SILATRIZOLE

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

December 05, 1995 to December 22, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study performed according to OECD test guideline 474 without any deviation.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Germany GLP Compliance Programme (inspected on 05/06/07 April 1995/signed on 1995-08-02)

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

422-940-4

EC Name:

-

Cas Number:

155633-54-8

Molecular formula:

C24H39N3O3Si3

IUPAC Name:

2-(2H-1,2,3-benzotriazol-2-yl)-4-methyl-6-[2-methyl-3-(2,2,4,6,6-pentamethyl-3,5-dioxa-2,4,6-trisilaheptan-4-yl)propyl]phenol

Test material form:

other: solid

Details on test material:

- Name of test material (as cited in study report): G4375
- Physical state: whitish solid
- Analytical purity: > 98%
- Impurities (identity and concentrations): Methanol (<100 ppm) and Isopropanol (<100 ppm)
- Purity test date: certificate of analysis 20/10/1995
- Lot/batch No.: DEF/C 95003/B- Expiration date of the lot/batch: September 1996 (retest date)
- Stability under test conditions: No data
- Storage condition of test material: room temperature, avoid UV irradiation

Test animals

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, CH-4414 Fullinsdorf
- Age at study initiation: 8-12 weeks old
- Weight at study initiation: Males: 35.2 ± 2.9 g, Females: 26.0 ± 2.4 g.
- Assigned to test groups randomly: yes
- Housing: Ind ividually in Makrolon type I cages with wire mesh top (EHRET GmbH, D-79302 Emmendingen) and granulated softwood bedding (Altromin, D-32791 Lage/Lippe).
- Fasting period before study: yes, 18h before treatment, the animals received no food but water ad libitum.
- Diet (e.g. ad libitum): ad libitum, pelleted standard diet (Altromin 1324, D-32791 Lage/Lippe)
- Water (e.g. ad libitum): ad libitum, tap water (Gemeindewerke, D-64380 Robdorf).
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3°C
- Humidity (%): 30 -70 %
- Air changes (per hr): no data (standard laboratory conditions)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: December 05, 1995 To: December 22, 1995

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: Methocel (Serva)+ Tween 80 (Fluka) (1% w/v + 1% v/v in deionised water).
- Justification for choice of solvent/vehicle: The vehicle was chosen according to its relative non-toxicity for the animals.
- Amount of vehicle (if gavage or dermal): 10 ml/kg bw

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Prepared on day of administration.
The test article-vehicle mixture, the vehicle alone, or the positive control was administered by oral route at a constant volume of 10 mL/kg body weight. All mice in the experimental and control groups were weighed immediately prior to dose administration, and the dose volume was based on individual body weights.

Duration of treatment / exposure:

Unique treatment by oral route (gavage), bone marrow cells were collected 24 and 48 hours after treatment

Frequency of treatment:

Unique treatment

Post exposure period:

24 and 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 200, 670 & 2000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

At least 6 males and 6 females. See Table 7.6.2/1:Animal assignment and doses in "Any other information on material and methods including tables"

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPA, Sigma-Aldrich, > 98%). Dissolved in deionised water.
- Justification for choice of positive control(s): Positive control recommended by OECD TG 474. The stability of CPA at room temperature is good. At 25°C only 3.5% of its potency is lost after 24 hours.
- Route of administration: Orally, once (10 ml/kg)
- Doses / concentrations: 30 mg/ kg bw

Examinations

Tissues and cell types examined:

Bone marrow cells; the incidence of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes was determined for each mouse and treatment group.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
No pre-experiment was performed as the compound did not show any toxicity in the acute toxicity test by oral route in rats at a dose of 2000 mg/kg bw. Therefore 2000 mg/kg bw has been used as the upper limit as recommended for non-toxic test articles. Two adequate spaced dose levels extending over a single log range were added.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
The test article-vehicle mixture, the vehicle alone, or the positive control was administered once by oral route at a constant volume of 10 mL/kg body weight.
All mice in the experimental and control groups were weighed immediately prior to dose administration, and the dose volume was based on individual body weights.
Samples of the bone marrow were taken at 24h for animals treated with the three doses of test item, for the animals treated with the vehicle and for the animals treated with the positive control. Samples were taken at 48h after treatment only for the animals treated with the highest dose level of test article.

