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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3'-(1,4-Phenylene)bis(5,6-diphenyl-1,2,4-triazine)
EC Number:
700-823-1
Cas Number:
55514-22-2
Molecular formula:
C36H24N6
IUPAC Name:
3,3'-(1,4-Phenylene)bis(5,6-diphenyl-1,2,4-triazine)
Details on test material:
- Name of test material (as cited in study report): WP30
- Substance type: mono-constituent substance
- Physical state: powder

- Analytical purity: 99.8 %
- Impurities (identity and concentrations): unknown impurity with MW 380: 0.19 %, unknown impurity with MW 558: 0.29 %,


- Purity test date: July 7th 2011
- Lot/batch No.: LP140

- Stability under test conditions: stable
- Storage condition of test material: at room temperature in a dry and ventilated place

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
Number: 130 Sprague-Dawley rats (65 males and 65 females) were received at CIT on 01 September 2011. Strain and sanitary status: Crl CD® (SD) IGS BR, Caesarian Obtained, Barrier Sustained-Virus Antibody Free (COBS-VAF®).
Breeder: Charles River Laboratories France, L’Arbresle, France. Age/Weight: at the beginning of the treatment period, the males were approximately 10 weeks old and weighed approximately 400 g (range 348 g to 451 g). The females were approximately 9 weeks old and weighed approximately 220 g (range 180 g to 246 g). The males and the females were sexually mature and were not siblings. The females were previously unmated. Acclimation: the animals were acclimated for a period of 5 days before the beginning of the treatment period. A larger number of animals than necessary were acclimated to permit selection and/or replacement of individuals. Allocation to study: during the acclimation period, the required number of animals (64 males and 64 females) was selected according to body weight and clinical condition.
They were then allocated to groups (by sex) according to a computerized stratification procedure bases on body weight, so that the average body weight of each group was similar (these data are not shown). Identification: each animal was individually identified by an ear tattoo (unique CIT identity number.
Environmental conditions:
From arrival at CIT, the animals were housed in a barriered rodent unit. The animal room conditions were set as follows: . temperature : 22 ± 2°C, . relative humidity : 50 ± 20%, light/dark cycle : 12h/12h (7:00 - 19:00), . ventilation : about 12 cycles/hour of filtered, non-recycled air. The temperature and relative humidity were recorded continuously. The animal room was disinfected before the arrival of the animals and cleaned regularly thereafter.Housing:The animals were individually housed, except during pairing, in polycarbonate cages (UAR, 43.0 cm x 21.5 cm x 18.0 cm) with stainless steel lids and containing autoclaved sawdust (SICSA, Alfortville, France). Toward the end of gestation and during lactation with their litter, autoclaved wood shavings (SICSA, Alfortville, France) was provided as nesting material, a few days before delivery and during the lactation period. Each cage contained an object (Nylabone) for enrichment of the environment for the rats. The cages were placed in numerical order on the racks.
Food and water:
The animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch No. 9615507 (SSNIFF Spezialdiäten GmbH, Soest, Germany), which was distributed weekly. The animals had free access to bottles containing tap water (filtered with a 0.22 µm filter).Contaminant analyses:The batches of diet, sawdust and wood shavings are analyzed by the suppliers for composition and contaminant levels. Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet, drinking water, sawdust or wood shavings at levels which could be expected to interfere with, or prejudice, the outcome of the study

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 % in water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Justification for use and choice of vehicle: validated analytical method demonstrating stability and homogeneity of the test item in the vehicle
- vehicle: 0.5% aqueous methylcellulose solution prepared using: . purified water, obtained by reverse osmosis

PREPARATION OF DOSING SOLUTIONS:
The test item dosage forms were prepared weekly, dosage form stability/homogeneity was confirmed from 10 to 100 mg/mL for a 9-day period) and were maintained at +4°C and protected from light prior to use and delivered in brown flasks.
On weeks 1, 3 and 5, concentration was checked for each concentration and homogeneity was checked on weeks 1 and 5.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical analysis of the dosage forms
1 Principle and validation of the method
A High Performance Liquid Chromatography with UV detection (HPLC/UV) analytical method for the determination of WP30 in dosage form samples was used.
The validation of the analytical method was conducted in CIT/Study No. 37878 VAA. The analytical method was validated for dosage forms ranging from 10 to 100 mg/mL in aqueous solution of methylcellulose at 0.5% (w/w).

