Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March 9 to May 7, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: guideline test with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Identification: Epikote P
Structure : Substance is reaction product of Epikote 828 (CAS 25068-38-6)
n= 0 (90 mol%): n= 1 (10 mol%), and biphenyl-4-ol (CAS 92-69-3)
Molecular formula : C45H44O6 (ca. 52.5% by weight) Lower molecular weight approximately 7.5%; higher molecular weight approximately 40% by weight
Molecular weight: 680.8 (main component); Approximately 900 (number average resin)
CAS Number : 161308-15-2 (main component)
Description : Pale yellow powder
Batch : 8401500-710036 (taken from label)
Purity: > 99.8%
Test substance storage: At room temperature in the dark
Stability under storage conditions :stable
Expiry date : 01 December 2010

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
Test System Rat: Crl:WI(Han) (outbred, SPF-Quality).
Rationale Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC). Source Charles River Deutschland, Sulzfeld, Germany.
Total number of animals 30 males, 30 females, (females were nulliparous and non-pregnant).
Age at start of treatment Approximately 6 weeks.
Identification Earmark and tattoo.
Randomization By computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Acclimatization period At least 5 days before the start of treatment under laboratory conditions.
Health inspection Prior to commencement of treatment to ensure that the animals were in a good state of health.
Animal husbandry
Room number 11.
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: 18.3 -22.1°C), a relative humidity of 30-70% (actual range: 40 - 89%) 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 sterilized 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. Certificates of analysis were examined and then retained in the NOTOX archives.
Diet : Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® SpezialdiSten GmbH, Soest, Germany). Results of analyses for nutrients (each batch) and contaminants (quarterly) were examined and archived. A sample (approximately 500 grams) of each batch was retained at room temperature until finalization of the study report.
Water Free access to tap water. Certificates of analysis (performed quarterly) were examined and archived.
Analysis of bedding, paper, diet and water did not reveal any findings that were considered to have affected study integrity.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
Formulations were placed on a magnetic stirrer during dosing.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation < 10%).
Analysis of the Group 4 formulation after storage yielded a relative difference of s 10%. Based on this, the Group 4 formulation was found to be stable during storage at room temperature for at least 6 hours. Analysis of the Group 2 formulation yielded a relative difference of 17% but since concentration had not decreased during storage, the Group 2 formulation was still considered stable during storage at room temperature for at least 6 hours.
Formulation analyses confirmed that formulations of test substance were prepared accurately and homogenously, and were stable over at least 6 hours.
Duration of treatment / exposure:
28 days. Main animals were dosed up to the day prior to necropsy, and Recovery animals were dosed up to the day prior to start of the recovery period.
Frequency of treatment:
Once daily, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 25, 250 and 1000 mg/kg/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
One control group and three treated groups were tested, each consisting of 5 males and 5 females. An extra 5 animals per sex in the control and high dose group were group were allowed 14 days of recovery.
Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability over 6 hours.
The following parameters were evaluated: clinical signs daily; functional observation tests in week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.
Positive control:
N/A

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. Since no clinical signs were observed in the range finding study, no time range was set within which clinical observations should be conducted after dosing. 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 L/R), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent). Since no clinical signs were observed in the range finding study, no time range was set within which functional observation tests should be conducted after dosing, motor activity test (recording period: 12 hours during overnight for individual animals, using a computerized monitoring system, Pearson Technical Services, Debenham, Stowmarket, England). Since the abovementioned measurements did not reveal treatment-related effects, the functional observation tests were not performed at the end of the recovery phase.
Body weights/Food consumption: weekly
Clinical laboratory investigations
Blood samples were collected from all animals under iso-flurane anaesthesia (Abbott Laboratories Ltd., Hoofddorp, The Netherlands) immediately prior to scheduled post mortem examination at the end of the treatment and recovery phase, between 7.00 and 10.30 a.m.. Animals were deprived of food overnight (for a maximum of 20 hours), but water was available. Blood samples were drawn from the retro-orbital sinus and collected into tubes (Greiner Bio-One, Bad Haller, Austria) prepared with EDTAfor haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into untreated tubes
for determination of bile acids.



Sacrifice and pathology:
Pathology
1. Necropsy
All animals surviving to the end of the observation period were deeply anaesthetized using iso-flurane vapor (Abbott Laboratories Ltd., Hoofddorp, The Netherlands) and subsequently exsanguinated. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Rats found dead were subjected to a full post mortem examination as soon as possible after death and always within 24 hours. 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):
Adrenal glands,Brain [cerebellum, mid-brain, cortex] ,Caecum Cervix Colon Duodenum Epididymides * Eyes (including optic nerve and harderian
gland) * Femur including joint Heart lleum Jejunum Kidneys Liver Lung, infused with formalin Lymph nodes - mandibular, mesenteric Ovaries Peyer's patches [jejunum, ileum] if detectable,Prostate gland Rectum Sciatic nerve Seminal vesicles including coagulating gland, Skeletal muscle Spinal cord -cervical, midthoracic, lumbar Spleen Sternum with bone marrow,Stomach Testes * Thymus Thyroid including parathyroid [if detectable] ,Trachea Urinary bladder Uterus Vagina All gross lesions
Some tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
2. 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 Uterus (including cervix) Prostate Seminal vesicles includir Ovaries Thyroid including parathyroid
3. Histotechnology
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded in paraffin wax (Klinipath, Duiven, The Netherlands) and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).
4. 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 animal no. 48 which died at blood sampling,
- all gross lesions.
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.
Histopathology was subjected to a peer review.


