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Toxicological information

Genetic toxicity: in vivo

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2007
Report Date:
2007

Materials and methods

Test guidelineopen allclose all
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

Test animals

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%

Administration / exposure

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.
Doses / concentrationsopen allclose all
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

Examinations

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.

Results and discussion

Test results
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.

Applicant's summary and conclusion

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).