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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

AHU377 did not show evidence of a mutagenic potential using standard mutagenicity criteria.

AHU377 is not clastogenic in human lymphocytes under the experimental conditions.

The substance did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of rats treated up to 2000 mg/kg/day.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Cultured peripheral human lymphocytes were used as test system.
Species / strain / cell type:
lymphocytes: human peripheral lymphocytes
Details on mammalian cell type (if applicable):
human peripheral lymphocytes
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
Rat liver microsomal enzymes S9
Test concentrations with justification for top dose:
In order to select the appropriate dose levels for the chromosome aberration test cytotoxicity data were obtained in a dose range finding test. AHU377 was tested in the absence and in the presence of 1.8% (v/v) S9-fraction.
The highest tested concentration was determined by the solubility of AHU377 in the culture medium at the 3 h exposure time. At the 24 h exposure time, AHU377 was tested beyond the limit of solubility to obtain adequate toxicity data.
In the dose range finding test blood cultures were treated with 3, 10, 33, 100 and 333 µg
AHU377/ml culture medium with and without S9-mix.
Vehicle / solvent:
dimethyl sulfoxide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
Cultured peripheral human lymphocytes were used as test system. Blood was collected from healthy adult, non-smoking male volunteers.
Blood samples

age 28, AGT = 14.5 h (Nov. 2003)
age 38, AGT = 17.0 h (Nov. 2003); without S9-mix age 25, AGT = 16.5 h (Feb. 2004); with S9-mix age 37, AGT = 14.2 h (Nov. 2003)

Blood samples were taken from healthy adult male volunteers by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. 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) (Merck), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively) (lnvitrogen 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 (Murex, Dartford, England) was added.
Rationale for test conditions:
The study procedures described in this report were based on the most recent OECD, EEC and ICH guidelines.
Evaluation criteria:
A test item was considered positive (clastogenic) in the chromosome aberration test if:
a) it induced a dose-related statistically significant (Chi-square test, 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 item was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, P < 0.05) increase in the number of cells with chromosome aberrations.
The preceding criteria are not absolute and other modifying factors might enter into the final evaluation decision.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, exclusive gaps) for each exposure group was compared to that of the solvent control using Chi-square statistics.
Key result
Species / strain:
lymphocytes: Cultured peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.
Conclusions:
AHU377 is not clastogenic in human lymphocytes under the experimental conditions described in this report.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
no guideline followed
Qualifier:
according to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Qualifier:
according to
Guideline:
other: (ICH Harmonised Tripartite Guidelines (S2B), 1997)
Qualifier:
according to
Guideline:
other: ICH Harmonised Tripartite Guideline (S2A), 1995
Principles of method if other than guideline:
The Salmonella mutagenicity test was done according to standard procedures used in Genetic
Toxicology and in vitro Safety Pharmacology, based on recommendations and descriptions of
methods in the literature (Maron DM and Ames BN, 1983) and in compliance with current
guidelines (OECD Guidelines, 1997) (ICH Harmonised Tripartite Guideline (S2A), 1995)
(ICH Harmonised Tripartite Guidelines (S2B), 1997).


GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Salmonella typhimurium strains TA1535, TA97a, TA98, TA100 and TA102
Species / strain / cell type:
S. typhimurium, other: TA1535, TA97a, TA98, TA100 and TA102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix
Test concentrations with justification for top dose:
concentrations:
• First experiment (plate incorporation): 8, 40, 200, 1000, 5000 μg/plate
• Second experiment (preincubation): 312.5, 625, 1250, 2500, 5000 μg/plate
In the plate incorporation test 0.1 ml and in the preincubation test 0.02 ml of test item stock
solution were used per plate, which means that of the above concentration values (in μg/plate)
10 times (plate incorporation) or 50 times (preincubation) higher concentrated (in μg/ml)
stock solutions were prepared with DMSO. DMSO was used as the negative control.
All test item concentrations used refer to the weight of the base.

