Calcium nitrite

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

4-16 January 1996

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Good-quality study, conducted to GLP. The test material was a 34% solution of hydrated calcium nitrite. The deviation from the current relevant OECD guideline (e.g. inclusion also of TA102 or E.coli) would not impact the overall conclusion.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

26 May 1983 version

Deviations:

yes

Remarks:

Current Guideline recommends the use of at least 5 tester strains; the study used only 4, omitting a strain for detecting cross-linking mutagens (e.g. TA102 or E. coli WP2 uvrA). Calcium nitrite was only tested at up to 1700 µg/plate (cf. 5000 µg/plate).

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29/12/1992 version

Deviations:

yes

Remarks:

Current Guideline recommends the use of at least 5 tester strains; the study used only 4, omitting a strain for detecting cross-linking mutagens (e.g. TA102 or E. coli WP2 uvrA). Calcium nitrite was only tested at up to 1700 µg/plate (cf. 5000 µg/plate).

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

1984 version

Deviations:

yes

Remarks:

Current Guideline recommends the use of at least 5 tester strains; the study used only 4, omitting a strain for detecting cross-linking mutagens (e.g. TA102 or E. coli WP2 uvrA). Calcium nitrite was only tested at up to 1700 µg/plate (cf. 5000 µg/plate).

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Calcium nitrite

EC Number:

237-424-2

EC Name:

Calcium nitrite

Cas Number:

13780-06-8

Molecular formula:

Ca.2HNO2

IUPAC Name:

calcium nitrite

Constituent 2

Test material form:

liquid

Method

Target gene:

Histidine

Species / strain

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 S9 fraction obtained from the livers of male Sprague-Dawley rats

Test concentrations with justification for top dose:

50, 150, 500, 1500 or 5000 µg DCI/plate [top dose level is equivalent to about 1700 µg calcium nitrite/plate]

In a preliminary toxicity test, concentrations of 5, 50, 500 or 5000 µg/plate were tested in strains TA1535, TA1537, TA98 and TA100, with and without metabolic activation. Both the mutagenic and cytotoxic effects of the test material on these strains were analysed. As only slight cytotoxicity was observed at the highest concentration, subsequent mutagenicity testing on the four bacterial strains also used 5000 µg/plate as the top concentration; other concentrations used were 50, 150, 500 and 1500 µg/plate (with and without metabolic activation).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: The test substance was supplied as an aqueous solution.

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: not applicable
- Exposure duration: 3 days
- Expression time (cells in growth medium): not applicable
- Selection time (if incubation with a selection agent): not applicable
- Fixation time (start of exposure up to fixation or harvest of cells): not applicable

SELECTION AGENT (mutation assays): no data

NUMBER OF REPLICATIONS: 3

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable

NUMBER OF CELLS EVALUATED: not applicable

DETERMINATION OF CYTOTOXICITY
- Method: measured by the presence of an incomplete background lawn.

Evaluation criteria:

A test substance is considered positive (mutagenic) in the test if it induced at least a 2-fold increase (with some evidence of a positive dose-response) in the number of revertants with respect to the number induced by the solvent control in two separate experiments, with any of the tester strains, either with or without metabolic activation.

A test substance is considered to be negative (not mutagenic) if the total number of revertants in any tested strain at any concentration is not greater than 1.5 times the solvent control value, with or without metabolic activation.

Statistics:

No statistical evaluation was performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Large, dose-related increases in the revertant colony numbers were observed in both mutation experiments

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 2

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 3

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Large, dose-related increases in the revertant colony numbers were observed in both mutation experiments

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data

RANGE-FINDING/SCREENING STUDIES: The test material caused slight cytotoxicity (incomplete bacterial lawn) at the highest tested concentration (5000 µg DCI/plate) in all four tester strains, both with and without S9.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: no data
- Negative (solvent/vehicle) historical control data: no data

Remarks on result:

other: Although the test material was applied at up to the recommended limit concentration of 5000 µg/plate, the highest actual tested concentration of calcium nitrite was 1700 µg/plate.

Applicant's summary and conclusion

Conclusions:

In an OECD Test Guideline 471 study, to GLP, calcium nitrite (hydrate) showed evidence of mutagenic activity in Salmonella typhimurium strains TA100 and TA1535 (but not in TA98 or TA1537) in the presence and absence of mammalian metabolic activation.

Executive summary:

The genotoxic potential of calcium nitrite (hydrate) was assessed in an in vitro bacterial reverse mutation (Ames) assay, conducted according to OECD Test Guideline 471 and to GLP.

 

In a preliminary toxicity test, Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 were exposed to test material concentrations of 0, 5, 50, 500 or 5000 µg/plate (equivalent to up to 1700 µg calcium nitrite/plate), both in the presence and absence of metabolic activation by rat liver fraction S9 (alongside appropriate vehicle controls). Only slight cytotoxicity was observed at the highest concentration only, therefore this was used as the highest test concentration in the mutagenicity assay.

Triplicate cultures of the four bacterial strains were exposed to the test material at concentrations of 50, 150, 500, 1500 or 5000 µg/plate, both with and without S9 (alongside appropriate vehicle and positive controls). These cultures were incubated for 72 hours at 37°C before being inspected for signs of cytotoxicity and for the incidence of revertant colonies. The main mutagenicity experiment was repeated using the same test material concentrations and following an identical procedure.

 

No signs of cytotoxicity were observed for any of the tested strains, at any of the tested concentrations, in either of the mutagenicity experiments. Large, dose-related increases in the number of revertant colonies were seen for S. typhimurium strains TA100 and TA1535 (with and without S9) in both mutagenicity experiments. No significant, dose-related increase was seen in S. typhimurium strains TA98 or TA1537 (both in the absence and presence of S9). The concurrent positive control substances demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations.

 

Under the conditions of this assay, calcium nitrite (hydrate) showed evidence of mutagenic activity in bacteria.