SILATRIZOLE

Toxicity to reproduction

Administrative data

Endpoint:

one-generation reproductive toxicity

Remarks:

based on generations indicated in Effect levels

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 December 1999 to 18 February 2000

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The methodology used for this study followed guidelines related to pharmaceutical products. This study followed the ICH guideline, but the design and endpoints are similar to a standard one- generation study with regard to fertility parameters. However, pre- and postnatal development was investigated in separate studies.

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:

2001

Report date:

2001

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: Note for Guidance on Reproductive Toxicology: Detection of Toxicity to Reproduction for Medicinal Products (CPMP/ICH/386/95), ICH Harmonized Tripartite Guideline.

Deviations:

no

Principles of method if other than guideline:

The objective of this study was to evaluate the potential toxic effects of the test substance, following daily oral administration (gavage) to male and female rats from before mating, through mating and until implantation. For males, this study permits the detection of functional effects (e.g. on libido or epididymal sperm maturation) and for females, this study permits the detection of effects on the estrous cycle, tubal transport, implantation and the development of pre-implantation stages of the embryo.

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
- Other name: Phenol,2-(2H-Benzotriazo!-2-yl)-4-methyl-6-[2-Methyl-3- [1,3,3,3-Tetramethyl-1-[(Trimethylsi!y!)Oxy]Disiloxanyl]Propyl]-
- Physical state: white crystalline powder, atmost odorless
- Analytical purity: 100% (w/w)
- Lot/batch No.: DEF/C 95004/B
- Expiration date of the lot/batch: 20 November 2001
- Stability under test conditions: The results of the analyses demonstrated a satisfactory stability of the dosages forms over a 9-day period at +4°C, away from light.
- Storage condition of test material: at room temperature and protected from light.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Strain: ico: Wi (ÎOPS AF/Han) from Charles River - Iffa Credo, l'ArbresIe, France.
- Age at study initiation: (P) males: 8 wks and females: 10 wks
- Weight at study initiation: (P) Males: 222-272 g; Females: 165-247 g
- Fasting period before study: no
- Housing: The animals were housed individually in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm). A metal tray containing autoclaved sawdust (SICSA, Alfortville, France), was placed under each cage.
- Diet (e.g. ad libitum): A04 C pelleted diet, batch Nos. 90730 and 90928 (UAR, Villemoisson-sur-Orge, France), ad libitum.
- Water (e.g. ad libitum): bottles containing filtered tap water using a 0.22 micron filter (Millipore SA, Vélizy, France), ad libitum.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ±2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): about 12 cycles/hour
- Photoperiod (hrs dark / hrs light): 12h/î2h

IN-LIFE DATES: From: 14 December 1999 To: 18 February 2000

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: 4% (w/w) aqueous methylcellulose with 1% (w/w) Tween 80

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was administered as a suspension in the vehicle. The test substance was ground to fine powder using a mortar and pestle, suspended in the
vehicle in order to achieve the concentrations of 12.5, 37.5 and 125 mg/mL and then homogenized using a magnetic stirrer. In addition, the 125 mg/mL dosage form was mixed using an ultra-turrax® apparatus during 3 minutes, from day 25 of treatment for the males or day 11 of treatment for the females, and until the end of the study, in order to improve its physical homogeneity. The test substance dosage forms were made up to 7 days of treatment in advance and were stored at +4°C and protected from light prior to use. They were delivered each day to the animal room, protected from light.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: 12.5, 37.5 and 125 mg/mL
- Amount of vehicle (if gavage): 8 mL/kg/day
- Lot/batch no. (if required):
. methylcellulose (Methocel (MC, réf. 64605; ÎO-25 mPAS), batch Nos. 366101/1 61198 and 366101/1 51398, was supplied by Fluka (St Quentin-Fallavier, France),
. Tween 80, batch No. 37H01641, was supplied by Sigma (St Quentin-Fallavier, France), . purified water, obtained by reverse osmosis using a milli-Ro apparatus (Millipore SA, Saint-Quentin-en-Yvelines, France).
- Purity: 4% (w/w) aqueous methylcellulose with 1% (w/w) Tween 80

Details on mating procedure:

- M/F ratio per cage: 1/1
- Length of cohabitation: until evidence of successfull mating (up to three weeks)
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- Each female was placed with the same male until mating occurred or 21 days had elapsed.
- Further matings after two unsuccessful attempts: no data
- After successful mating each pregnant female was caged (how): no data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The suitability of the proposed dosage form preparation was confirmed by the analysis of homogeneity and stability of dosage forms for up to 9 days at +4°C prepared using this procedure between 12.5 and 200 mg/ml in a previous 13/26 week toxicity study {CIT/Study No. 16539 TCR} with the batch No. DEF/C 95004 B. The raw data were archived with this latter study.
During the treatment period, a check of the concentrations was performed by CIT on suspensions prepared for use in the study as follows: the concentration of samples of each control and test substance dosage form prepared for use in weeks 1, 4 and 8 was determined.