2 Composition of an analytical sequence Each analytical sequence consisted of at least: . a blank sample (diluent only), . ten standard samples at nominal concentration, prepared from two independent standard solutions, . study samples prepared from aliquots of the dosage forms.
The standard samples bracketed the dosage form samples. The blank sample was checked for the absence of chromatographic interference.

3 Analytical sequence suitability rules The acceptance of the analytical sequence depended on the precision of the standard samples and
on the agreement of the standard sample results. Acceptance criteria are defined in CIT Standard Operating Procedures (SOPs).
The acceptance criteria include: . precision of the standard solutions [%RSD (Relative Standard Deviation) must pass defined acceptance criteria],
agreement of the standard samples (the mean response factor of standard samples prepared from standard solution #1 must agree with the mean response factor of standard samples prepared from standard solution #2).

4 Study sample re-analysis rules No re-analysis was performed since the acceptance criteria were met for each series.

5 Determination of dosage form homogeneity and stability The test item dosage forms were prepared weekly (according to CIT/Study No. 37879 AHS, dosage
form stability/homogeneity was confirmed from 10 to 100 mg/mL for a 9-day period) and were maintained at +4°C and protected from light prior to use and delivered in brown flasks.
The homogeneity of each test item dosage form prepared for use was determined in weeks 1 and 5

6 Determination of WP30 concentration in dosage forms The concentration of the test item in samples of each control and test item dosage forms prepared for use were determined in weeks 1, 3 and 5 . Before day 1 and whenever possible for the other formulations, these analyses were performed prior to administration of the dosage forms to the animals.
Acceptance criterion: . measured concentration = nominal concentration ± 15%.
Duration of treatment / exposure:
Three groups of 10 male and 10 female Sprague-Dawley rats received the test item, WP30, daily, by oral (gavage) administration, before mating (2 weeks), during mating (3 weeks at least for the males and up to 3 weeks for the females) and, for the females, throughout gestation and until day 5 p.p., at dose-levels of 100, 300 or 1000 mg/kg/day. A group of 10 males and 10 females received the vehicle control, 0.5% (w/w) aqueous methylcellulose, under the same experimental conditions. In addition, three groups of eight males and eight females received also the test item for 5 weeks at dose-levels of 100, 300 or 1000 mg/kg/day for blood plasma concentration measurements on study day 1 and at the end of the treatment period (study week 5). The dosing volume was 10 mL/kg/day.
Frequency of treatment:
Three groups of 10 male and 10 female Sprague-Dawley rats received the test item, WP30, daily, by oral (gavage) administration, before mating (2 weeks), during mating (3 weeks at least for the males and up to 3 weeks for the females) and, for the females, throughout gestation and until day 5 p.p., at dose-levels of 100, 300 or 1000 mg/kg/day. A group of 10 males and 10 females received the vehicle control, 0.5% (w/w) aqueous methylcellulose, under the same experimental conditions. In addition, three groups of eight males and eight females received also the test item for 5 weeks at dose-levels of 100, 300 or 1000 mg/kg/day for blood plasma concentration measurements on study day 1 and at the end of the treatment period (study week 5). The dosing volume was 10 mL/kg/day.
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 or 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
Three groups of 10 male and 10 female Sprague-Dawley rats received the test item, WP30, daily, by oral (gavage) administration, before mating (2 weeks), during mating (3 weeks at least for the males and up to 3 weeks for the females) and, for the females, throughout gestation and until day 5 p.p., at dose-levels of 100, 300 or 1000 mg/kg/day. In addition, three groups of eight males and eight females received also the test item for 5 weeks at dose-levels of 100, 300 or 1000 mg/kg/day for blood plasma concentration measurements on study day 1 and at the end of the treatment period (study week 5). The dosing volume was 10 mL/kg/day.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels were selected in agreement with the results obtained in a 2-week rat toxicity study where there were no treatment-related effects up to 1000 mg/kg/day.

