treatment of 24 and 48 h .

No biologically relevant effects of CN-Nitcal on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that CN-Nitcal does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations.

Conclusions:

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

CN-Nitcal did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Finally, it is concluded that this test is valid and that CN-Nitcal is not clastogenic in human lymphocytes under the experimental conditions described in the report.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 April 2010 to 02 June 2010

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study was performed with a substance analogue and the data are read across.

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Species strain
- Type and identity of media:
-RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 33, 100, 333, 666 and 1011 µg/mL
Without S9-mix, 24 hours treatment: 33, 100, 333, 666 and 1011 µg/mL
Experiment 1:
Without and with S9-mix, 3 hours treatment: 1, 3, 10, 33, 100, 333, 666 and 1011 µg/mL
Experiment 2
Without and with S9-mix, 24 hours treatment: 1, 3, 10, 33, 100, 333, 666 and 1011 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 medium
- Justification for choice of solvent/vehicle:Test compound was soluble in RPMI 1640 medium and RPMI 1640 medium has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9; 15 µg/mL for the 3 hours treatment period and 5 µg/mL for the 24 hours treatment period

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9; 7.5 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplo cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more then MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: No precipitation was observed up to and including the top dose of 1011 µg/mL (= 0.01 M)

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest test substance concentration of 1011 μg/ml

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
No toxicity was observed up to and including the highest tested dose level in both experiments.

Conclusions:

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

Mutation frequencies in cultures treated with positive control chemicals were increased by 16- and 8.6-fold for MMS in the absence of S9-mix, and by 13- and 16-fold for CP in the presence of S9-mix. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.

In the absence of S9-mix, Potassium nitrate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Potassium nitrate did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9 for metabolic activation.

It is concluded that Potassium nitrate is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in this report.

Reference 4

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:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

(1997)

Deviations:

yes

Remarks:

No duplicates, did not affect the outcome of the study

Principles of method if other than guideline:

Ishidate M Jr, Sofuni T. The in vitro chromosomal aberration test using Chinese Hamster Lung (CHL) fibroblast cells in culture. In: Ashby, editor. Progress in mutation research. Vol 5. New York: Elsevier Science Publishers; 1985. p. 427-32.

GLP compliance:

yes

Remarks:

National Institute of Environmental Research. GLP Notice No. 2008-44 for hazard assessment with chemicals in environment. Gwacheon (Korea): Ministry of Environment. 2008.

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: cultivated CHO-K1 obtained from the Korean Cell Line Bank (KCLB 10061)
- Culture medium: F-12 medium (Gibco BRL, NY, USA)

Metabolic activation:

with and without

Metabolic activation system:

obtained from MoltoxTM, Annapolis, Maryland, USA)

Test concentrations with justification for top dose:

Cell proliferation suppression test / preliminary test: 0.156, 0.3125, 0.625, 1.25, 2.5, 5 and 10 mM

Main test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 0, 0.156, 0.313, 0.625, 1.25, 2.5, 5 and 10 mM
Without S9-mix, 48 hr exposure; 48 hr fixation: 0, 0.156, 0.3125, 0.625, 1.25, 2.5, 5 and 10 mM
With S9-mix, 6 hr exposure; 24 hr fixation: 0, 0.156, 0.3125, 0.625, 1.25, 2.5, 5 and 10 mM

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Distilled water
- Justification for choice of solvent/vehicle: Distilled water was according to the results of a solubility test

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: mitomycin C (0.0004 mg/mL)

Remarks:

without S9

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: cyclophosphamide (0.01 mg/mL)

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium (in a 60 mm plate)

DURATION
- Preincubation period: 72 hr
- Exposure duration: 6 hr (with S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colcemid
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: no duplicates, two samples were made from each plate.