The results of the analyses demonstrated a satisfactory stability of the dosages forms over a 9-day period at +4°C, away from light. Throughout the treatment period, a satisfactory agreement was observed between the nominal and measured concentrations of the test substance in the dosage forms administered since the deviations from nominal concentrations were in an acceptable range of ±5%.

Duration of treatment / exposure:

- males: 29 days before mating and during the mating period (until sacrifice),
- females: 15 days before mating, during the mating period, during pregnancy until day 7 post-coitum inclusive (or until sacrifice, for un-mated females).

Frequency of treatment:

1/day, 7 days/week

Details on study schedule:

Male animals were dosed for 29 days before mating and during the mating period (until sacrifice) and female animals were dosed for 15 days before mating, during the mating period, during pregnancy until day 7 post-coitum inclusive (or until sacrifice, for un-mated females).
Parental males and females were paired within their respective dose groups for up to twenty one days.
At final sacrifice of the males, sperm was sampled from the epididymides for count, motility, viability and morphology of spermatozoa. Sperm heads were counted in the testes and the daily sperm production was calculated.
On day 15 post-coitum, all females were killed, the concepti were removed by hysterectomy and the dams were examined macroscopically. Litter parameters were recorded on ail pregnant females: number of corpora lutea, dead and live concepti, scars, early and late resorptions and implantation sites.
The parent males and females were submitted to a macroscopic post mortem examination and the testes and epididymides were weighed.

Doses / concentrations

Remarks:

Doses / Concentrations:
100, 300 or 1000 mg/kg bw/day
Basis:
nominal conc.

No. of animals per sex per dose:

24 males and 24 females per dose group for a total of 96 males and 96 females (see table 7.8.1/1: Animal assignment & doses).

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose-levels were determined following the results of two previous studies performed at 100, 300 and 1000 mg/kg/day in rats: a 13/26 week toxicity study (CIT/Study No. 16539 TCR) and a study for effects on embryo-fetal development {Becker H. and Biedermann K. 1996/RCC No. 607184). In the 13/26 week study, no signs of toxicity were noted at any dose-level; only slightly lower food consumption was recorded at 1000 mg/kg/day in the teratology study during the phase of treatment (day 6 to day 15 post-coitum). Consequently, the same dose-levels were chosen for the present study.
- Rationale for animal assignment (if not random): during the acclimation period, the required number of animals (96 males and 96 females) was selected according to body weight and/or clinical condition and allocated by sex to the groups, according to body weight, using a stratification procedure.

Positive control:

No

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily including weekends during the treatment period.
- Cage side observations checked in table [No.?] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day, at approximately the same time (including evidence of abortion/resorption for the females).

BODY WEIGHT: Yes
- Time schedule for examinations:
The body weight of each male was recorded on the first day of treatment (day 1), then twice a week until sacrifice.
The body weight of each female was recorded on the first day of treatment (day 1), then twice a week until mated (or until sacrifice) and on days 0, 4, 8, 11 and 15 post-coitum.

FOOD CONSUMPTION:
The quantity of food consumed by each male was recorded once a week, over a 7-day period, from the first day of treatment until the end of premating period.
The quantity of food consumed by each female was measured during the premating period (weekly from the first day of treatment) and during pregnancy (over the periods day 0-day 8 and day 8-day 15 post-coitum). During the mating period, the food consumption was not recorded.
Food intake per animal and per day was calculated.

Oestrous cyclicity (parental animals):

The estrous cycle stage was determined from a fresh vaginal lavage (stained with methylene blue), each morning during the mating period, until the females were mated.

