Registration Dossier

Administrative data

Endpoint:
in vivo mammalian germ cell study: cytogenicity / chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Although the study was conducted according to GLP and well documented methods, " Practical guide 10: How to avoid unnecessary testing on animals", Section 3.3.2 states it is important that the reliability indicator (Klimisch score) reflects the assumptions of similarity. Thus, a score of 1 (reliable without restrictions) should normally not be used for results derived from read-across.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1990
Report Date:
1990

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
other: Merck Sharp & Dohme Research Laboratories, Genetic Toxicity, SOP, Assay for Chromosal Aberrations in Mouse Bone Marrow
Deviations:
yes
Remarks:
see below:
Principles of method if other than guideline:
Fewer than fifty mitotic cells were availalbe for scoring for one animal in the 150 mg/kg L-158,086 dose group (animal #1307) at the 48 hour sacrifice; one animal in the 500 mg/kg group at the 48 hr sacrifice; two animals in the 1.0 mg/kg Mitomycin C group at the 24 hr sacrifice. Fewer than fifty mitotic cells were scored for one animal in the 3.5 mg/kg Mitomycin dose group which had no mitotic cells available for scoring.
GLP compliance:
yes
Type of assay:
chromosome aberration assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report):L-158,086


- Analytical purity:99.8%

- Lot/batch No.:158,086-005H010

- Stability under test conditions: stable

Test animals

Species:
mouse
Strain:
other: Crl:CD-1R(ICR) BR
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:Charles River Breeding Farms, Raleigh, N.C.
- Age at study initiation:Thirty days
- Weight at study initiation: 20.0-28.6 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Fasting period before study: not specified
- Housing: 4 to 12 mice per cage
- Diet (e.g. ad libitum): purina certified rodent chow- ad libitum
- Water (e.g. ad libitum): tap water - ad libitum
- Acclimation period: 7 days

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
dH20
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

Test substance (oral gavage)

1500,500 and 150 mg/kg levels were prepared as solutions in dH20. The top levle was a clear yellow solution.
High dose: 150 mg/mL in dH20
Middle: 50 mg/mL by dilution from high dose
Low: 15 mg/mL by dilution from high dose

Positive control (intraperitoneal injection)
substance: mitomycin c
vehicle: saline
concentration: 350 microg/ml and 100 microg/ml

negative control (oral gavage):
dH20
Duration of treatment / exposure:
animals sacrificed at 6, 24 and 48 hours (positive control at 24 hrs)
Frequency of treatment:
single dose at time 0 for all treatment groups
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
1500 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
500 mg/kg
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
150 mg/kg
Basis:
nominal conc.
No. of animals per sex per dose:
Negative control: 36 mice
1500 mg/kg: 30
500 mg/kg:24
150 mg/kg:24
positive control 12
Control animals:
yes, concurrent vehicle
Positive control(s):
mitomycin C;

- Route of administration:intraperitoneal injection
- Doses / concentrations: 1 mg/kg

Examinations

Details of tissue and slide preparation:
Slide preparation:
a) fixed celss were dropped on to clean , wet slides and allowed to dry
b) as soon as fixed cells were spread on each slide, that slide was coded with a random number which had been correlated with the animal number. Random numbers were generated by coputer by the cytogen system
c) all slides were stained wth Giemsa and coverslips were applied to them.
d) all slides were checked for quality and chromosome spreading.

Slide analysis:
Slide analysis was done according to criteria in the SOP.
Evaluation criteria:
P<0.001
Statistics:
Statistical analysis was carried out by the Biometrics Department, MSDRL, West Point, PA and is described in SOP. The proportion of aberrant cells for each mouse was used as a data point. Individual dose level results were compared by a least significatn difference procedure with concurrent negative controls.

Also, a trend test was used to investigate evidence for a dose relation. The proportion of animals with aberrant cells in each group was also analyzed.

Results and discussion

Test results
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls valid:
yes
Negative controls valid:
yes
Positive controls valid:
yes
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Dose range:808-3000 mg/kg
- Solubility:
- Clinical signs of toxicity in test animals:ataxia, decreased activity, bradypnea and ptosis. The signs were seen at the 3000 mg/kg dose level at 15 min and at 2308 mg/kg within 90 minutes. No signs were seen at lower doses on the first day. There were three deaths at 2308 mg/kg and five deaths at 3000 mg/kg. Deaths occured in about 3 to 4.25 hours and were precede by a loss of righting reflex. Surviving mice appeared normal at 3 to 4 hours. On the second day ataxia and bradypnea were seen at the 1775 mg/kg dose. By the end of hte second day all survivors appeared normal. The LD50 was calculated to be 2248 mg/kg.
- Evidence of cytotoxicity in tissue analyzed:
- Rationale for exposure:
- Harvest times:
- High dose with and without activation:
- Other:

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay):
- Induction of micronuclei (for Micronucleus assay):
- Ratio of PCE/NCE (for Micronucleus assay):
- Appropriateness of dose levels and route:
- Statistical evaluation:

Any other information on results incl. tables

Mean Proportions (%) of Aberrant Cells
Sacrifice Time Control 150mg/kg 500mg/kg 1500 mg/kg
6 hr 0.5 0 0 0.5
24 hr 0.83 0.5 1 1
48 hr 0.67 1.32 0.52 0.25
Combined 0.67 0.59 0.51 0.58

Results of Statistical Tests (P-values) on the Rankit-Transformed Proportions: Doses compared to the negative control (a)
Sacrifice Time Trend 150 mg/kg 500mg/kg 1500mg/kg
6 hr 0.35 0.060N 0.060N 0.5
24 hr 0.248 0.316N 0.458 0.315
48 hr 0.132N 0.134 0.424N 0.246N
Combined 0.399N 0.394 0.275N 0.458N

(a): a one-tailed LSD procedure was computed for the individual L-158,086 dose group vs. negative control comparison. The P-values are adjusted for multiple comparisons against a common control. This adjustment is applied to only those P-values <=0.05

N: Indicates a reverse effect, i.e., the mean proportion of aberrant celss is lower for the L-158 ,086 treatment group than for the negative control group

In vivo assay for chromosomal aberrations in bone marrow cells from mice treated with L-158,086

Proportions (Pa) of Animals with at Least One Aberrant Cell

Proportions (Pa) of Animals with at Least One Aberrant Cell
Sacrifice Time Control 150 mg/kg 500 mg/kg 1500 mg/kg
6 hr 3/12 0/8 0/8 2/8
24 hr 4/12 2/8 2/8 4/8
48 hr 3/12 3/8 2/8 1/8
Combined 10/36 5/24 4/24 7/24

Resutls of Statistical Tests (p-values)

Proportions (Pa) of Animals with at Least One Aberrant Cell
Sacrifice Time Trend 150 mg/kg 500 mg/kg 1500 mg/kg
6 hr 0.307 0.193N 0.193N 0.693
24 hr 0.172 0.545N 0.545N 0.388
48 hr 0.187N 0.455 0.693 0.465N
Combined 0.374 0.385N 0.249N 0.566

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information): negative
There was no evidence for induction of chromosom aberrations in animals treated with L-158,086 under the conditions of this test. This result also applies to the read-across substance losartan free acid.