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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 05-Sep-2014 to 20-Aug-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
adopted 21 September 1998
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
Directive 96/54/EC, 30 September 1996
Deviations:
no
GLP compliance:
yes
Limit test:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, B.V., Kreuzelweg 53, 5961 NM Horst / Netherlands
- Age at study initiation: Ca. 7 weeks
- Weight at study initiation: Actual Body Weight Ranges (at Acclimatization) were for the Males 162 - 203 g (mean: 181 g) and for the Females: 125 - 151 g (mean: 138 g)
- Fasting period before study: The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- Housing: In groups of up to four in Makrolon type-4 cages with wire mesh tops and standard softwood bedding (J. Rettenmaier & Söhne GmbH & Co. KG, 73494 Rosenberg / Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) including paper enrichment (Enviro-dri from Lillico, Biotechnology, Surrey / UK).
- Diet: Pelleted standard Harlan Teklad 2914C (batch no. 20/14 and 46/14) rat / mouse maintenance diet (Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was available ad libitum. The feed batches were analyzed for contaminants.
- Water: Community tap-water from Itingen was available ad libitum in water bottles. Bacteriological assay, chemical and contaminant analyses of respective samples were performed
- Acclimation period: 7 days under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12-hour fluorescent light / 12-hour dark cycle with at least eight hours music during the light period.

IN-LIFE DATES: From: 11-Sep-2014 (start of 7-day Acclimatization) To: 18/19-Dec-2014 (Necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
300
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared with the test item as delivered by the Sponsor.
The vehicle was pre-warmed to a temperature of approximately 40 °C. DGEBADA was weighed into a glass beaker on a tared precision balance and approximately 80 % of the warm vehicle was added (w/v). The mixture was mixed thoroughly using a homogenizer until a clear to colloid-like yellowish solution was obtained. Having obtained a homogeneous mixture, the remaining vehicle was added until the required final volume was reached. Separate formulations were prepared for each concentration. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.
The dose formulations were stored for up to eight days in glass beakers at room temperature (20 ± 5 °C).

