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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test; no evidence of mutagenic activity

Gene Mutation; negative

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:
Apr 1991 - June 1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
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:
Rat-liver microsomal fraction S9 was prepared in advance from male RAI rats
Test concentrations with justification for top dose:
The concentration range of CGP 48933 to be tested in the mutagenicity test was determined in a preliminary toxicity test. Thus, CGP 48933 was tested for mutagenic effects without microsomal activation at five concentrations in the range of 312.5 - 5000 μg/plate. In the experiment carried out with microsomal activation five concentrations in the range of 312.5 - 5000 μg/plate were tested. In order to confirm the results, the experiments were repeated with the same concentration ranges.
Six concentrations of CGP 48933 ranging from 20. 6-5000 μg/plate were tested with strains s. typhimurium TA 100 and E.coli WP2uvrA to determine the highest concentration to be used in the mutagenicity assay. The experiments were performed with and without microsomal activation. From the results obtained, the highest concentration suitable for the mutagenicity test was selected to be 5000 μg/plate without and with microsomal activation.
Vehicle / solvent:
CGP 48933 was dissolved in dimethylsulfoxide at room temperature.
The highest concentration of CGP 48933 was determined in a preliminary solubilisation test to be so mg/ml. Lower concentrations of the test substance were obtained by-appropriate dilution of the stock solution with dimethylsulfoxide. No precipitates or aggregates were noted.
Details on test system and experimental conditions:
This test system permits the detection of gene mutations induced by the test material or its metabolites in histidine-requiring strains of Salmonella typhimurium and in a tryptophan-requiring strain of Escherichia coli. When the Salmonella strains are exposed to a mutagen, some of the bacteria in the treated population, through chemical interaction with the compound, undergo genetic changes which cause them to revert to a non-histidine-requiring state and thus to grow in the
absence of exogenous histidine. Similarly, after mutation, the E.coli bacteria are able to grow in tryptophan-free medium. Mutagenic effects of the test ubstance are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone reverse-mutation to histidine prototrophism or tryptophan prototrophism, respectively. Different tester strains are used because of differing sensitivities to known mutagens. The following bacterial
strains have been used in this study:
Strain Type of Mutation
s. typhimurium TA 100 b ase-pair substitution
s. typhimurium TA 1535 base-pair substitution
E. coli WP2uvrA base-pair substitution
s. typhimurium TA 98 frame-shift
s. typhimurium TA 1537 frame-shift
Rationale for test conditions:
The concentration range of CGP 48933 to be tested in the mutagenicity test was determined in a preliminary toxicity test.
Statistics:
A statistical analysis was not performed. At present the use of statistical methods concerning this particular test system is not generally recommended
Key result
Species / strain:
S. typhimurium, other: TA 98, TA 100, TA 1535 and TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Based on the results of these experiments and on standard evaluation criteria, it is concluded that CGP 48933 and its metabolites did not induce gene mutations in the strains of S. typhimurium and E.coli used.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Nov 1991 - Apr 1992
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)
GLP compliance:
yes
Type of assay:
other: 6-thioguanine and can be quantified by comparison of the numbers of 6-TG resistant colonies in the treated and control cultures
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
V79 Chinese hamster cells . (clone 65/3) were originally derived from embryonic lung tissue.
Metabolic activation:
with and without
Metabolic activation system:
Rat-liver post mitochondrial supernatant (S9 fraction) was prepared in advance from male RAI rats
Test concentrations with justification for top dose:
cytotoxicity test
In the two parts of the experiment, 12 concentrations of the test substance and two vehicle (dimethylsulfoxide) controls were tested. The highest concentration was determined by the solubility of the test substance in the solvent (dimethylsulfoxide). Lower concentrations were prepared by serial dilution -by a factor of 0.5.
Range with activation:
2.71-5550.00 μg/ml
Range without activation:
2.71-5550.0Q μg/ml

