SILATRIZOLE

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 31, 1995 to January 18, 1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to OECD test guideline No. 414 and in compliance with GLP, without major 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 1

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

Version / remarks:

(1981)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

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 (powder)
- Analytical purity: 98% minimum
- 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: No data
- Stability under test conditions: in the vehicle (bi-distilled water with 4% Methylcellulose and 1% Tween 80): stable at least 4 hours
- Storage condition of test material: at room temperature (approx.20°C) protected from light.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL, Biological Research Laboratories, Ltd. Wölferstrasse 4, 4414 Füllinsdorf / Switzerland
- Age at study initiation:
Dose-range finding test: 10 weeks old at pairing
Main test: 11 weeks minimum
- Weight at study initiation:
Dose-range finding test: 212 - 269 grams at Day 0 post coitum.
Main test: 207-294 grams at Day 0 post coitum
- Fasting period before study: no
- Housing:
The rats were housed individually in Makroton cages (type-3) with wire mesh tops and standardized granulated softwood bedding (Lignocel, Schill AG, CH-4132 Muttenz / Switzertand).
- Diet (e.g. ad libitum): Pelleted standard Kliba 343 rat/mouse maintenance diet ("Kliba", Klingentalava- muehle AG, CH 4303 Kaiseraugst / Switzerland) was available ad libitum (Batch no. 69/95 and 71/95).
- Water (e.g. ad libitum): Tap water in bottles was available ad libitum. Results of bacteriological, chemical and contaminant analyses scheduled to be conducted at Ieast once yearly by RCC (contaminant analyses only) and by the Official Chemist of the Kanton Basel-Landschaft (bacteriological and chemical analyses).
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Humidity (%): 40-70 %
- Air changes (per hr): 10 to 15
- Photoperiod (hrs dark / hrs light): 12h dark/ 12h light

IN-LIFE DATES (main study): From 08 December 1995 to 18 January 1996

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: Bi-distilled water with 4% Methylcellulose and 1% Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The mixtures of the test article and vehicle were prepared daily before administration. The test article was weighed into a glass beaker on a tared precision balance (Mettler PE 360) and the vehicle added (w/w). The mixtures were prepared using a mortar and pestle and a magnetic stirrer. During the daily administration period, homogeneity was maintained using a magnetic stirrer.

VEHICLE
- Amount of vehicle (if gavage): 10 ml/kg body weight, with a daily adjustment of the individual volume to the actual body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Homogeneity and stability of the test article/vehicle mixtures were determined during the dose range-finding study. Samples were taken immediately after preparation and again 4 hours later. During the dosing period of this study, additional sample for confirmation of the concentration, homogeneity and stability were taken at the initiation and the termination of the dosing period (mean deviation from nominal concentration: 15% maximum). Analyses were performed by the RCC Analytical Chemistry Laboratory using a method developed by RCC. The concentration of the test material in formulations was determined by High-performance liquid chromatography (HPLC).
CONCENTRATIONS:
The mean concentrations of the homogeneity samples were found to be 97.5%, 99.5% and 100.1% of the nominal concentrations of 10 mg/ml, 30 mg/ml and 100 mg/ml, respectively.
HOMOGENEITY:
The homogeneity varied in the range from -3% to +2% of the mean concentrations.
STABILITY:
The test item was found to be stable in the vehicle at room temperature for at least four hours

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1 male ; 1 female
- Length of cohabitation: overnight
- The males used for mating were delivered on the same date as the females. Therefore the fertility could not be proofed previously.
- Further matings after two unsuccessful attempts: no
- Verification of same strain and source of both sexes: no data
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 post coitum (pregnancy)

Duration of treatment / exposure:

From Day 6 to 15 post coitum.

Frequency of treatment:

Daily

Duration of test:

15 Days

No. of animals per sex per dose:

Range-finding study: 6 females/dose
Main test: 25 females/dose

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: determined in RCC study 365534

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were checked at least twice daily for any mortalities.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: The animals were observed at least twice daily for signs of reaction to treatment and/or symptoms of ill health,

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded daily, from day 0 until day 21 post coitum.

FOOD CONSUMPTION : Yes
Food consumptions were recorded for the following periods: days 0-6, 6-11, 11-16 and 16-21 post coitum

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: all internal organs, with emphasis on the uterus, uterine contents, position of fetuses in the uterus and number of corpora lutea
When considered appropriate, macroscopic changes in the dams were photographed and samples of tissue fixed in neutral phosphate buffered 4% formaldehyde solution for possible microscopic examination.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

For the reproduction data, group mean values were calculated both on a per dam basis and on a percentage per group basis. Only dams with live fetuses on day 21 post coitum were included in the calculations.

Fetal examinations:

- External examinations: Yes: all per litter
- Soft tissue examinations (viscera and brain): Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No

Statistics:

The following statistical methods were used to analyze body weights, food consumption and reproduction and skeletal examination data:
- Univariate one-way analysis of variance was used to assess the significance of intergroup differences if the variables could be assumed to follow a normal distribution
- The Dunnett-test (many-one t-test), based on a pooled variance estimate, was applied for the comparison between the treated groups and the control group.
- The Steel test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- Fisher's Exact test for 2x2 tables was applied if the variables could be dichotomized without loss of information.
Individual values, means, standard deviations and t-statistics were rounded off before printing.