DIET PREPARATION
- Not relevant due to exposure via gavage

VEHICLE
- Justification for use and choice of vehicle: Considered non-toxic to the test animals and ability to form a homogeneous mixture with the test item
- Concentration in vehicle (nominal): 0, 20, 60 and 100 mg/mL (at 0, 100, 300 and 1000 mg/kg bw/day, respectively)
- Amount of vehicle (by gavage): 5 mL/kg body weight
- Lot/batch no.: BCBN0917V (Source: Sigma-Aldrich Chemie GmbH, Steinheim / Germany, Expiry Date: 30-Sep-2016)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At experimental start, on day 1 and during weeks 4, 8 and 13, duplicate samples of the control group as well as three samples (top, middle and bottom) of about 1 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration. Duplicate samples of about 1 g of each concentration were taken to confirm stability (4 hour and 10 days).
These representative samples were dispatched to the analytical laboratories internally (at room temperature and back up samples, on dry ice) and either stored frozen at -20 ± 5 °C until analysis or directly analyzed.
The test item was used as analytical standard.
Stock solutions of DGEBADA in acetonitrile were prepared for external calibration. For example, 20.92 mg of DGEBADA was weighed into a 50 mL volumetric flask and filled to about 75 % of final volume with acetonitrile. The mixture was sonicated for at least five minutes and brought to volume with acetonitrile to yield a solution with a concentration of 418.4 µg/mL. Aliquots of this stock calibration solution were diluted with acetonitrile to obtain calibration solutions with nominal concentrations ranging from 12.55 to 200.8 µg/mL. On each occasion calibration solutions derived from two stock solutions were used for calibration.
Each sample was transferred into an appropriate volumetric flask. The sample vial was successively rinsed with at least two portions of acetonitrile and the rinsings were combined in the volumetric flask. The flask was filled to about 75 % of the target volume with acetonitrile and dissolution was achieved by sonication for at least five minutes. The flask was filled to the mark with acetonitrile. Sample solutions were further diluted with acetonitrile into the calibration range.
Typical HPLC systems from Merck-Hitachi series 7000 were used:
- Computerized System: EZ Chrom Elite; Agilent Technologies
- Column: Kinetex C18; 50 x 4.6 mm, 5 µm
- Pre-Column: Phenomenex 4 x 3 mm
- Column Temperature: Ambient
- Eluent A: Purified water / acetonitrile (50/50, v/v)
- Eluent B: Acetonitrile
- Gradient:
Time [min]; Eluent A [%]; Eluent B [%]
0; 100; 0
4; 100; 0
8; 5; 95
10; 5; 95
10.1; 100; 0
15; 100; 0
- Flow: 1 mL/min
- Wave Length: 230 nm
- Injection Volume: 10 µL
The linearity of the analytical system used for sample analyses was demonstrated with a good relationship between peak areas measured and calibration solution concentrations. All calibration points used met the acceptance limit of ±20 % variation from the calibration curve derived by linear regression analysis. The coefficients of determination (R²) were higher than 0.99.
The DGEBADA concentrations in the dose formulations ranged from 82.8 to 104.5 % with reference to the nominal and were within the accepted range of ±20 %, except for samples prepared on 11 December 2014 that were in the range from 126.5 to 136.0 %.
The homogeneous distribution of DGEBADA in the preparations was approved because single results found did not deviate more than 4.5 % from the corresponding mean and met the specified acceptance criterion of =15 %. Accordingly the effects were assigned to the nominal concentrations.
In addition, the test item was found to be stable in application formulations when kept up to ten days at room temperature due to recoveries which met the variation limit of 10 % from the time-zero (homogeneity) mean value.
Duration of treatment / exposure:
92/93 days
Frequency of treatment:
Daily, at approximately 24 hour intervals
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 males and 10 females
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected by the Sponsor.
- Rationale for animal assignment: Randomly allocated to groups by body weight.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Observations for viability / mortality were recorded twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule:
Daily Observations: The animals were observed for clinical signs once before commencement of administration as well as daily on Days 1 - 92/93 (twice daily during Days 1 - 3) during the treatment period. From Day 34 onwards, symptoms were also recorded immediately after administration.
Weekly Detailed Observations: The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed once before commencement of administration and once weekly (weeks 1 to 12) thereafter.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly (on Days 1, 8, 15, 29, 57 and 91)
Body weights were recorded weekly during acclimatization, treatment and recovery periods and before necropsy, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.
- Body weight observations checked in tables 4 and 5 were included.

FOOD CONSUMPTION: Yes
The food consumption was recorded once during the acclimatization period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the Harlan Laboratories computer.

FOOD EFFICIENCY: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During week 13
- Dose groups that were examined: All animals of the control and high dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood Sampling after 13 Weeks: 18/19-Dec-2014
- Anaesthetic used for blood collection: Yes, light isoflurane anaesthesia
- Animals fasted: Yes. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: All animals
- Parameters examined: Glucose; Urea; Creatinine; Bilirubin, total; Cholesterol, total; Triglycerides; Phospholipids; Aspartate aminotransferase; Alanine aminotransferase; Lactate dehydrogenase; Alkaline phosphatase, Gamma-glutamyl-transferase; Creatine kinase; Sodium; Potassium; Chloride; Calcium; Phosphorus; Protein, total; Albumin; Globulin; Albumin/Globulin ratio; Coagulation Prothrombin time (= Thromboplastin time); Activated partial Thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood Sampling after 13 Weeks: 18/19-Dec-2014
- Animals fasted: Yes. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- How many animals: All animals
- Parameters examined: Glucose; Urea; Creatinine; Bilirubin, total; Cholesterol, total; Triglycerides; Phospholipids; Aspartate aminotransferase; Alanine aminotransferase; Lactate dehydrogenase; Alkaline phosphatase; Gamma-glutamyl-transferase; Creatine kinase; Sodium; Potassium; Chloride; Calcium; Phosphorus; Protein, total; Albumin; Globulin; Albumin/Globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: Urine Sampling after 13 Weeks: 18/19-Dec-2014
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum.
- Parameters examined: Urine volume (18 hour); Specific gravity (relative density); Colour; Appearance; pH value; Nitrite; Protein; Glucose; Ketones; Urobilinogen; Bilirubin; Erythrocytes; Leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes, relevant parameters from a modified Irwin screen test were evaluated.
- Time schedule for examinations: During week 13
- Dose groups that were examined: All dose groups, all animals
- Battery of functions tested: grip strength / locomotor activity
Sacrifice and pathology:
Sacrifice:
After 13 Weeks: 18/19-Dec-2014
All animals were weighed and necropsied. Descriptions of all macroscopical abnormalities were recorded. All animals were anesthetized by intraperitoneal injection of pentobarbitone and killed by exsanguination.