Mutagenicity test
original experiment:
Range with activation:
51.41-1388.00 μg/ml
Range without activation:
205.56-5550.00 μg/ml
confirmatory experiment:
Range with activation:
51.41-1388.00 μg/ml
Range without activation:
205.56-5550.00 μg/ml
Vehicle / solvent:
dimethylsulfoxide
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-dimethylnitrosamine
ethylmethanesulphonate
Details on test system and experimental conditions:
cytotoxicity test: The cultures were exposed to the test substance for five hours in the presence and for 21 hours in the absence of a metabolic activation system.
Mutagenicity test
Depending on the toxicity of the test compound 2.5-5.0x10E6 cells of passage 26 (original experiment) and passage 28 (confirmatory experiment) were plated in 30 ml growth medium into 175 cm2 flasks and incubated overnight. The growth medium was replaced for five hours by 27 ml treatment medium and 3.0 ml S9 activation mixture, or for 21 hours by 30 ml treatment medium alone.
In each assay, cultures were treated in duplicate with four test chemical concentrations, a positive and a negative (dimethylsulfoxide) control. In the non-activated part of the experiment, the positive control was the ultimate mutagen ethylmethansulphonate (EMS, Merck) at a concentration of 300 nl/ml. In the
part with metabolic activation the positive control was the prornutagen N-ni troso-dimethylamine ( DMN, Merck) at a concentration of 1. 0 μl/ml.
Rationale for test conditions:
The test system allows the ·detection of base-pair substitutions, frameshift mutations and deletions induced by the test substance or by its metabolites. Mutagenic effects are manifested by the appearance of cells resistant to 6-thioguanine and can be quantified by comparison of the numbers of 6-TG resistant colonies in the treated and control cultures. To ensure that any mutagenic effect · of metabolites of the test substance found in mammals is also detected, an experiment is performed, in which the metabolic turnover of the test material is simulated in vitro by the addition of an activation mixture, containing rat-liver post mitochondrial fraction S9 and co-factors, to the cell cultures.
Evaluation criteria:
All mutant frequencies are normalized to a virtual efficiency of 100% at the end of the expression period cloning If the cloning efficiency of the survivor II cultures is lower than 15%, the corresponding mutant frequency is usually not calculated, owing to the high statistical insignificance of the result. For every concentration a mean mutant factor, which is defined as the ratio of the mean mutant frequencies of the treated cultures with the mean mutant frequencies of the sol vent control cultures will be calculated. If the mean mutant-frequency value of a treated culture is smaller than the mean mutant-frequency value of the solvent control cultures it will be calculated as 1.00. The sensitivity of the test is restricted to a lower limit of the mutant frequency of 4x10E-6 . If the mean mutant frequency measured is below this limit, it will be reported to be smaller than 4x10E-6 and the mutant factor will be calculated assuming a mutant frequency of 4x10E-6 .
Key result
Species / strain:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
True negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
Based on the results of two independently performed experiments and under the given experimental conditions, it is concluded that CGP 48 933 and its metabolites did not show any mutagenic potential in this forward mutation system.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Micronucleus test; Negative