In general, for this test system, the criteria for a concentration selection are as follows: ideally,
the highest concentration of a test item should be slightly bacteriotoxic. Qualitative
information on survival is obtained by careful microscopic observation of the light
background lawn of cell growth on the plates. The absence, or the change in appearance of the
background lawn of growth is taken as evidence of a bacteriotoxic effect. A decrease below
the negative control level in the number of colonies per plate (< 60%) is also taken as an
indication of bacteriotoxicity. However, limited solubility in the test-compatible vehicles
and/or precipitation on the agar may prevent testing at bacteriotoxic concentrations.
Generally, for this test system, concentrations of > 5 mg/plate are not used, even if
bacteriotoxicity and solubility would allow this.
Using the criteria given above, the highest concentration and four lower concentrations were
chosen on the basis of the results from previous experiments with the test item. The data from
these experiments are not shown in the present report.
Vehicle / solvent:
The test item, AHU377, was dissolved and diluted in DMSO to obtain the following
concentrations:
• First experiment (plate incorporation): 8, 40, 200, 1000, 5000 μg/plate
• Second experiment (preincubation): 312.5, 625, 1250, 2500, 5000 μg/plate
Untreated negative controls:
yes
Remarks:
Dimethylsulfoxide
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
benzo(a)pyrene
mitomycin C
other: 2-Aminoanthracene
Details on test system and experimental conditions:
The test item, AHU377, was dissolved and diluted in DMSO to obtain the following
concentrations:
• First experiment (plate incorporation): 8, 40, 200, 1000, 5000 μg/plate
• Second experiment (preincubation): 312.5, 625, 1250, 2500, 5000 μg/plate
In the plate incorporation test 0.1 ml and in the preincubation test 0.02 ml of test item stock
solution were used per plate, which means that of the above concentration values (in μg/plate)
10 times (plate incorporation) or 50 times (preincubation) higher concentrated (in μg/ml)
stock solutions were prepared with DMSO. DMSO was used as the negative control.
All test item concentrations used refer to the weight of the base.
Experiment 1 was conducted using the plate incorporation method, without preincubation,
experiment 2 as preincubation tests (20 minutes at 37ºC).
For the plate incorporation method, two ml of molten top agar containing 0.6% agar, 0.5%
NaCl, 0.05 mM biotin and 0.05 mM L-histidine were mixed successively with the following
components:
0.1 ml test item solution (or, for the controls, vehicle or positive control solution)
0.1 ml overnight culture (about 108 cells)
0.5 ml S9-mix or 0.5 ml of phosphate buffer
This mixture was poured on the surface of a minimal agar plate (Vogel-Bonner Medium E)
and allowed to solidify at room temperature.
For the preincubation method, the following components were mixed successively on ice
(with S9-mix) or at room temperature (with phosphate buffer):
0.5 ml S9-mix or 0.5 ml of phosphate buffer
0.1 ml overnight culture (about 108 cells)
0.02 ml test item solution (or vehicle, or 0.1 ml positive control solution)
This mixture was incubated at 37°C for 20 minutes (in a water bath).
Then two ml of molten top agar containing 0.6% agar, 0.5% NaCl, 0.05 mM biotin and 0.05
mM L-histidine were added, poured on the surface of a minimal agar plate (Vogel-Bonner
Medium E) and allowed to solidify at room temperature.
For each concentration of the test item, and for each positive and negative control group, three
plates were used. The plates were incubated in the dark at approximately 37°C with 50% to
70% humidity. Temperature and humidity in the incubation room were monitored by the Rees
Environmental Monitoring System (EnRep; Rees Scientific, Trenton, NJ, USA). After 3 days,
the colonies were counted. Upon counting, the plates were checked microscopically for the
presence of a light background lawn of growth. Colony counting was done with an image
analyzer (DOMINO; Perceptive Instruments, Halstead, UK), unless this was impossible
because of precipitation or other technical reasons. The data were recorded and printed by a
computer system (AMOS/TARAS, version 3.1.; Datasign, Basel, Switzerland).
Evaluation criteria:
Generally, the data obtained from a Salmonella mutagenicity test are interpreted as follows:
the results of a test are valid if the negative control data lie within the range of the historical
control data obtained in our laboratory, and the positive controls induce a positive
effect as defined by the criteria given below. The sterility tests of the test-item solutions, the
vehicle, the phosphate buffer and the S9-mix used should provide acceptable results.
Further, a test item is judged to be mutagenic in the plate test if it produces, in at least one
concentration and one strain, a response equal to twice (or more) the negative control
incidence. The only exception is strain TA102, which has a relatively high spontaneous
revertant number, where an increase by a factor of 1.5 above the negative control level is
taken as an indication of a mutagenic effect. Negative and positive findings have to be
reproducible in at least two independent experiments. If a clear decision is not possible after
two experiments, further tests are performed using only the critical strains and test item
concentrations lying close to the critical range previously identified.
The results have greater significance if a concentration-related increase in the number of
revertant colonies is observed. When conclusions have to be drawn from borderline effects,
the variations between individual plates are also taken into consideration.
The results obtained in a plate test are usually evaluated with the criteria described.
Key result
Species / strain:
S. typhimurium, other: TA1535, TA97a, TA98, TA100 and TA102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
other: The positive control data showed the mutagenicity pattern expected, except for 2 cases.
Additional information on results:
In experiment 1 mitomycin C was not inducing
an increased revertant rat in Salmonella typhimurium strain TA102. Since this strain
responded normally to 2-aminoanthracene, the lack of a response to mitomycin C is not
considered to compromise the validity of the study. The revertant rates induced 2-
nitrofluorene in experiment 2 lay slightly below the historical control range. However, since
this deviation was very small, the experiment was considered valid.
Conclusions:
From the results obtained, the test item AHU377 did not show evidence of a mutagenic potential under the conditions described above and using standard mutagenicity criteria.
Endpoint:
in vitro gene mutation study in mammalian cells
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro gene mutation study in mammalian cells does not need to be conducted because adequate data from a reliable in vivo mammalian gene mutation test are available
Justification for type of information:
Please refer to section 7.6.2.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Rats treated with LCZ696/AHU 377 at all doses exhibited group mean %PCE and frequencies of micronucleated PCE that were similar to the values for the vehicle control group. There were no instances of statistically significant increases in micronucleus frequency for any of the groups receiving the test item.