GROSS PATHOLOGY: Yes
Organ weights: The organs were weighed before fixation and recorded on the scheduled dates of necropsy. Relative organ weights were calculated on the basis of the body weight-to-brain weight.

HISTOPATHOLOGY: Yes
Samples of the tissues and organs listed in table 1, below, were collected from all animals at necropsy and fixed in neutral phosphate buffered 4% formaldehyde solution unless indicated otherwise.

Seminology and Spermatid Count
- Time schedule: After Treatment, 18/19-Dec-2014
- How many animals: Sperm analysis was performed in all males.
Motility: At necropsy of males, a sperm sample from the left caudal epididymis was obtained from each male. The sample was diluted with a pre-warmed (about 37 - 40 °C) physiological medium, and shortly after being obtained, one hundred sperm cells were counted in duplicates (two sperm samples) microscopically for determination of percentage of not motile, stationary motile and progressively motile sperm.
Morphology: The sperm cells in the original physiological medium for motility determination were used for morphological assessment after Eosin staining and fixation. The slides were stored at room temperature until analysis. Five hundred (500) sperm cells per sample were evaluated microscopically and classified into the following categories:
Code Description
A Normal, complete sperm
B Normal head, abnormal tail
C Normal head only, tail detached
D Abnormal head only, tail detached
E Abnormal head, normal tail
The percentages of categories A to E were determined. In the absence of treatment-related effects in the control males and high-dose males, the males of the low- and middle-dose were not assessed.
Sperm Count
The left testis and the left cauda epididymis were taken for determination of homogenization resistant sperm cells (HRS) in testes and epididymis. The testes and cauda epididymis were frozen at -20 ± 5 °C pending evaluation. For evaluation the testes and cauda epididymis were thawed and processed separately. Testes and cauda epididymis were weighed and placed in Triton-X-100 solution and homogenized with a blender (Ultra Turrax) and an ultrasonic water bath. Sperm heads were counted microscopically using a modified Neubauer chamber. Dilution factors for each sample were documented in the raw data. These evaluations were performed in the first instance only for group 1 and 4 males. In the absence of a treatment-related effect the remaining frozen tissues were not evaluated.

