Nitric acid, ammonium calcium salt

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Nitric acid, ammonium calcium salt is not mutagenic in Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and, TA 100 and E. Coli WP2 uvr A with and without metabolic activation. Tests were performed according to OECD 471 and EU B.13/14 guidelines. No cytotoxicity was observed but test was performed up to limit concentrations (5000 microg/plate). No chromosomal aberrations were induced in human lymphocytes tested with and without metabolic activation according to OECD 473 and B.10 guidelines. 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. However 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. A mouse lymphoma assay has been performed with nitric acid, ammonium calcium salt (according to OECD guideline 476 and EC guideline B.17), showing no effects on the thymidine kinase locus in L5178Y mouse lymphoma cells with and without metabolic activation. Test concentrations were up to doses showing precipitation and cytotoxicity.

Justification for selection of genetic toxicity endpoint

Three in vitro studies are available, all with a klimisch score 1, all relevant for showing no adverse effect.

Short description of key information:

Nitric acid, ammonium calcium salt, appeared to be not mutagenic in all three in vitro tests, i.e. the Ames test, a chromosome aberration study, and a Mouse Lymphoma Assay all with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 05-18, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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; 5 µg/plate in saline for TA1535

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix; 60 µg/plate in mili-Q water for TA1537

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9-mix; 10 µg/plate in DMSO for TA98

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9-mix; 650 µg/plate in DMSO for TA100

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

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

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

09-Apr-2010 to 30-May-2010

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)

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

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

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26-Aug-2010 to 09-Nov-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)

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):

- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 ¿g/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: 100, 333, 2510, 3330 and 5000 µg/mL
Without S9-mix, 24 hours treatment: 100, 333, 2510, 3330 and 5000 µg/mL
Experiment 1:
Without S9-mix, 3 hours treatment: 50, 100, 200, 300, 350, 375, 400 and 450 µg/mL
With S9-mix, 3 hours treatment: 10, 100, 600, 1000, 2000, 2500, 2750 and 3000 µg/mL
Experiment 2
Without S9-mix, 24 hours treatment: 300, 320, 350, 360, 370, 380, 390 and 400 µg/mL
With S9-mix, 3 hours treatment: 10, 100, 600, 1000, 2000, 2750, 3000 and 3250 ¿g/mL

Vehicle / solvent:

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

Negative solvent / vehicle controls:

yes

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

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: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/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 than 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:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

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 1250 µg/mL and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 2510 µg/mL in the absence of S9, 3 hours treatment; at dose levels of 3330 µg/mL in the presence of S9, 3 hours treatment; at dose levels of 333 µg/mL in the absence of S9, 24 hours treatment

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:
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 88 and 80% compared to the total growth of the solvent controls after the 3 and 24 hours treatment period, respectively.

In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 80 and 67% compared to the total growth of the solvent controls after the 3 hours treatment period in the first and second experiment, respectively.

Conclusions:

It is concluded that CN-Nitcal is not mutagenic in the mouse lymphoma L5178Y test system under the experimental conditions described in the report.

Executive summary:

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.

 

Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses.

 

In the absence of S9-mix, CN-Nitcal 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, CN-Nitcal 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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

No in vivo studies are required, as all in vitro studies showed no genotoxicity.

Justification for classification or non-classification

Based on all information available, nitric acid, ammonium calcium salt does not have to be classified for genotoxicity according to the CLP Regulation.