Sperm parameters (parental animals):

Before sacrifice, each male was anesthetized (using isoflurane), and sperm was sampled from the tail of left epididymis (after weighing) for the sperm analysis.
Parameters examined in [P] male parental generations: Testicular sperm, testis weight, epididymis weight, daily sperm production, sperm count in testes, Epididymal sperm motility, Epididymal sperm count, Sperm viability, Epididymal sperm morphology.

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in offspring: number and distribution of dead and live concepti (embryos).

GROSS EXAMINATION OF DEAD PUPS: no

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were asphyxiated using carbon dioxide and exsanguinated after most of the females had been killed.
- Maternal animals: All surviving animals were asphyxiated using carbon dioxide on day 15 post-coitum and submitted to hysterectomy.

GROSS NECROPSY
Gross necropsy consisted of the principal thoracic and abdominal organs (with particular attention paid to the reproductive organs) was performed on all animals. The number of corpora lutea and implantation sites was recorded whenever possible for all females.

HISTOPATHOLOGY / ORGAN WEIGHTS
The body weight of all males was recorded but the testes and the epididymides were separately weighed (wet), as soon as possible after dissection.
All the macroscopic lesions and the following tissues from all males and females were sampled and preserved in 10% buffered formalin (except for testes and epididymides which were fixed in Bouin's fluid): head of left epididymis, right testis, right epididymis, seminal vesicles, ovaries, uterus, prostate, vagina.

Postmortem examinations (offspring):

Not examined.

Statistics:

Data other than organ weights: Mean values were compared by one-way variance analysis and Dunnet's test (mean values being considered as normally distributed, variances being considered as homogenous). Percentage values were compared by Fisher's exact probability test.
For organ weight data, a sequence was used based on the number of animals in each group.
- If the number of animals in each group is ≥ 5, values following a normal distribution were analyzed for homogeneity of variance using Bartlett test or Fisher test followed by Dunn test or Man-Whitney test if the variances were heterogeneous or followed by a student test or a Dunnett test if the variances were homogeneous.
- If the number of animals in each group is not ≥ 5 or if the distribution of data cannot be normalized, comparison of treated and control groups using original (untransformed) values (including organ weight) using a Dunn test followed by a Dunn test or a Mann-Whitney test.

Reproductive indices:

The following calculations were performed for each group:
Mating index: (Number of mated animals / Number of paired animals) x 100
Fertility index: (Number of pregnant female partners / Number of mated pairs) x 100
Pré-implantation loss: [(Number of corpora lutea - Number of implantation sites) / Number of corpora lutea] x 100
Post-implantation loss: [(Number of implantation sites - Number of live concepti) / Number of implantation sites] x 100

Offspring viability indices:

Number of live or dead concepti.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

No microscopic examination was deemed necessary.

Other effects:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment-related clinical signs were noted in parent animals. No mortality occurred in any group.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
- The body weight gain and food consumption were similar in the control group and the 100 and 300 mg/kg bw/day groups.
- The slight differences from controls noted in the mean body weight gain for males given the test substance at 300 mg/kg/day, were considered to be of no toxicological significance, since they
were slight, only occasionally statistically significant (days 8 to 11 and days 11 to 15), transient and not dose-related. This conclusion was also supported by the absence of any other apparent effects at this dose-level and at a higher dose-level. The slight difference from controls noted in the mean food consumption of the males given the test substance at 300 mg/kg/day (-7%, p<0.001, between day 15 to 22), was considered to be of no toxicological significance since it was slight, transient and not dose-related.
- A lower mean body weight gain was recorded in females given 1000 mg/kg/day during the premating period which was principally due to a mean body weight loss recorded between day 4 and day 11 inclusive (-4 g). This difference from controls, partially compensated over the period day 11 to day 15 (+14 g vs. +6 g in controls) was attributed to treatment with the test substance.
- For females of the top dose group, a slightly lower mean food consumption was recorded over the premating period (-10% and -5%, p<0.001, between days 1 to 8 and days 8 to 15 respectively). This difference from control, correlated with a lower body weight gain, was attributed to treatment with the test substance.

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
The estrous cycle was not affected by the treatment with the test substance at any dose-level. At hysterectomy there was no evidence of an effect of the test substance on ovulation, pre- or post-implantation of concepti in any treated group.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
There was no effect of the test substance on the evaluated sperm parameters in any treated group.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
The pre-coital time was similar in all groups (between 2.4 and 4.0 days). The mating index was 100% in all groups. The fertility index was similar in the control and treated groups (between 83% and 93%, without dose-relationship). The gestation index was 100% in all female groups.