- Rationale for selecting satellite groups: Satellite animals were not mated and were allocated for toxicokinetic investigations only

Positive control:
None

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked in table were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly until mating and then body weight were recorded at designated intervals

OPHTHALMOSCOPIC EXAMINATION: YES:
- pupillary reflex, visual stimulus response on five males and females per group at the end of the study

HAEMATOLOGY: Yes
- Prior to sacrifice, blood samples were also taken from these animals for analysis of hematology.

CLINICAL CHEMISTRY: Yes
- Prior to sacrifice, blood samples were also taken from these animals for analysis of blood biochemistry parameters.

URINALYSIS: Yes
- Prior to sacrifice, blood samples were also taken from these animals for analysis of urinalysis.

NEUROBEHAVIOURAL EXAMINATION: Yes
A Functional Observation Battery including touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, rectal temperature and motor activity was performed on five males and females per group at the end of the study


Sacrifice and pathology:
Sacrifice (principal animals) On completion of the treatment period, after at least 14 hours fasting, all surviving F0 males and females were deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and sacrificed by exsanguination. . males: after the end of the pairing period (at least 5 weeks of treatment in total), . females: on day 6 p.p.. The female which did not delivered on day 25 p.c. was sacrificed without overnight fasting by inhalation of carbon dioxide gas followed by cervical dislocation (after checking body weight as an indication of a possible un-noticed delivery) Animals prematurely sacrificed or found dead Males (principal and satellite animals) There was no premature sacrifice in males. The male found dead was submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs.
Females
The female (W27025) sacrificed for no delivery was submitted for a macroscopic post-mortem examination of the principal thoracic and abdominal organs. The pregnancy status was determined. For apparently non-pregnant female, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique. Organ weights (adults) The body weight of each animal sacrificed as scheduled was recorded before sacrifice, and the organs specified in the Tissue Procedure Table were weighed (wet) as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated. For the female (W27025) sacrificed on day 25 p.c. due to no delivery, the organs specified in the Tissue Procedure Table were weighed Macroscopic post-mortem examination Principal parent animals A complete macroscopic post-mortem examination was performed on all parent animals including that was found dead during the study. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues. The numbers of corpora lutea and implantation sites were also recorded for females sacrificed as scheduled on day 6 p.p.. The tissues specified in the Tissue Procedure Table were preserved in 10% buffered formalin (except for the testes and epididymides which were fixed in modified Davidson's fixative). Preparation of histological slides All tissues required for microscopic examination were trimmed based on the RITA guidelines (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin (except testes and epididymides which were stained with hematoxylin/PAS). This tissue processing was performed at CIT.
Microscopic examination
A microscopic examination was performed on: . all tissues listed in the tissue procedure table from the first five males sacrificed and the first five females that delivered and were sacrificed on day 6 p.p. of the control and high-dose groups (groups 1 and 4) and for the male rat (W26923) that was found dead, . all macroscopic lesions of all the animals of the low- and intermediate-dose groups (groups 2 and 3) sacrificed on completion of the treatment period, . the female rat (W27025) sacrificed because of no delivery to investigate possible causes. Special emphasis was paid to the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Other examinations:
The males were sacrificed during study week 6. Body weights and selected organs weights were recorded and a complete macroscopic post-mortem examination performed, with particular attention paid to the reproductive organs. A microscopic examination was also conducted on selected organs from the first five males in the control group and the high-dose group. Microscopic examination was conducted on all macroscopic lesions from all groups.
Dams were sacrificed on day 6 p.p.. Body weights and selected organs weights were recorded and a complete macroscopic examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was then conducted on selected organs from the first five females to deliver in the control group and the high-dose group and on any macroscopic lesions from all groups.
Statistics:
The following statistical tests were done:
-For hematology, biochemistry and urinanalysis parameters, the normality of data distribution was checked. This was followed by assessment of homogenity of variance or by logarithmic transformation of values
For Organ weights and depending of normaity of each group, a non-parametric (Kruska-Wallis followed by a Dunn test ) or a parametric (one way analysis of variance followed by a Dunnett test) approach was selected.
The other data were compared by one-way analysis of variances and Dunnett test or by Fisher exact probability test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effect observed