DETAILS OF SLIDE PREPARATION: At the scheduled sacrifice times, 6 mice per sex per treatment were sacrificed by cervical dislocation. Immediately following sacrifice, the femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a syringe. The cell suspension was centrifuged at 1500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grunwald (Merck, D-64293 Darmastadt) /Giemsa (Gurr, BDH Limited Poole, UK). Cover slips were mounted with EUKITT (Kindler, D-79110 Freiburg). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS: Evaluation of the slides was performed using Nikon microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE; exception CPA = 2000 PCEs) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 PCEs. The analysis was performed with coded slides.

Evaluation criteria:

A test article is classified as mutagenic if it induces either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the test points.
A test article producing neither a dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant positive response for at any of the test points is considered non-mutagenic in this system.
This can be confirmed by means of the nonparametric Mann-Whitney test (evaluation of statistical significance at the five per cent level p< 0.05).
However both biological and statistical significance should be considered together.

Criteria for a Valid Test: The negative controls are in the range of the historical control data (0.03-0.26 % PCEs with micronuclei), the positive controls show substantially increase values and more than 80% of animals are evaluable.

Statistics:

Described in "Evaluation Criteria" above

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY: no range-finding study (choice of doses based on the absence of toxicity observed in an acute oral toxicity study)

RESULTS OF DEFINITIVE STUDY (See Table 7.6.2/2 in Remarks on results including table)
- Induction of micronuclei: There was no significant enhancement in the frequency of the detected micronuclei in comparison to the corresponding negative controls at any preparation interval after administration of the test article and with any dose level used. CPA induced a significant increase in micronucleated polychromatic erythrocytes (p ≤ 0.05, nonparametric Mann-Whitney test).
- Ratio of PCE/NCE: The mean number of normochromatic erythrocytes was not substantially increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that the test item had no cytotoxic properties in the bone marrow.
- Appropriateness of dose levels and route: doses levels were chosen according to an acute oral toxicity study where no toxic effects have been observed.

Any other information on results incl. tables

Table 7.6.2/2: Summary table of Bone Marrow Micronucleus Study

Treatment	Dose (mg/kg bw)	Sex	Time (h)	Number of mice	PCE/Total Erythrocytes	Micronucleated Polychromatic Erythrocytes
						PCEs with micronuclei (%)	Number per 10000 PCEs scored
Vehicle	0	M&F	24	5	1000/845	0.03	3
Test substance (G4375)	200	M&F	24	5	1000/817	0.07	7
	670	M&F	24	5	1000/797	0.04	4
	2000	M&F	24	5	1000/913	0.02	2
CPA	30	M&F	24	5	1000/927	0.68	68
Test substance (G4375)	2000	M&F	48	5	1000/757	0.09	9

 

Complete data results (individual n° of micronucleated PCE) are attached in "Background attached material "

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
Under the conditions described in this report, Silatrizole (encoded "G4375") did not induce a significant increase in micronucleated polychromatic erythrocytes in mice. Silatrizole was concluded to be negative in the mouse micronucleus assay.

Executive summary:

In an in vivo mouse micronucleus assay performed according to the OECD test Guideline 474 and in compliance with GLP, the test articleSilatrizole (encoded "G4375") was orally administered to mouse (at least 6/sex/dose) at doses of 200, 670 and 2000 mg/kg bw. A vehicle control group and a positive control group treated with cyclophosphamide (6 animals/sex) were also added. Samples of bone marrow ware taken at 24 hours in 5 animals per sex of each groups (including control groups). At 48 hours, samples were taken only from 5 animals per sex dosed with 2000 mg/kg bw of test item.

 

The mean number of normochromatic erythrocytes was not substantially increased after treatment with the test article as compared to the mean values of NCEs of the corresponding negative controls, indicating that the test item had no cytotoxic properties in the bone marrow.

There was no significant enhancement in the frequency of the detected micronuclei in comparison to the corresponding negative controls at any preparation interval after administration of the test article and with any dose level used. The positive control showed a statistically significant increase of induced micronucleus frequency.

 

The results of the assay indicate that under the conditions described in this report, Silatrizole did not induce a significant increase in micronucleated polychromatic erythrocytes mice. Silatrizole was concluded to be negative in the mouse micronucleus assay. The test substance is considered as non-mutagenic according to the OECD TG 474 Mammalian Erythrocyte Micronucleus Test.

This study is considered as acceptable and satisfies the requirement for this endpoint.