Histotechnique: All organ and tissue samples were processed, embedded and cut at an approximate thickness of 4 micrometers and stained with haematoxylin and eosin.
Histopathology: Slides of all organs and tissues collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist. In addition, test item-related findings noted in the livers and mesenteric lymph nodes of high-dose males and females and in the pituitary glands of high-dose males trigger the evaluation of these organs in the middle- and low-dose groups. Attempts were made to correlate gross observations with microscopic findings. The stage of oestrus was evaluated, as the stage of spermatogenesis and histopathology of the interstitial cell structure. A peer review was performed by W. Henderson.
Immunohistochemistry: Slides of the kidney from all male animals of the control and high-dose groups were immunostained with an anti-alpha-2-microglobulin-antibody. The slides were examined by the principal investigator for histopathology. Slides of the kidney from all male animals of the control and high-dose groups were shipped to: Stewart Jones, Propath UK Limited, Willow Court, Netherwood Road, Hereford / United Kingdom
Other examinations:
no
Statistics:
The following statistical methods were used to analyse body weight, food consumption, grip strength, locomotor activity, clinical laboratory data, ophthalmoscopy, sperm analysis, organ weights and ratios as well as macroscopic findings:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnett-test when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
No fatalities occurred. Increased salivation in males and females treated in groups 3 and 4 from week 5 onwards were considered to be incidental.
Mortality:
no mortality observed
Description (incidence):
No fatalities occurred. Increased salivation in males and females treated in groups 3 and 4 from week 5 onwards were considered to be incidental.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
In group 4 the males had test item related lower mean body weight from day 15 of treatment. Differences in mean body weights of both sexes in group 3 & 4 were considered to be within the range of typical variation.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The mean daily food consumption was not affected in males and females.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
The mean daily food consumption was not affected, but in group 4 the males had test item related lower mean body weight gain from day 15 of treatment.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no test item-related ophthalmoscopic changes.
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Group 3 & 4, both sexes: Lower mean platelet counts. Group 4 males: higher relative neutrophil & monocyte and lower mean relative lymphocyte counts; females: elevated mean relative monocyte and mean absolute neutrophil counts
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Group 2-4 males, group 3&4 females: Increased cholesterol and phospholipid levels. Group 4 males: reduced glucose, potassium, protein and globulin levels & hepatic enzyme activation. Group 4 females: Aminotransferase activities increased
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no test item-related differences in the urinalysis parameters of males and females at all dose levels.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
No effects in the functional observation battery. Mean fore- and hind limb grip strength values increased. Locomotor Activity reductions in males of group 2 and both sexes of groups 3&4
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Group 2-4: Lower mean absolute and relative prostate weights;
Group 3&4 males: Higher mean relative kidney weights;
Group 4 females: Lower mean absolute thymus weight, mean thymus-to body weight ratio and mean thymus-to-brain weight ratio
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related adverse macroscopical findings at any dose level.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test-item related findings were present in the mesenteric lymph nodes of females at 300 mg/kg/day and both sexes at 1000 mg/kg/day, and in the liver (males and females) and pituitary (males only) at 1000 mg/kg bw/day. The most prominent liver findings were observed in the female rat no. 72.
In female no. 72 treated with 1000 mg/kg/day, liver findings consisted of moderate periportal hepatocellular hypertrophy (enlarged hepatocytes with deep eosinophilic cytoplasm and variably-sized nuclei), moderate centrilobular hepatocellular vacuolation (presence of numerous fine coalescing cytoplasmic vacuoles) and slight pigment deposition (presence of brown pigment within Kupffer cells and macrophages in portal areas). Other findings recorded in this animal were slight tubular vacuolation in the kidneys (presence of fine vacuoles in the proximal tubular epithelium), minimal to slight decreased lymphocytes in the lymphoid organs (mesenteric lymph node, spleen and thymus) and reduced number/size of corpus lutei in the ovaries. Tubular vacuolation in the kidneys and reduced number/size of corpus lutei in the ovaries were the histological correlates of the pale renal discoloration and reduced ovarian size observed at necropsy, respectively.
In the liver, minimal focal/multifocal hepatocellular necrosis (presence of a few scattered collections of a few necrotic hepatocytes) was observed in two of ten males and one of ten females at 1000 mg/kg bw/day. In the mesenteric lymph nodes, minimal to slight decreased lymphocytes were recorded from 300 mg/kg bw/day in females and at 1000 mg/kg bw/day in males and females.
In the pituitary, minimal multifocal cell hypertrophy was recorded in seven of nine males; the change was characterized by the presence of increased number of scattered enlarged pale eosinophilic cells in the pars distalis.
In the remaining animals treated with 1000 mg/kg/day, findings in the liver included minimal focal/multifocal hepatocellular necrosis (presence of scattered collections of a few necrotic hepatocytes) were noted in two of ten males and one of ten females. In four of ten males and four of ten females, minimal decreased number of lymphocytes was recorded in the mesenteric lymph nodes. .
Following the immunostaining with an anti alpha 2u-globulin antibody, an overall similar (low or medium) staining level were observed in the kidneys of males at 0 and 1000 mg/kg bw/day.
There were no microscopic findings in prostate, seminal vesicles or coagulating glands to account for the decreased weights recorded at necropsy.
All other findings were considered to be of spontaneous origin, unrelated to the test item. Of note, squamous papilloma in the forestomach and deciduoma in the uterus were recorded at 1000 mg/kg bw/day in one of ten males and one of ten females, respectively. In the absence of any other associated changes in the stomach and uterus, these isolated findings, known to occur spontaneously in rats, were considered to be unrelated to the test item.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no test item-related deaths at any dose level. Increased salivation was noted during daily observations in males and females treated with 300 mg/kg bw/day and 1000 mg/kg bw/day from week 5 onwards or in detailed weekly observations. Rats treated with 100 mg/kg bw/day were unaffected. All other findings were considered to be incidental. There were no test item-related findings noted during the weekly detailed observations.