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the experimental conditions of this study: . the No Observed Effect Level (NOEL) for systemic parental toxicity was considered to be 1000 mg/kg/day
Executive summary:

The objective of this study was to evaluate the potential toxic effects of the test item, WP30, following daily oral (gavage) administration to male and female rats from before mating, during mating and, for the females, throughout gestation until day 5post-partum (p.p.)inclusive. This study provides initial information on possible health hazards (including neurological and immunological effects) likely to arise from repeated exposure over a limited period of time. It can also indicate effects on male and female reproductive performance, such as gonadal function, mating behavior, conception, development of the conceptus and parturition.

Methods

Three groups of 10 male and 10 female Sprague-Dawley rats received the test item, WP30, daily, by oral (gavage) administration, before mating (2 weeks), during mating (3 weeks at least for the males and up to 3 weeks for the females) and, for the females, throughout gestation and until day 5p.p., at dose-levels of 100, 300 or 1000 mg/kg/day. A group of 10 males and 10 females received the vehicle control, 0.5% (w/w) aqueous methylcellulose, under the same experimental conditions. In addition, three groups of eight males and eight females received also the test item for 5 weeks at dose-levels of 100, 300 or 1000 mg/kg/day for blood plasma concentration measurements on study day 1 and at the end of the treatment period (study week 5). The dosing volume was 10 mL/kg/day. 

Animals were checked daily for clinical signs, mortality, and detailed clinical observations were conducted weekly. In males, body weights and food consumption were recorded weekly until mating and then body weight were recorded at designated intervals. In females, body weights and food consumption were recorded weekly until mating and then at designated intervals throughout gestation and lactation. The animals were paired for mating after 2 weeks of treatment and the dams were allowed to litter and rear their progeny until day 5p.p.... A Functional Observation Battery including touch response, forelimb grip strength, pupillary reflex, visual stimulus response, auditory startle reflex, tail pinch response, righting reflex, landing foot splay, rectal temperature and motor activity was performed on five males and females per group at the end of the study. Prior to sacrifice, blood samples were also taken from these animals for analysis of hematology, urinalysis and blood biochemistry parameters. 

The males were sacrificed during study week 6. Body weights and selected organs weights were recorded and a complete macroscopicpost-mortemexamination performed, with particular attention paid to the reproductive organs. A microscopic examination was also conducted on selected organs from the first five males in the control group and the high-dose group. Microscopic examination was conducted on all macroscopic lesions from all groups. 

Dams were sacrificed on day 6p.p..Body weights and selected organs weights were recorded and a complete macroscopic examination was performed, with particular attention paid to the reproductive organs. A microscopic examination was then conducted on selected organs from the first five females to deliver in the control group and the high-dose group and on any macroscopic lesions from all groups.

 

Results

The WP30 concentrations in the administered dosage forms were within an acceptable range of variation (± 15%). WP30 was not detected in control samples.

ToxicokineticsOn determination of blood plasma concentration for toxicokinetic calculation, none of the satellite male and female rats had significant blood plasma levels on day 1 or at the end of the treatment period (blood plasma level < 0.500 ng/mL, the limit of quantification), with the exception of two satellite males and five satellite females which had blood plasma levels slightly higher than the limit of quantification on study day 1 or at the end of the treatment period. 

Overall, it was considered that there was no significant systemic exposure to the test item.

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