L-Valine, N-(1-oxopentyl)-N-[[2'-(2H-tetrazol-5-yl)[1,1'-biphenyl]-4-yl]methyl]-, phenylmethyl ester

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dec 1996 - Aug 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1997

Report date:

1997

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian bone marrow chromosome aberration test

Test material

Test material information

Constituent 1

Reference substance name:

Tetrazolyl-valinesteramid (3-methyl-2-{pentanoyl-[2'-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-amino}-butyric acid benzyl ester)

EC Number:

604-047-3

Cas Number:

137863-20-8

Molecular formula:

C31 H35 N5 O3

IUPAC Name:

Tetrazolyl-valinesteramid (3-methyl-2-{pentanoyl-[2'-(1H-tetrazol-5-yl)-biphenyl-4-ylmethyl]-amino}-butyric acid benzyl ester)

Test animals

Species:

rat

Strain:

other: Tif: RAif (SPF) rats

Details on species / strain selection:

Young adult male and female rats (Tif: RAif (SPF)) were reared under SPF conditions by Novartis Pharma AG.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Young adult male and female rats (Tif: RAif (SPF)) were reared under SPF conditions by Novartis Pharma AG. The air-conditioned animal rooms were maintained at a temperature of 20.0 - 21.0°C and a relative humidity of 24 - 46%. The room was illuminated for 12 hours daily. The rats were fed a standard diet of NAFAG No. 890 and tap water ad libitum. They were kept on location for at least five days prior to being used in the study. Shortly before use the health status of the animals was checked by the laboratory personnel according to veterinary/scientific standards. The animals were housed 5/cage (micronucleus test), 3/cage (reserve animals) or individually (tolerability test) and marked with color pens. They were distributed to the different treatment and control groups by random selection with the aid of a computer generated random list.
Prior to application of the test substance or the control substances, the weight of the animals was determined. The age range at the time of application was 7 to 8 weeks.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

Arachis oil

Details on exposure:

The test substance, the negative and positive controls were dosed at a volume of 10 ml/kg body weight by gavage.
Oral by stomach tube, 10 ml/kg body weight

Duration of treatment / exposure:

The test substance was administered once by gavage to groups of 5 male and 5 female Tif: RAif (SPF) rats at three different doses. Additional groups of animals were treated with the vehicle alone or with the positive control cyclophosphamide ( 40 mg/kg). From the high dose group and from the negative control group animals were sacrificed 16, 24 and 48 hours thereafter. From the intermediate and the low dose group and from the positive control group animals were sacrificed
24 hours after application. Dose-finding was carried out in a preceding tolerability test.

Frequency of treatment:

The following numbers of animals/sex/dose were treated and analyzed:

Preparation of bone marrow High dose (HD) Intermediate dose (ID) Low dose (LD) Positive control Negative control
after treatment 1250mg/kg 625 mg/kg 312.5 mg/kg (CPA) (vehicle)
16 hours 5m+5f 5m+5f
24 hours 5m+5f 5m+5f 5m+5f 5m+5f 5m+5f
48 hours 5m+5f 5m+5f
Reserve* 3m+3f
*) BM of reserve animals prepared only in case of premature death in the HD group

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw/day

Remarks:

tolerability test

Doses / concentrations 2

Dose / conc.:

1 250 mg/kg bw/day

Doses / concentrations 3

Dose / conc.:

312.5 mg/kg bw/day

Doses / concentrations 4

Dose / conc.:

625 mg/kg bw/day

No. of animals per sex per dose:

The following numbers of animals/sex/dose were treated and analyzed:

Preparation of bone marrow High dose (HD) Intermediate dose (ID) Low dose (LD) Positive control Negative control
after treatment 1250mg/kg 625 mg/kg 312.5 mg/kg (CPA) (vehicle)
16 hours 5m+5f 5m+5f
24 hours 5m+5f 5m+5f 5m+5f 5m+5f 5m+5f
48 hours 5m+5f 5m+5f
Reserve* 3m+3f
*) BM of reserve animals prepared only in case of premature death in the HD group

Control animals:

yes, concurrent vehicle

Positive control(s):

the positive control cyclophosphamide ( 40 mg/kg)

Examinations

Evaluation criteria:

The results of the experiments were evaluated with respect to the mean number of PCEs with micronuclei.
The groups compared differed by treatment, sampling time and sex of the animals. The data from females and males were pooled for evaluation. In case of significant sex differences data have to be evaluated for each sex separately in addition.
Assay acceptance criteria
1. The result of the experiment should not be influenced by a significant technical error or a recognized artifact.
2. The high dose of the test substance applied should be the maximum tolerated dose causing no death in a group of four animals in the range finding-test. In the absence of lethality in the tolerability test, the high dose should be up to a maximum of 2000 mg/kg body weight or the
highest applicable dose due to the limited solubility of the test substance.
3. The quality of the slides must allow a clear differentiation between PCEs and NCEs.
4. The mean number of micronucleated PCEs in the negative control groups should not exceed the value of 0.20%.
5. The positive control should fulfill the criteria for an active substance.

Statistics:

The significance of differences was assessed by the Chi-Squared-Contingency-Test (df=1, p<0.05).

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Any other information on results incl. tables

The dose finding experiment with PBS 859 DS showed that treatment with 2000 mg/kg led to severe signs of toxicity, such as ventral recumbency, ataxia, reduced locomotor activity, dyspnea, hunched posture, and loss in body weight and led to the death of an animal. At 1250 mg/kg, similar, but less severe signs was observed. This dose was confirmed by treating a second pair of rats. Based on these results, doses of 312.5, 625 and 1250 mg/kg were chosen for the micronucleus test.

In the micronucleus test, the percentage of PCEs was not decreased in any of the dose groups. No signs of bone marrow toxicity were observed. At the 16 hours sampling time in the group treated with 1250 mg/kg of the test substance the mean percentage of micronucleated PCEs was 0.13 compared to a negative control value of 0.11. At the 24 hours sampling time in the groups treated with the test substance the mean percentage of micronucleated PCEs was 0.16 (1250 mg/kg), 0.13 (625 mg/kg) and 0.18 (312.5 mg/kg) respectively. The respective negative control value for this group was 0.15%. At the 48 hours sampling time in the group treated with 1250 mg/kg of the test substance the mean percentage of micronucleated PCEs was 0.15 compared to a negative control value of 0.12. In the positive control (24 hours) the mean percentage of micronucleated PCEs ( 1.19) was significantly increased.

Applicant's summary and conclusion

Conclusions:

In all dosage groups assessed at the different periods post treatment, no statistically significant increase in the number of micronucleated polychromatic erythrocytes was observed when compared with the respective negative control group.