NUMBER OF CELLS EVALUATED: 200 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: the ratio of cell proliferation

OTHER EXAMINATIONS:
- Determination of polyploidy: yes

Evaluation criteria:

Results were evaluated as being positive only when the percentage of chromosomal aberrations was ≥ 10% (≥ 20 abnormalities in 200 cells observed).

Statistics:

Statistical analysis of the results was not performed.

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:

(according to the guideline max. 0.01M for non-cytotoxic compounds)

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Cell proliferation suppression test / preliminary test:
- The substance showed no suppression of cell proliferation after 24 hours of treatment (89.40 - 240.26% at each concentration).

Conclusions:

A chromosome aberration study with ammonium nitrate was performed according to OECD 473 guideline and GLP principles, in cultured CHO-K1 cells in one experiment. It is concluded that ammonium nitrate is not clastogenic in CHO-K1 cells.

Executive summary:

A chromosome aberration study with ammonium nitrate was performed according to OECD 473 guideline and GLP principles, in cultured CHO-K1 cells. The short (6 hours) and continuous (24 and 48 hours) treatment was performed in one experiment. No toxicity was observed up to the highest tested concentration of 10 mM.

Ammonium nitrate did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix. No effects of ammonium nitrate on the number of polyploid cells was observed both in the absence and presence of S9-mix. Therefore it can be concluded that ammonium nitrate does not disturb mitotic processes and cell cycle progression under the experimental conditions described in this report.

Based on the results it can be concluded that ammonium nitrate is not clastogenic in cultured CHO-K1 cells.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In accordance with column 2 of REACH Annex VII to X, no in vivo genotoxicity studies are required, as the in vitro toxicity study results are negative. An in vivo chromosome aberration study with ammonium nitrate is available showing negative results. However, as the study is not documented properly, it cannot be used for assessment.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Adequacy of study:

supporting study

Reliability:

4 (not assignable)

Rationale for reliability incl. deficiencies:

documentation insufficient for assessment

Remarks:

Not a guideline study. The information given is limited to the above mentioned. The aberration frequency at 5.2 mg/kg bw is without biological meaning and may be a mistake in the table. It is not clear whether a metaphase-arresting agent was added before sacrifice.

Principles of method if other than guideline:

Only one sex used, sampling earlier than 1.5 normal cell cycle; no positive controls.

GLP compliance:

not specified

Type of assay:

chromosome aberration assay

Species:

mouse

Strain:

not specified

Sex:

male

Details on test animals or test system and environmental conditions:

- Sex: male
- Age: sexually-mature
- Number: 6/dose
- Controls: 9 animals vehicle only

Route of administration:

oral: gavage

Duration of treatment / exposure:

15 days

Frequency of treatment:

daily

Post exposure period:

Sampling times and number of samples: 6 h after last administration

Dose / conc.:

0 mg/kg bw (total dose)

Dose / conc.:

5.2 mg/kg bw (total dose)

Dose / conc.:

20.9 mg/kg bw (total dose)

Dose / conc.:

104 mg/kg bw (total dose)

Dose / conc.:

417 mg/kg bw (total dose)

No. of animals per sex per dose:

6/dose

Control animals:

other: 9 animals vehicle only (vehicle = water)

Tissues and cell types examined:

Chromosome preprations were prepared using the standard Ford method from bone marrow cells; 100 metaphases were examined per animal.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

No increase in aberrations was found in the treated groups relative to the negative control.

Additional information on results:

EFFECT ON MITOTIC INDEX OR PCE/NCE RATIO: not indicated

GENOTOXIC EFFECTS: No increase in aberrations was found in the treated groups relative to the negative control.

ABERRATION FREQUENCY:
- Number of metaphases with aberrations (gaps excluded): -0.5, 0.3, 0.7, 0.8 and 1.1 per 100 metaphases for 0, 5.2, 20.9, 104 and 417 mg/kg bw, respectively

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on the available data, ammonium nitrate does not have to be classified for genotoxicity according to Regulation (EC) No 1272/2008.