Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18 June 2007 to 20 August 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
impurity
Test material form:
solid: particulate/powder
Details on test material:
The test material is a UVCB substance.
Specific details on test material used for the study:
Identification AD-2000
Form White powder
Batch L-620150
Storage At room temp in the dark
Stability under storage condition Stable
Expiry date 1 January 2009

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rat: Wistar Cri:(WI) BR (outbred, SPF-Quality).
Recognised by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).
Source: Charles River Deutschland, Sulzfeld, Germany.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Test system: Rat: Wistar Cri:(WI) BR (outbred, SPF-Quality).
Total number of animals: 20 males, 20 females, (females were nulliparous and non-pregnant).
Age at start of treatment: Approximately 6 weeks
Randomisation: By computer-generated random algorithm according to body weight, with all animals within± 20% of the sex mean.
Acclimation period: At least 5 days before the start of treatment under laboratory conditions.

Conditions
Animals were housed in a controlled environment, in which optimal conditions were considered to be approximately 15 air changes per hour, a temperature of 21.0 ± 3.0°C (actual range: 21.2- 23.7°C), a relative humidity of 30-70% (actual range: 43- 83%) and 12 hours artificial fluorescent light and 12 hours darkness
per day.
Cleaning procedures in the room might have caused the temporary
fluctuations above the optimal maximum level of 70% for relative humidity. Temporary fluctuations from the light/dark cycle (with a maximum of 1 hour) occurred due to performance of functional observations in the room. Based on laboratory historical data, these fluctuations were considered not to have affected the
study integrity.

Accommodation
Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm; during overnight activity monitoring individual housing in Mill type; height 15 cm.) with sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage­ enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). No cage-enrichment was provided during overnight activity monitoring.

Diet
Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiaten GmbH, Soest, Germany).
Results of analyses for nutrients and contaminants of each batch were examined and archived.

Water
Free access to tap water. Certificates of analysis (performed quarterly) were examined and archived.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
Oral gavage, using a plastic feeding tube.
Formulations were placed on a magnetic stirrer during dosing.
Vehicle:
propylene glycol
Details on oral exposure:
Once daily, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose.
Vehicle: Propylene glycol (Merck, Darmstadt, Germany), specific gravity 1.036
Formulations (w/w) were prepared daily within 5 hours prior to dosing, and were homogenised to visually acceptable levels. Adjustment was made for specific gravity of the vehicle. No correction was made for the purity of the test substance.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Accuracy and homogeneity were determined for formulations prepared for use during treatment.
Duplicate samples (approximately 500 mg for Groups 1 -- 3 and approximately 200 mg for Group 4), which were taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 100, 100, 250 or 500 mi. For determination of accuracy, samples were taken at 50% height or at 90%, 50% and 10% height. The latter set of samples was also used for the determination of the homogeneity of the formulations. The flasks were filled up to the mark with methanol. In order to dissolve the test substance the solutions were ultrasonicated for 15 minutes. The solutions were further diluted by a factor of 100 with 75/25 (v/v) methanoi/Milli-O water to obtain concentrations within the calibration range.

For quantitation, an LC/MS technique was used and the concentrations were determined by measuring three major peaks in the chromatogram at m/z 369.4, 481.5 and 497.4.

Calibration curves were constructed using five concentrations. For each concentration, two responses were used. Linear or quadratic regression analysis was performed using the least squares method with a 1/concentration2 weighting factor. The coefficients of correlation (r) were > 0.99.


The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were between
90-114% of target. In the Group 1 formulations, no test substance was detected.


Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose.
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
5 ml/kg bw with vehicle
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
5 ml/kg bw with vehicle
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
5 ml/kg bw with vehicle
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
5 ml/kg bw with vehicle
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control:
None required

Examinations

Observations and examinations performed and frequency:
Mortality/Viability: At least twice daily. The time of death was recorded as precisely as possible.
Clinical signs: At least once daily, detailed clinical observations were made in all animals. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, degree and duration were recorded.
All symptoms were recorded and graded according to fixed scales: Maximum grade 1: grade 0 = absent, grade 1 = present
Maximum grade 3 or 4: grade 1 = slight, grade 2 = moderate,
grade 3 = severe, grade 4 = very severe
Functional observations: During week 4 of treatment, the following tests were performed on all animals (abbreviations mentioned in the respective tables indicated between brackets): hearing ability (HEARING), pupillary reflex (PUPIL UR), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0=normal/present, score 1 =abnormal/absent). motor activity test (recording period: 12 hours during overnight for individual animals, using a computerised monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
Body weights: On Days 1, 8, 15, 22 and 31
Food consumption: On Days 1, 8, 15, 22, and 31.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Clinical investigations:
Blood samples were collected from all surviving animals under iso-flurane anaesthesia (Abbott Laboratories Ltd., Zwolle, The Netherlands) immediately prior to scheduled post mortem examination, between 7.00 and 10.30 a.m.. The animals were fasted overnight (with a maximum of 20 hours) before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One, Bad Hailer, Austria) prepared with EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.9 ml)and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL).


Sacrifice and pathology:
Pathology

Necropsy

All animals surviving to the end of the observation period were deeply anaesthetised using iso­ flurane vapour (Abbott Laboratories Ltd., Zwolle, The Netherlands) and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution, Klinipath, Duiven, The Netherlands):


Identification marks: not processed
Adrenal glands
Aorta
Brain [cerebellum, mid-brain, cortex] Caecum
Cervix
(Clitoral gland) Colon Duodenum Epididymides
(Eyes with optic nerve [if detectable] and
Harderian gland)
(Female mammary gland area) (Femur including joint)
Heart Ileum Jejunum Kidneys (Larynx)
(Lacrimal gland, exorbital) Liver
Lung, infused with formalin
Lymph nodes - mandibular, mesenteric
(Nasopharynx) Oesophagus

Ovaries
Pancreas
Peyer's patches [jejunum, ileum] if detectable
Pituitary gland (Preputial gland) Prostate gland Rectum
(Salivary glands - mandibular, sublingual) Sciatic nerve
Seminal vesicles (Skeletal muscle) (Skin)
Spinal cord -cervical, midthoracic, lumbar
Spleen
Sternum with bone marrow
Stomach Testes Thymus
Thyroid including parathyroid [if detectable] (Tongue)
Trachea
Urinary bladder
Uterus
Vagina
All gross lesions

Organ Weights

The following organ weights and terminal body weight were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus

Histopathology:
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all Main group 1 and 4 animals,
-all tissues from all animals of all dose groups which died spontaneously,
-all gross lesions
Statistics:
The following statistical methods were used to analyse the data:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test1 (many-to­ one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to
follow a normal distribution.
- The exact Fisher-test was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of
exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs of toxicity noted during the observation period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No mortality occurred during the study period that was considered to be related to treatment with the test substance.

Two control females (no. 21 and 23) and one female receiving 1000 mg/kg/day (no. 37) were found dead on Day 15 or Day 29. In all three animals there was evidence of a gavage accident and the death of these animals was considered unrelated to the test substance.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights and body weight gain of treated animals were considered to have been unaffected by treatment with the test substance.

No toxicological significance was ascribed to the slightly lower body weight (statistically significant) in females at 150 mg/kg/day at the start of the study (Day1). Body weight gain during the study remained well within normal limits for female rats of this age and strain.
A slightly reduced body weight gain was noted in females at 50 mg/kg/day throughout
treatment. The minor change in body weight gain (not statistically significant) occurred in the absence of a dose-related distribution and means remained well within normal limits for female rats of this age and strain.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption before or after allowance for body weight was similar between treated and control animals.
Food efficiency:
no effects observed
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
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects at highest dose tested

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Wistar rats were treated with the test substance for 31 consecutive days by daily oral gavage at dose levels up to 1000 mg/kg/day.

No treatment-related toxicologically significant changes were noted in any of the parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination).

From the results presented in this report a definitive No Observed Adverse Effect Level
(NOAEL) for the test substance of 1000 mg/kg/day was established.