Rubidium nitrate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Link to relevant study records

Reference

Endpoint:

in vitro transformation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 November 2016 to 23 February 2017

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:

OECD Guidelines for Testing of Chemicals No. 476 "In Vitro Mammalian Cell Gene Mutation Tests using the Hprt and xprt genes”. Adopted 29 July 2016.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell transformation assay

Specific details on test material used for the study:

Identification:
Rubidium nitrate
Physical State / Appearance:
White powder
Batch:
215091B002
CAS No.:
13126-12-0
Purity:
Treated as 100%
Expiry Date:
01 September 2017
Storage Conditions:
Room temperature, in the dark, over silica gel
Intended use / Application:
Catalyst
No correction for purity was made.

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type. The cells have a stable karyotype with a modal chromosome number of 22 (Howard-Flanders, 1981).
The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.

Metabolic activation:

with and without

Metabolic activation system:

The S9 mix was prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration.

Test concentrations with justification for top dose:

Preliminary Cytotoxicity Test - The concentrations of test item used were 5.78, 11.56, 23.13, 46.25, 92.5, 185, 370, 740 and 1480 μg/mL.
Main test - The dose range of test item was 92.5 to 1480 μg/mL in both the absence and presence of metabolic activation.

Vehicle / solvent:

Identity:
MEM Medium
Supplier:
Sigma
Batch number
RNBF2732 (Preliminary Toxicity Test) RNBF2732 and RNBF7600 (Main Test)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

The V79 cell line has been used successfully in in vitro experiments for many years. The high proliferation rate (doubling time 12 - 16 h in stock cultures) and a good cloning efficiency of untreated cells (as a rule more than 50 %) make it an appropriate cell line to use for this study type. The cells have a stable karyotype with a modal chromosome number of 22 (Howard-Flanders, 1981).
The V79 cell stocks were obtained from Harlan CCR in 2010 and originated from Labor für Mutagenitätsprüfungen (LMP); Technical University; 64287 Darmstadt, Germany.
Laboratory stock cell cultures will be periodically checked for stability and absence of mycoplasma contamination. The stock of cells is stored in liquid nitrogen. For use, a sample of cells will be removed before the start of the study and grown in Eagles Minimal Essential (MEM) (supplemented with sodium bicarbonate, L-glutamine, penicillin/streptomycin, amphotericin B, HEPES buffer and 10% fetal bovine serum (FBS)) at approximately 37 °C with 5% CO2 in humidified air.
Cell stocks spontaneously mutate at a low but significant rate. Before a stock of cells is frozen for storage the number of pre-existing HPRT-deficient mutants must be reduced. The cells are cleansed of mutants by culturing in HAT medium for four days. This is MEM growth medium supplemented with Hypoxanthine (13.6 μg/mL, 100 μM). Aminopterin (0.0178 μg/mL, 0.4 μM) and Thymidine (3.85 μg/mL, 16 μM). After four days in medium containing HAT, the cells are passaged into HAT free medium and grown for four to seven days. Bulk frozen stocks of these “HAT” cleansed cells are frozen down prior to use in the mutation studies, with fresh cultures being removed from frozen before each experiment.
Lot Numbers 25/08/16 and 22/09/16 was used in this study, and were pre-prepared in house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 tested for its capability to activate known mutagens in the Ames test and the certificates of efficacy are presented in Appendix 2.
The S9 mix was prepared by mixing S9 with a phosphate buffer containing NADP (5 mM), G6 P (5 mM), KCl (33 mM) and MgCl2 (8 mM) to give a 20% or 10% S9 concentration. The final concentration of S9 when dosed at a 10% volume of S9-mix was 2% for the Preliminary Toxicity Test and the Mutagenicity Test.

Statistics:

When there is no indication of any increases in mutant frequency at any concentration then statistical analysis may not be necessary. In all other circumstances comparisons will be made between the appropriate vehicle control value and each individual concentration, using Student’s t-test. Other statistical analysis may be used if they are considered to be appropriate.

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:

valid

Positive controls validity:

valid

Conclusions:

The test item did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.

Executive summary:

The test substance was tested to assess the potential mutagenicity of a test substance on the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus of the V79 cell line according to OECD 476 TG.

Chinese hamster (V79) cells were treated with the test item at six concentrations, in duplicate, together with vehicle (MEM medium) and positive controls in both the absence and presence of metabolic activation.

The concentrations used in the main test were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be the 10 mM limit dose as recommended by the OECD 476 guidelines. The concentrations of test item plated for cloning efficiency and expression of mutant colonies were as follows:

4-hour without S9 Final concentration of BOTP (μg/mL) 92.5 , 185, 370, 740, 1110, 1480

4-hour with S9 (2%) Final concentration of BOTP (μg/mL) 92.5 , 185, 370, 740, 1110, 1480

The vehicle (MEM medium) controls gave mutant frequencies within the range expected of V79 cells at the HPRT locus.

The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system.

The test substance was shown to be non-mutagenic to V79 cells at the HPRT locus under the conditions of the test.t item did not induce any toxicologically significant or dose-related (linear-trend) increases in the mutant frequency at any of the dose levels in the main test, in either the absence or presence of metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

According to the Endpoint specific Guidance (Chapter R.7a, version 4.1, October 2015) bacterial mutagenesis assays of inorganic metal compounds are frequently negative due to limited capacity for uptake of metal ions and/or the induction of large DNA deletions by metals in bacteria potentially leading to an increased death rate in mutants. The high prevalence of false negatives for metal compounds might suggest that mutagenesis assays with mammalian cells, as opposed to bacterial cells, would be the preferred starting point for testing for this class of Annex VII substances. Therefore, mutagenicity data in mammalian cells is provided instead.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

The test substance did not induce any significant or dose-related increases in mutant frequency per survivor in either the presence or absence of metabolic activation. The test item was therefore considered to be non-mutagenic to V79 cells at the HPRT locus under the conditions of this test.