There were no instances of statistically significant increases in micronucleus frequency for any of the groups receiving the test item.

It is concluded that the substance did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of rats treated up to 2000 mg/kg/day

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: gene mutation
Remarks:
Rat bone marrow micronucleus test after oral administration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jan - Aug 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
other: ICH Tripartite Harmonised Guideline on Genotoxicity: Specific Aspects of Regulatory Tests, 1995 (European Agency, 1995).
Qualifier:
according to
Guideline:
other: Hayashi, Tice & MacGregor et al 1994; Richold, Chandley & Ashby et al, 1990
GLP compliance:
yes
Type of assay:
other: the ratio of PCE to NCE (expressed as %PCE)
Species:
rat
Strain:
Wistar
Details on species / strain selection:
out-bred young adult male and female Han Wistar Crl:WI (Han) rats

The micronucleus test in the rat has been used in a number of collaborative trials, and correlates quite well with other systems as long as high enough doses and multiple sampling times are used.
The test item was given as two administrations, 24 hours apart and animals were sampled 24 hours after the final administration. In this way, cells exposed to the test item over the period 24 to 48 hours prior to sampling were examined. This has been shown to be of sufficient duration for the expression of any genotoxic potential (Purchase & Ray, 1981; Heddle, Stuart & Salamone, 1984; Ashby, Tinwell & Gulati et al, 1990; Ashby & Mirkova, 1987).
Sex:
male/female
Details on test animals and environmental conditions:
out-bred young adult Han Wistar Crl:WI (Han) rats