BODY WEIGHT AND WEIGHT GAIN
At 1000 mg/kg bw/day, the males had lower mean body weight and lower mean body weight gain from day 15 of treatment. These were considered to be test item related. The differences in the mean body weights of both sexes treated with 100 and 300 mg/kg bw/day and in females treated with 1000 mg/kg bw/day were considered to be within the range of typical variation.

FOOD CONSUMPTION
The mean daily food consumption of the test item-treated rats was not affected.

FOOD EFFICIENCY
The mean daily food consumption was not affected, but in group 4 the males had test item related lower mean body weight gain from day 15 of treatment.

OPHTHALMOSCOPIC EXAMINATION
There were no test item-related ophthalmoscopic changes.

HAEMATOLOGY
In males treated with 1000 mg/kg bw/day, higher relative neutrophil and monocyte counts, lower mean relative lymphocyte count, lower platelet count were noted when compared with the controls. Females treated with 1000 mg/kg bw/day had elevated mean relative monocyte counts, reduced platelets and increased mean absolute neutrophil count. Males and females treated with 300 mg/kg bw/day had only lower mean platelet counts when compared with the controls. Males and females treated with 100 mg/kg bw/day were unaffected.

CLINICAL CHEMISTRY
Test item-related differences in clinical biochemistry included increased mean cholesterol and mean phospholipid levels in males at all dose levels. At 1000 mg/kg bw/day, parameters that were reduced in males included glucose levels, potassium levels, protein and globulin levels. Males also showed differences in several hepatic enzymes indicative of increased activation, including aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase. Increased activities of the aspartate and alanine aminotransferases were noted in females at 1000 mg/kg bw/day. These values are generally associated with increased metabolism in the liver and represent an adaptive response. Hence, these changes are considered to be test item related. Higher cholesterol and phospholipid levels seen in females at 1000 mg/kg/day and 300 mg/kg bw/day were also considered to be related to the test item. Increased potassium levels were noted in males treated with 300 mg/kg bw/day.

URINALYSIS
There were no test item-related differences in the urinalysis parameters of males and females at all dose levels.

NEUROBEHAVIOUR
There were no test item-related findings in the functional observation battery at week 13. The mean fore- and hind limb grip strength values of the test item-treated rats compared favourably with those of the respective control rats. Test item related reductions of locomotor activity was noted in males treated with 100 mg/kg/day and 300 mg/kg bw/day and both sexes treated with 1000 mg/kg bw/day.

ORGAN WEIGHTS
Lower mean absolute and relative prostate weights noted at 100, 300 and 1000 mg/kg/day were dose-related and considered to be related to the treatment with the test item. Other test item related differences in organ weights included higher mean relative kidney weights in males at 300 mg/kg/day and 1000 mg/kg/day, and lower mean absolute thymus weight, mean thymus-to body weight ratio and mean thymus-to-brain weight ratio in females at 1000 mg/kg bw/day.