Indices:

Percentage pre-implantation loss was calculated as:
(Number of corpora lutea - Number of implantations)/ Number of corpora lutea x 100
Percentage post-implantation loss was calculated as:
(Number of implantations - Number of live foetuses)/ Number of implantations x 100
Sex ratio of males/females (%) was calculated as:
(number of males/females)/ number of implantations x 100

Historical control data:

Historical control were provided in the study (Reproduction data of dams, spontaneous abnormal findings of fetuses (external, visceral or skeletal examination), skeletal examination of fetuses (stage of development) on fetus basis or on litter basis, Necropsy findings)

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
There were no deaths during the course of this study, no clinical signs or symptoms were observed and no abnormal findings were attributed to treatment with the test item. Treatment with the test article caused slightly reduced food consumption during the dosing period in the dams at 1000 mg/kg bw/day, with statistical significance during days 6-11 post coitum (Dunnett-test, p<0.05). This finding was considered to be incidental; no effects were noted upon the mean body weights.
The differences amongst the reproduction parameters of the dams dosed with the test item and the vehicle control group gave no indication of test article related effects. The small differences noted were within the normal the normal range of deviations for animals of this strain and age and were considered to be incidental.
No indications of test article related macroscopic changes were noted during necropsy. Common abnormal macroscopic findings were noted in four females of the groups treated with 0, 100 and 1000 mg/kg bw/day, respectively and in one female of group treated with 300 mg/kg bw/day.

Effect levels (maternal animals)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Basis for effect level:

other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
The foetal parameters - external examination, visceral or skeletal examinations, sex ratios and body weights - yielded no reference to test article related effects.
In the fetuses of the group at 300 mg/lk bw/day, one had a rudimentary tail and in another the left fore paw was caudaly flexed. In the group at 1000 mg/kg bw/day one fetus had the left hindleg caudally malpositioned. Those findings were considered to be spontaneous changes commonly noted in fetuses of this rat strain.
The marginally increased mean body weights in group at 300 mg/kg bw/day was considered to be an effect of the different number of fetuses per dams (15.6 in vehicle control group Vs 14.8 in group treated at 300 mg/kg bw/day).
No abnormalities were noted during visceral examination.
The common abnormal findings noted were within the normal range of variation for fetuses of this rat strain and therefore considered to be incidental.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other:

Remarks on result:

other: No significant effects upon offspring viability, growth or development.

Fetal abnormalities

Abnormalities:

not specified

Overall developmental toxicity

Developmental effects observed:

not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

The oral administration of the test material at dose levels up to 1000 mg/kg bw/d, to pregnant rats from Day 6 to Day 15 of gestation resulted in no significant systemic effects on the adults. There were no significant effects on any of the uterine parameters examined. There were no significant effects upon offspring viability, growth or development. The No Observed Adverse Effect Level (NOAEL) for adult toxicity and developmental toxicity was 1000 mg /kg bw/d.

Executive summary:

In a developmental toxicity study conducted according to the OECD guideline No. 414 and in compliance with GLP, the test material diluted in bi-distilled water with 4% Methylcellulose and 1% Tween 80 was administered to 25 mated females Sprague-Dawley rats/dose by gavage at dose levels of 0, 100, 300 and 1000 mg/kg bw from day 6 post coitum through day 15 post coitum. The dosages were based on a range-finding study. Individual clinical observations, bodyweight and food consumption were recorded during the study. The females were sacrificed on Day 21 of gestation, examined macroscopically and the uterine contents examined. The number of corpora lutea, implantation number, foetal and placental weights, foetal sex, and external appearance were recorded. All live foetuses were preserved, processed and subsequently examined for skeletal or visceral anomalies.

There were no deaths during the course of this study, no clinical signs or symptoms were observed and no abnormal findings were attributed to treatment with the test item. Treatment with the test article caused slightly reduced food consumption during the dosing period in the dams at 1000 mg/kg bw/day, with statistical significance during days 6-11 post coitum (Dunnett-test, p<0.05). This finding was considered to be incidental; no effects were noted upon the mean body weights.

The differences amongst the reproduction parameters of the dams dosed with the test item and the vehicle control group gave no indication of test article related effects. The small differences noted were within the normal the normal range of deviations for animals of this strain and age and were considered to be incidental.

The foetal parameters - external examination, visceral or skeletal examinations, sex ratios and body weights - yielded no reference to test article related effects.

 

The oral administration of the test material at dose levels up to 1000 mg/kg bw/day, to pregnant rats from Day 6 to 15 of gestation resulted in no significant systemic effects on the adults. There were no significant effects on any of the uterine parameters examined. There were no significant effects upon offspring viability, growth or development.

The No Observed Adverse Effect Level (NOAEL) for adult toxicity and developmental toxicity was 1000 mg /kg bw/day.

 

Under the test conditions, the test material is not classified according to the annex VI of the Regulation (EC) No. 1272/2008 (CLP).

 

This study is acceptable and satisfies the requirement for developmental toxicity endpoint.