They were housed in cages that conform to the Code of Practice for the housing and care of animals used in scientific procedures (Home Office, London, 1989). They were housed in groups of the same sex. Aspen wood chips (Datesand Ltd, Manchester) were used for bedding. Bottled water (public supply) and diet, (Special Diets Services Ltd, RM1.(E).SQC.) were provided ad libitum. Additionally, in order to enrich the environment and enhance the welfare of the animals, they were provided with wooden Aspen chew blocks and/or nesting material. All of the above are routinely monitored and are not known to contain any biological or chemical entity which might interfere with the test system.
The temperature and relative humidity were between 19 to 25°C and 33 to 70% respectively (see Deviations from study plan). The holding rooms were illuminated by fluorescent light for 12 hours out of each 24 hour cycle and are designed to receive at least 15 fresh air changes per hour.
Animals were acclimatised for at least 5 days prior to dosing and were identified by numbered ear-tag. Range-finder animals were allocated to groups of up to three but were not randomised. In the micronucleus experiment, rats were individually identified and randomised to groups of six animals using a system of random numbers. Cages were suitably labelled (using a colour-coded procedure) to clearly identify the study number, study type, start date, number and sex of animals, together with a description of the dose level and proposed time of necropsy. Checks were made on the first day of treatment to ensure group weights differed from the overall mean by no more than 5%
Route of administration:
oral: gavage
Vehicle:
0.5% CMC
Details on exposure:
In a range-finder experiment, which were used to select an appropriate maximum dose level for the micronucleus test, in accordance with current recommendations (OECD, 1997; Fielder, Allen & Boobis et al, 1993).
Both male and female rats were used in the range-finder experiment. In the absence of substantial inter-sex differences in toxicity (a difference in MTD of 2-fold or greater), or likely sex-specific human exposure, only a single sex need to be tested in the micronucleus experiment (OECD, 1997). Based on the range-finder results, male animals only were tested in the micronucleus test.
Dosing preparations were made by formulating LCZ696 (with the aid of a pestle and mortar and silverson mixer) in 0.5% (w/v) aqueous sodium carboxymethylcellulose, type 7HF (0.5% CMC) to give the concentrations specified for each experiment in the table below. The test item preparations were protected from light, maintained as an even suspension (by stirring) and used within 2 hours of formulation as follows:
Experiment Concentration of dosing preparation Dose administered
(mg/mL) * (mg/kg) *
Range-finder 50 500
70 700
100 1000
140 1400
200 2000
Micronucleus experiment 50 500
100 1000
200 2000
_____________________________________________________________________
* Prepared daily and administered on two consecutive days
Duration of treatment / exposure:
Animals were dosed once daily for two consecutive days with the test item or vehicle. The positive control was given as a single administration at 20 mg/kg, on the second day of dosing.
Animals were treated in the micronucleus experiment as follows overleaf:

Micronucleus experiment treatment details

Treatment group Dose administered Dose volume Number of animals treated (b)
(mg/kg/day) (a) (mL/kg)
Vehicle 0 10 6M
LCZ696 500 10 6M
LCZ696 1000 10 6M
LCZ696 2000 10 6M
Positive control, CPA 20 10 6M