GROSS PATHOLOGY
There were no test item-related adverse macroscopical findings at any dose level.

HISTOPATHOLOGY: NON-NEOPLASTIC
At 1000 mg/kg/day, moderate hepatocellular hypertrophy/vacuolation in one of ten females was noted and considered to be associated with changes in kidneys, lymphoid organs and ovaries consistent with stress/altered metabolic status.
Further changes seen at 1000 mg/kg/day included
• Minimal multifocal hepatocellular necrosis in the liver, minimal decreased lymphocytes in the mesenteric lymph nodes, and minimal multifocal cell hypertrophy in the pituitary gland (pars distalis) of males.
• Minimal multifocal hepatocellular necrosis the liver of a few rats (in two of ten males and one of ten females)
• Minimal to slight decreased lymphocytes in the mesenteric lymph nodes of four of ten males and four of ten females,
• Minimal multifocal cell hypertrophy in the pituitary gland (pars distalis) of seven of nine males.
In addition, minimal decreased lymphocytes in the mesenteric lymph nodes was observed in one of ten females at 300 mg/kg bw/day.
There were no test item-related effects in the testis, the integrity of the various cell types present within the different stages of the sperm cycle (spermatogenesis), the quantification of the different stages (quantitative staging) and the integrity of interstitial cells being unaffected.

- Seminology
The mean sperm counts of samples taken from the testis and from the epididymis of test item treated rats were unremarkable when compared with the controls. The evaluation of sperm motility showed that the number of progressive sperm cells was significantly lower (p<0.01) in males at all dose levels and that mean number of stationary sperm cells was according significantly higher at all dose levels (p<0.05% at 100 mg/kg/day and p<0.01% at 300 and 1000 mg/kg/day) when compared with control males. The number of non-motile sperm was significantly higher in males (p<0.05) at 1000 mg/kg bw/day. The reduction of sperm motility was considered to be a test item-related change.
There were no test item-related differences in the sperm morphology between sperm cells of the control group and rats treated with 1000 mg/kg bw/day.

There was no test item-related effect in the testis, the integrity of the various cell types present within the different stages of the sperm cycle (spermatogenesis) and the integrity of interstitial cells were unaffected. Following quantification of the different stages (quantitative staging), all stages were recorded in each group, there was no biologically significant quantitative difference in the distribution of stages among control and treated groups.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
< 100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
behaviour (functional findings)
organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
male reproductive system
Organ:
ventral prostate gland
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Any other information on results incl. tables

Locomotor Activity

Reductions seen in the mean locomotor activity of the males treated with 100 mg/kg/day and 300 mg/kg bw/day and both sexes treated with 1000 mg/kg bw/day were considered to be test item-related. The mean locomotor activity of the females treated with 100 or 300 mg/ kg bw/day were considered to be unaffected.

Table 2: Locomotor activity of male test animals

Allocation and

Dose Levels



mg/kg bw/day

Group 1

Control Males

0

Group 2

Males



100

Group 3

Males



300

Group 4

Males



1000

6 minutes

330

315 (-4.5 %)

292 (-11.5 %)

293 (-11.2 %)

12 minutes

206

150 (-27.2 %)

189 (-8.3 %)

181 (-12.1 %)

18 minutes

165

116 (-29.7 %)

156 (-5.5 %)

87 (-47.3 %)

24 minutes

152

116 (-23.7 %)

65 (-57.2 %)

60 (-60.5 %)

30 minutes

144

74 (-48.6 %)

28 (-80.6 %)

22 (-84.7 %)

Total

997

771 (-22.7 %)

730 (-26.8 %)

643 (-35.5 %)

Bold values denote statistically significant changes.