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:
Oct 1991 - March 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
mammalian bone marrow chromosome aberration test
Species:
rat
Strain:
other: Tif: RAIf (SPF)
Details on species / strain selection:
Young adult male and female rats under SPF conditions by CIBA-GEIGY. (Tif: RAif (SPF) were reared They were obtained from the animal farm at least one day prior to being used in the test. The air-conditioned animal rooms were maintained at a temperature of 19-23°C and a relative humidity of 40-70%. The room was
illuminated for 12 hours daily. The rats were fed a standard diet of NAFAG No. 890 and tap water ad libitum. Maximal 5 animals were housed per cage, identified by cage card and individual ear tags.
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
Arachis oil
Details on exposure:
58 female ( f) and 58 male (m) rats were treated. The test substance and the negative control were dosed at a volume of 10 ml/kg body weight by gavage. The positive control CPA (20 mg/kg) was administered intraperitoneally.
In the first step of the tolerability test the maximum dose of 3125. o mg/kg of CGP 48 933 as well as the two lower doses of 781.3 mg/kg and 195.3 mg/kg caused no death and no symptoms of toxicity in the treated animals. In the second step of the tolerability test two animals were treated with the dose of
3125.0 mg/kg. None of the animals died and therefore this dose was selected as the highest dose to be administered in the micronucleus test.
Duration of treatment / exposure:
The test was performed with doses of 312 5. o mg/kg b. w, 1562.5 mg/kg b.w. and 781.3 mg/kg b.w. at each of three sampling times of 16, 24 and 48 hours.
Frequency of treatment:
The test was performed with doses of 312 5. o mg/kg b. w, 1562.5 mg/kg b.w. and 781.3 mg/kg b.w. at each of three sampling times of 16, 24 and 48 hours.
Post exposure period:
16, 24 and 48 hours
Dose / conc.:
3 125 mg/kg bw/day
Dose / conc.:
1 562.5 mg/kg bw/day
Dose / conc.:
781.3 mg/kg bw/day
No. of animals per sex per dose:
58 female (f) and 58 male (m) rats were treated

Treatment High Intermed. Low Positive Negative
dose (HD) dose (ID) dose (LD) control control
(1/2 HD) (1/4 HD) (CPA) (vehicle)
Number/sex 15m + 15f 15m + 15f 15m + 15f 15m + 15f 15m + 15f
Control animals:
yes, concurrent vehicle
Positive control(s):
The positive control was cyclophosphamide - CPA
Tissues and cell types examined:
Bone marrow was harvested from the shafts of both femurs in fetal calf serum. Nucleated cells were removed by a cellulose column step.
Details of tissue and slide preparation:
A 10 μm-filter was attached to the nozzle of a 20 ml syringe. Into this syringe 1. 5 g of a mixture ( 1: 1 w/w) of microcrystalline cellulose (Sigmacell type 50) and a-cellulose fibers was placed. Thereafter, the bone marrow suspension was applied to the top of the column and eluted with Hank's BSS buffer. The eluate containing the resuspended in erythrocytes was centrifuged and the cells fetal calf serum. Smears prepared from were this suspension were stained with May-Grunwald/Giemsa solution and mounted.
Evaluation criteria:
The results of the experiments are evaluated with respect to the mean number of PCEs with micronuclei. The groups compared differ by treatment, sampling time and sex of the animals. If there is no significant difference between animals of either sex, the data from females and males are pooled for evaluation.
Statistics:
The significance of differences was assessed by the Chi-SquareTest (Level of significance alfa=0.05, DF=1).
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
not specified
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
It is concluded that under the given experimental conditions no evidence for clastogenic or aneugenic effects was obtained in rats treated with CGP 48 933.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

In 2010 a meta-analysis (Sipahi et al 2010) suggested that angiotensin receptor blockers could be associated with increased cancer risk – over 85% of the patients included in the meta-analysis received telmisartan. In response to that publication, Novartis completed a meta-analysis with individual patient data with valsartan that showed no increased risk of cancer or cancer related death (approximately 44,000 patients with a mean valsartan exposure of almost 3 years/patient from four trials: Val-HeFT VALUE, VALIANT and NAVIGATOR). Novartis submitted these data to the FDA and EMA and both authorities concluded that treatment with angiotensin receptor blockers do not increase a patient’s risk of developing cancer

In 2011 another meta-analysis of 70 randomized clinical trials (over 320,000 patients) concluded that, in patients receiving angiotensin receptor blockers, there was no difference in the risk of cancer (odds ratio 1·01, 95%CI 0.93 to ·1.09) or in the risk of cancer-related mortality (odds ratio 1·00, 95%CI 0.87 to 1.15) (Bangalore et al 2011).

Additional information

Justification for classification or non-classification

There was no evidence of mutagenicity or clastogenicity in the studies.