a Doses administered once daily for two consecutive days, approximately 24 hours apart (except positive control)
b Animals sampled 24 hours after final dose administration CPA Cyclophosphamide, administered as a single dose
M Male
Frequency of treatment:
Doses administered once daily for two consecutive days, approximately 24 hours apart (except positive control)
Post exposure period:
In the micronucleus experiment post-dosing observations were performed immediately after each dose administration and at least twice in the 4 hours following dosing. Observations were also recorded at least once on the day of bone marrow sampling.
Dose / conc.:
0 mg/kg bw/day
Dose / conc.:
500 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
Dose / conc.:
20 mg/kg bw/day
Remarks:
Positive control, CPA Cyclophosphamide, administered as a single dose
No. of animals per sex per dose:
6 males
Control animals:
yes, concurrent vehicle
Positive control(s):
CPA Cyclophosphamide, administered as a single dose
Tissues and cell types examined:
The slides from all control and dose groups were arranged in numerical order by sampling time and analysed by a person not connected with the dosing phase of the study.
Initially the relative proportions of polychromatic erythrocytes (PCE), seen as bright orange enucleate cells, and normochromatic erythrocytes (NCE), seen as smaller dark green enucleate cells, were determined until a total of at least 1000 cells (PCE plus NCE) had been analysed.
Counting continued (but of PCE only) until at least 2000 PCE had been observed. All PCE containing micronuclei observed during these two phases of counting were recorded. The vernier coordinates of all cells containing micronuclei were recorded to a maximum of six per 2000 cells scored.
Details of tissue and slide preparation:
Test item- and vehicle-treated rats were sampled in groups, 24 hours after the second administration; CPA-treated rats were sampled 24 hours after the single dose. Rats were killed by an overdose of sodium pentobarbitone, given via intraperitoneal injection and subsequently ensured by cervical dislocation, in the same sequence used for dosing
One femur from each animal was exposed, removed, cleaned of adherent tissue and the ends removed from the shanks. Using a syringe and needle, bone marrow’s were flushed from the marrow cavity with 2 mL foetal bovine serum into appropriately labelled centrifuge tubes.
A further 3 mL of foetal bovine serum was added to the tubes, which were then centrifuged at 200 x 'g' for approximately five minutes; the serum was aspirated to leave one or two drops and the cell pellet. The pellet was mixed into this small volume of serum in each tube by using a Pasteur pipette, and from each tube one drop of suspension was placed on the end of each of two slides labelled with the appropriate study number, sampling time, sex, date of preparation and animal number. The latter served as the code so analysis could be conducted "blind". A smear was made from the drop by drawing the end of a clean slide along the labelled slide.
Slides were allowed to air-dry and then fixed for 10 minutes in absolute methanol. Slides were dried and stored at room temperature until required for staining. One slide from each set was taken (any remaining slides were kept in reserve). Prior to staining the slides were fixed again for 10 minutes in absolute methanol. After rinsing several times in distilled water, slides were stained for 5 minutes in 12.5 µg/ml acridine orange made up in 0.1 M phosphate buffer pH 7.4. Slides were rinsed in phosphate buffer, then allowed to dry and stored in the dark at room temperature prior to analysis.


Evaluation criteria:
The frequencies of micronucleated PCE in vehicle control animals were compared with the historical negative control data to determine whether or not the assay was acceptable.
Statistics:
For each group, inter-individual variation in the numbers of micronucleated PCE was estimated by means of a heterogeneity x² test (Lovell, Anderson & Albanese et al, 1989).
The numbers of micronucleated PCE in each treated group were then compared with the numbers in vehicle control groups by using a 2 x 2 contingency table to determine x² (Lovell, Anderson & Albanese et al, 1989). Probability values of P.S0.05 were to be accepted as significant. A further statistical test (for linear trend) was used to evaluate possible dose-response relationships.
Although the heterogeneity x² test provided evidence of significant (P.S0.05) variability between animals within at least one group, a clearly negative results was obtained. Consequently non-parametric analysis (e.g. the Wilcoxon rank sum test; Lehmann, 1975) was not performed.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
not examined
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Rats treated with LCZ696 at all doses exhibited group mean %PCE and frequencies of micronucleated PCE that were similar to the values for the vehicle control group. There were no instances of statistically significant increases in micronucleus frequency for any of the groups receiving the test item.
Conclusions:
There were no instances of statistically significant increases in micronucleus frequency for any of the groups receiving the test item.
It is concluded that LCZ696 did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of rats treated up to 2000 mg/kg/day (the maximum recommended dose for this study).
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Mode of Action Analysis / Human Relevance Framework

Chromosome defects are recognised as being the basis of a number of human genetic diseases (Mitelman, 1991).Chemicals can be testedin vitrofor their ability to produce chromosome aberrations in cells with which they have direct contact in a static system. Chemicals may react differentlyin vivo, however, where metabolic systems other than liver cytochrome P448/P450 operate, and where the dynamic processes of absorption, metabolism and excretion are involved. Chemicals should therefore also be tested for chromosome damaging activityinvivo.

Additional information

Justification for classification or non-classification

Ames test; no evidence of mutagenic activity with and without metabolic activation.

Chromosome aberration; Not clastogenic with and without metabolic activation

The substance did not induce micronuclei in the polychromatic erythrocytes of the bone marrow of rats treated up to 2000 mg/kg/day