Other examinations:
no data
Statistics:
The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test 1 (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
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:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
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
Details on results:
Observations
1. Mortality
No mortality occurred during the study period that was considered to be related to treatment with the test substance.
One female (no. 48, receiving 250 mg/kg/day) died during blood sampling, which was considered to be an accidental death. Deaths at blood sampling are considered to be of incidental nature.
2. Clinical signs
No clinical signs of toxicity were noted during the observation period.
Incidental findings that were noted included salivation, rales, scabs in the neck or left shoulder area, a wound in the neck area and a broken tail apex. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance. No clinical signs were observed for females at 25 mg/kg/day, males at 250 mg/kg/day and females at 1000 mg/kg/day of the main groups and for control males and females of the recovery groups.
3. Functional observations
Variations noted in motor activity data between treated and control animals occurred in the absence of a dose-related response, similar changes in the opposite sex, and/or supportive clinical signs. Therefore, they were considered to be of no toxicological relevance. Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
4. Body weights
Body weights and body weight gain of treated animals remained in the same range as controls over the study period.
2.Food consumption
Food consumption before or after allowance for body weight was similar between treated and control animals.
Clinical laboratory investigations
1. Haematology
Haematological parameters of treated rats were considered not to have been affected by treatment.
Minor statistically significant differences when compared to control animals included lower white blood cell counts in males at 25 mg/kg/day and lower haemoglobin levels and haematocrit values in males at 250 mg/kg/day at the end of treatment and lower mean corpuscular haemoglobin concentration (MHCH) in males at 1000 mg/kg/day at the end of recovery. These changes were considered of no toxicological significance as they remained within the range considered normal for rats of this age and strain, occurred in the absence of a treatment-related distribution and/or occurred only at the end of the recovery period.
2. Clinical biochemistry
Clinical biochemistry parameters of treated rats were considered not to have been affected by treatment.
Female no. 56 at 1000 mg/kg/day showed the following changes in clinical biochemistry parameters compared to other females: lower aspartate aminotransferase (ASAT), alkaline phosphatase (ALP), albumin and chloride levels and higher total protein, urea, creatinine and cholesterol levels at the end of treatment and/or recovery. These changes and other findings in this female did not occur in any of the other animals of this study. Therefore, these findings were considered incidental of nature and not to be related to toxic properties of the test substance.
Minor statistically significant differences when compared to control animals were considered of no toxicological significance as they remained within the rangejconsidered normal for rats of this age and strain, occurred in the absence of a treatment-related distribution and/or occurred only at the end of the recovery period. These differences included: lower alanine aminotransferase (ALAT) levels in males at 25 mg/kg/day and females at 250 mg/kg/day at the end of treatment, lower alkaline phosphatase (ALP) levels in females at 1000 mg/kg/day at the end of recovery, higher albumin levels in males at 250 mg/kg/day at the end of treatment, lower total bilirubin in females at 1000 mg/kg/day at the end of treatment, higher urea levels in females at 25 mg/kg/day at the end of treatment, higher creatinine levels in females at 25 and 250 mg/kg/day at the end of treatment, lower bile acid levels in males at 1000 mg/kg/day at the end of recovery, higher sodium levels in males at 25 mg/kg/day at the end of treatment, lower chloride levels in females at 25 mg/kg/day at the end of treatment and lower calcium levels in males at 25 mg/kg/day at the end of treatment.
Pathology
1. Macroscopic examination
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of toxicity caused by the test substance.
Female no. 56 at 1000 mg/kg/day showed enlarged ovaries and pale discolouration of kidneys which were also enlarged. The macroscopic abnormalities and other findings in this female did not occur in any of the other animals of this study. Therefore, these findings were considered incidental of nature and not to be related to toxic properties of the test substance. Other incidental findings among control and treated animals included several reddish foci on the glandular mucosa of the stomach, bent tail apex, red/brown foci on the clitoral gland, fluid in the uterus, reddish discolouration of mandibular lymph nodes, reddish discolouration of the thymus and a constricted spleen. These findings are occasionally seen among rats used in these types of studies and, therefore, they were considered changes of no toxicological significance.
2. Organ weights
No toxicologically relevant changes were noted in organ weights and organ to body weight ratios.
Female no. 56 at 1000 mg/kg/day showed higher liver and kidney weights and higher liver, kidney and ovaries to body weight ratios. The changes in organ weights and other findings in this female did not occur in any of the other animals of this study. Therefore, these findings were considered incidental of nature and not to be related to toxic properties of the test substance.
Several minor differences in organ weights and organ to body weight ratios achieved statistical significance when compared to control animals. These changes included lower prostate weight and prostate to body weight ratio in males at 25 mg/kg/day, lower adrenals to body weight ratio in males at 250 mg/kg/day, higher spleen weight and spleen to body weight ratio in females at 250 mg/kg/day at the end of treatment. These changes were considered of no toxicological significance as they remained within the range considered normal for rats of this age and strain and occurred in the absence of a treatment-related distribution.
Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.
3. Microscopic examination
There were no microscopic findings recorded which could be attributed to treatment with the test substance.
Female no. 56 at 1000 mg/kg/day showed bilateral, marked, progressive nephropathy at the end of recovery. Whilst this finding is unusual in a rat of this age, it was regarded as not treatment-related.
All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Effect levels

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: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the results given in this report, the NOAEL for test substance in the 28-day oral test is evaluated to be 1000 mg/kg/day.
Executive summary:

This repeated dose toxicity study was understood to assess the toxic potential of test article, which followed OECD guideline of 407 under the GLP conditions. Wistar rats were treated with test substance for 28 consecutive by daily oral gavage at dose levels of 0, 25, 250 and 1000 mg/kg/day followed by a 14-day treatment-free recovery period, each consisting of 5 males and 5 females.

No 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).

Therefore, a No Observed Adverse Effect Level (NOAEL) and No Observed Effect Level (NOEL) for EPIKOTE P of 1000 mg/kg/day was established based on the results presented in this report.