ORGAN WEIGHTS (PARENTAL ANIMALS)
There was no noteworthy difference from controls in the testes or epididymides weight in any treated male group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related macroscopic post-mortem findings were noted in the parent animals.

Effect levels (P0)

Key result

Dose descriptor:

NOEL

Remarks:

(fertility)

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

body weight and weight gain

food consumption and compound intake

gross pathology

reproductive function (oestrous cycle)

reproductive function (sperm measures)

reproductive performance

Remarks on result:

other: There were no toxicologically significant effects

Results: F1 generation

General toxicity (F1)

Clinical signs:

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Details on results (F1)

VIABILITY:
There were no noteworthy or treatment-related differences from controls in any treated group.

Effect levels (F1)

Key result

Dose descriptor:

NOEL

Remarks:

(early embryonic development)

Generation:

F1

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

mortality

Remarks on result:

other: There were no noteworthy or treatment-related differences from controls in any treated group.

Overall reproductive toxicity

Reproductive effects observed:

not specified

Any other information on results incl. tables

None

Applicant's summary and conclusion

Conclusions:

There were no toxicologically significant effects upon fertility and no effects upon early embryonic development. The No Observed Effect Level (NOEL) for fertility and early embryonic development is therefore 1000 mg/kg bw/day.

Executive summary:

In a study of fertility and early embryonic development to implantation performed in compliance with GLP, the test material Silatrizole (encoded "G4375") diluted in 4% aqueous methylcellulose with 1% Tween 80 (w/w) was administered orally, by gavage, to groups of twenty-four male and twenty-four female rats, each day as follows: throughout premating period (29 days), mating period and until final sacrifice, in the males, throughout premating period (15 days), mating and pregnancy periods until day 7 post-coitum inclusive) in the females. The dose levels were 100, 300 and 1000 mg/kg bw/d with a similar sized control group receiving vehicle alone.

Mortality and clinical signs were checked daily. Body weight and food consumption were recorded at designated intervals throughout the study.

A mating trial was carried out on all animals: male and female animals were paired until mating, for a maximum of 3 weeks. The estrous cycle stages were recorded daily during this period. At final sacrifice of the males, sperm was sampled from the epididymides for count, motility, viability and morphology of spermatozoa. Sperm heads were counted in the testes and the daily sperm production was calculated.

On day 15 post-coitum, all females were killed, the concepti were removed by hysterectomy and the dams were examined macroscopically. Litter parameters were recorded on ail pregnant females: number of corpora lutea, dead and live concepti, scars, early and late resorptions and implantation sites. The parent males and females were submitted to a macroscopic post mortem examination and the testes and epididymides were weighed.

 

No treatment-related clinical signs were noted in parent animals. No mortality occurred in any group. The body weight gain and food consumption were similar in the control group and the 100 and 300 mg/kg/day groups. Slightly lower body weight gain (-4 g) and lower food consumption (-10% and -5%, p<0.001, between days 1 to 8 and days 8 to 15 respectively) were recorded in females given the test substance at 1000 mg/kg/day during the premating period. The pre-coital time was similar in all groups (between 2.4 and 4.0 days). The mating index was 100% in ail groups. The fertility index was similar in the control and treated groups (between 83% and 93%, without dose-relationship). The gestation index was 100% in all female groups. There was no effect of the test substance on the evaluated sperm parameters in any treated group. The estrous cycle was not affected by the treatment with the test substance at any dose-level. At hysterectomy there was no evidence of an effect of the test substance on ovulation, pre- or post-implantation of concepti in any treated group. No treatment-related macroscopic post-mortem findings were noted in the parent animals. There was no noteworthy difference from controls in the testes or epididymides weight in any treated male group.

 

When the test substance, was administered daily by oral gavage at 100, 300 or 1000 mg/kg bw/day to male and female rats, from the premating period, during the mating period and until sacrifice (males) or until day 7 post-coitum (females), no signs of paternal or maternal toxicity were noted at any dose-level, except a slightly lower body weight gain and food consumption during the premating period in females of the top dose-group. The gonadal function, mating behavior and fertility of parent animals, estrous cycle, tubal transport and implantation were not affected at any dose-level in females.

The No Observed Effect Level (NOEL) for fertility and early embryonic development is therefore 1000 mg/kg bw/d.

 

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