Table 3: Locomotor activity of female test animals

Allocation and

Dose Levels



mg/kg bw/day

Group 1

Control Females

0

Group 2

Females



100

Group 3

Females



300

Group 4

Females



1000

6 minutes

349

371 (+6.3 %)

290 (-16.9 %)

316 (-9.5 %)

12 minutes

198

208 (+5.1 %)

158 (-20.2 %)

147 (-25.8 %)

18 minutes

113

116 (+2.7 %)

85 (-24.8 %)

51 (-54.9 %)

24 minutes

24

67 (+179.2 %)

42 (+75.0 %)

11 (-54.2 %)

30 minutes

31

72 (+132.3 %)

43 (+38.7 %)

14 (-54.8 %)

Total

714

835 (+16.9 %)

617 (-13.6 %)

538 (-24.6 %)

Bold values denote statistically significant changes.

Body Weights

At 1000 mg/kg bw/day, the males had lower mean body weight and lower mean body weight gain from day 15 of treatment; the differences to the control males attained statistical significance (p<0.05 or p<0.01) from days 57 and 36 of treatment, respectively. These were considered to be test item-related.

At 300 mg/kg bw/day, the mean body weight and mean body weight gain of males were reduced from day 57 and day 15 of treatment, respectively. These differences were not statistically significant. Males at 100 mg/kg bw/day were not affected.

Table 4: Male Body Weights (g) and Difference to Control (%)

Allocation and

Dose Levels

mg/kg bw/day

Group 1

Control*

0

Group 2



100

Group 3



300

Group 4



1000

Day 1

216

214 (-0.9 %)

217 (+0.5 %)

217 (+0.5 %)

Day 8

261

261 (0.0 %)

264 (+1.1 %)

260 (-0.4 %)

Day 15

283

284 (+0.4 %)

287 (+1.4 %)

275 (-2.8 %)

Day 29

335

338 (-0.9 %)

336 (+0.3 %)

319 (-4.8 %)

Day 57

394

384 (-2.5 %)

379 (-3.8 %)

358 (-9.1 %)

Day 91

431

420 (-2.6 %)

412 (-4.4 %)

386 (-10.4 %)

Bold values denote statistically significant changes.

There were no test item-related differences in the mean body weights or mean body weight gain of females at any dose level.

Table 5: Female Body Weights (g) and Difference to Control (%)

Allocation and

Dose Levels

mg/kg bw/day

Group 1

Control*

0

Group 2



100

Group 3



300

Group 4



1000

Day 1

153

155 (+1.3 %)

153 (0.0 %)

154 (+0.7 %)

Day 8

175

175 (0.0 %)

177 (+1.1 %)

173 (-1.1 %)

Day 15

181

182 (+0.6 %)

182 (+0.6 %)

177 (-2.2 %)

Day 29

201

196 (-2.5 %)

203 (+0.5 %)

192 (-4.5 %)

Day 57

226

220 (-2.7 %)

223 (-1.3 %)

211 (-6.6 %)

Day 91

239

235 (-1.7 %)

239 (0.0 %)

220 (-7.9 %)

Bold values denote statistically significant changes.

Applicant's summary and conclusion

Conclusions:
In conclusion based on the results of this study, no-observed adverse effect levels (NOAEL) for systemic effects were below the lowest dose level of 100 mg/kg body weight/day of DGEBADA.
Executive summary:

The subchronic oral (gavage) 90-day repeated dose toxicity of the test item 4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, esters with acrylic acid (CAS 55818-57-0, DGEBADA) to Wistar rats of both sexes was investigated in a GLP-compliant dose-effect study according to the OECD TG 408 (1998) and EU B.29 (1996) protocols.

The test item was administered daily by oral gavage at dose levels of 0, 100, 300 and 1000 mg/kg body weight/day (named groups 1 to 4, respectively) using a volume of 5 mL/kg body weight. The control group was treated with the vehicle, PEG 300, only. The groups comprised 10 animals per sex which were sacrificed after 92/93 days of treatment. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during the acclimatization and treatment periods. Functional observational battery, locomotor activity and grip strength were performed during week 13. At the end of the dosing period, blood samples were withdrawn for haematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Sperm samples were collected at necropsy to assess count, motility and morphology. Histological examinations were performed on organs and tissues from all control and high dose animals and all gross lesions from all animals.

The administration of the test item to the test animals resulted in no test item-related deaths, no differences in mean food consumption, weekly observations (weeks 1 - 13) or functional observational battery (week 13), no differences of toxicological relevance in the fore- and hind limb grip strength values, no test item-related differences in the ophthalmoscopy and no test item-related effects on urine parameters.

Test item-related clinical signs included salivation in both sexes treated with 300 mg/kg bw/day and 1000 mg/kg bw/day. This finding was first noted in week 5 and was frequently seen throughout the rest of the study. This finding is generally associated with a bitter taste of the dosing solutions and is a secondary, non-adverse effect. Clearly lower mean body weight development was noted from day 15 of treatment in males treated with 1000 mg/kg bw/day.

Reduced locomotor activity was noted in males at all dose levels and in females treated at 1000 mg/kg bw/day.

In males treated with 1000 mg/kg bw/day, higher relative neutrophil and monocyte counts, lower mean relative lymphocyte count and lower platelet count were noted when compared with the controls. Females treated with 1000 mg/kg bw/day had elevated mean relative monocyte counts, reduced platelets and an increased mean absolute neutrophil count. Males and females treated with 300 mg/kg bw/day had only lower mean platelet counts when compared with the controls. Males and females treated with 100 mg/kg bw/day were unaffected.

Test item-related differences in clinical biochemistry parameters were noted in males at all dose levels. Increased mean cholesterol and mean phospholipid levels were noted in males at all dose levels, and is often associated with changes in lipid metabolism. Males also showed increased enzyme activation of aspartate aminotransferase, alanine aminotransferase and alkaline phosphatase. Similar findings were seen in females treated with 1000 mg/kg bw/day: increased activities of the aspartate and alanine aminotransferases. These differences, while test item related, are generally considered to be metabolic adaptation and of no toxicological relevance.

However, parameters that were reduced in males at 1000 mg/kg bw/day included glucose levels, potassium levels, protein and globulin levels. Although these values remained within the limits of the historical control values, they were considered to be related to the treatment with the test item. In females, significantly higher cholesterol and phospholipid levels (both p<0.01) were seen at 1000 mg/kg/day and 300 mg/kg/day, exceeded the upper limits of the historical control data and were considered to be test item-related.

The reduction of sperm motility noted at all dose levels was considered to be related to the test item.

Lower mean absolute and relative prostate weights noted at 100, 300 and 1000 mg/kg/day, and higher mean relative kidney weights in males at 300 mg/kg/day and 1000 mg/kg/day were considered to be test item-related effects. Lower mean absolute thymus weight, mean thymus-to body weight ratio and mean thymus-to-brain weight ratio in females at 1000 mg/kg bw/day were considered to be a stress reaction.

The test-item induced microscopic findings in the liver of a few rats at 1000 mg/kg bw/day (minimal hepatocellular necrosis, moderate hepatocellular hypertrophy/vacuolation); they were considered to be the histological correlates of the increased liver enzymes recorded in clinical biochemistry. The most prominent liver microscopic findings were observed in the female rat no. 72 (1000 mg/kg bw/day) and were associated with changes in kidneys, lymphoid organs and ovaries consistent with stress/altered metabolic status.

The pathogenesis of the multifocal cell hypertrophy observed in the pars distalis of the pituitary gland of males cannot be ascertained. However, the pituitary findings and the decreased prostate/seminal vesicles weights may represent an indicator of mild disruption of testosterone production/levels.

The pathogenesis underlying the decreased lymphocytes in the mesenteric lymph nodes is unknown in the absence of changes in the other lymphoid organs.

In conclusion based on the results of this study, no-observed adverse effect levels (NOAEL) for systemic effects were below the lowest dose level of 100 mg/kg body weight/day of DGEBADA.