4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 3-aminomethyl-3,5,5-trimethylcyclohexylamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

Adopted 21 September 1998

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

1

Reference substance name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 3-aminomethyl-3,5,5-trimethylcyclohexylamine

EC Number:

500-101-4

EC Name:

4,4'-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with 3-aminomethyl-3,5,5-trimethylcyclohexylamine

Cas Number:

38294-64-3

Molecular formula:

(C15 H16 O2 . C10 H22 N2 . C3 H5 Cl O)x

IUPAC Name:

Reaction product of 3-aminomethyl-3,5,5-trimethylcyclohexanamine with oligomerisation products of 4,4'-propane-2,2-diyldiphenol with 2-(chloromethyl)oxirane

Test material form:

liquid: viscous

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han™:RccHan™:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS (UK) Limited, Oxon, UK
- Age at study initiation: 6-8 weeks
- Weight at study initiation: 218 to 270 g (males), 158 to 193 g (females)
- Housing: in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding
- Diet (e.g. ad libitum): pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited., Oxon, UK), ad libitum
- Water (e.g. ad libitum): mains drinking water, ad libitum
- Acclimation period: 9 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 13 May 2016 (first day of treatment) to 09 September 2016 (final day of necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations were therefore prepared weekly for the first week and then fortnightly thereafter and stored at approximately 4 ºC in the dark and under nitrogen.

VEHICLE
- Concentration in vehicle: 1.67, 16.67, 33.33 mg/mL
- Amount of vehicle (if gavage): 6 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results showed the formulations to be stable for at least twenty-two days. Measured concentrations were within ±10% of nominal concentrations, confie´rming accurate formulation.

Duration of treatment / exposure:

90 d + 28 d recovery

Frequency of treatment:

daily, 7 d/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

200 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on previous dose range finding study (Envigo Research Limited Study Number YY28YK; Fourteen Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat)
- Rationale for animal assignment (if not random): randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups
- Rationale for selecting satellite groups:
- Post-exposure recovery period in satellite groups: 28 d

Examinations

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to thirty minutes post dosing and one hour after dosing. During the treatment-free period, animals were observed daily.
- Detailed individual clinical observations were performed for each non-recovery animal using a purpose built arena. The following parameters were observed:
Gait, Tremors, Twitches, Convulsions, Bizarre/Abnormal/Stereotypic behavior, Salivation, Pilo-erection, Exophthalmia, Lachrymation, Hyper/Hypothermia, Skin color, Respiration, Palpebral closure, Urination, Defecation, Transfer arousal, Tail elevation

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

FOOD CONSUMPTION:
- for each cage group at weekly intervals

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily, for each cage group, by visual inspection of the water bottles for any overt changes

OPHTHALMOSCOPIC EXAMINATION: Yes
- The eyes of all control and treated animals were examined pre-treatment and all non-recovery control and non-recovery high dose animals were examined before termination of treatment (during Week 12).

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 90 (non-recovery animals), Day 118 (recovery animals)
- Anaesthetic used for blood collection: No
- Animals fasted: No
- How many animals: 10/dose group
- Parameters: Hemoglobin (Hb), Erythrocyte count (RBC), Hematocrit (Hct), Erythrocyte indices (mean corpuscular hemoglobin (MCH), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC)), Total leukocyte count (WBC), Differential leukocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos), basophils (Bas)), Platelet count (PLT), Reticulocyte count (Retic), Prothrombin time (CT), Activated partial thromboplastin time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 90 (non-recovery animals), Day 118 (recovery animals)
- Animals fasted: No
- How many animals: 10/dose group
- Parameters: Urea, Glucose, Total protein (Tot.Prot.), Albumin, Albumin/Globulin (A/G) ratio (by calculation), Sodium (Na+), Potassium (K+), Chloride (Cl-), Calcium (Ca++), Inorganic phosphorus (P), Aspartate aminotransferase (ASAT), Alanine aminotransferase (ALAT), Alkaline phosphatase (AP), Creatinine (Creat), Total cholesterol (Chol), Total bilirubin (Bili), Bile acids , Gamma glutamyl transpeptidase, Triglycerides (Trigs)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to the start of treatment and at weekly intervals thereafter, all non-recovery animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performance tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.
- Dose groups that were examined: all dose groups
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
organ weights: Adrenals, Brain, Epididymides, Heart, Kidneys, Liver, Ovaries, Spleen, Testes, Thymus, Uterus (with cervix)

HISTOPATHOLOGY: Yes
samples preserved:
Adrenals, Aorta (thoracic), Bone & bone marrow (femur including stifle joint), Bone & bone marrow (sternum), Brain (including cerebrum, cerebellum and pons), Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes, Gross lesions, Heart, Ileum (including Peyer’s patches), Jejunum, Kidneys, Liver, Lungs (with bronchi), Lymph nodes (mandibular and mesenteric), Mammary gland, Muscle (skeletal), Ovaries, Pancreas, Pituitary, Prostate, Rectum, Salivary glands (submaxillary), Sciatic nerve, Seminal vesicles (including coagulating gland), Skin, Spinal cord (cervical, mid thoracic and lumbar), Spleen, Stomach, Testes, Thymus, Thyroid/Parathyroid, Tongue, Trachea, Urinary bladder, Uterus (with cervix), Vagina

All tissues from non-recovery control and 200 mg/kg bw/day dose group animals and any animals that died during the study were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.
Since there were indications of possible treatment-related mesenteric lymph node and adrenal changes, examination was subsequently extended to include similarly prepared sections of the mesenteric lymph nodes and adrenals from animals in the low, intermediate and recovery dose groups.

Statistics:

Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Hematology, Blood Chemistry, Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analyzed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed as follows:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analyzed using Bartlett’s test. Intergroup variances were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Incidences of increased salivation were evident in all animals of both sexes treated with 200 mg/kg bw/day from Day 1 (females) and Day 2 (males) until the termination of treatment. Episodes of noisy respiration were also evident in all animals of both sexes treated with 200 mg/kg bw/day from Day 1 (females) and Day 2 (males) onwards. Isolated incidences of a stained snout, labored respiration, decreased respiratory rate, hunched posture and fur loss were also evident in some females treated with 200 mg/kg bw/day and a stained snout or sneezing was also evident in two males treated with 200 mg/kg bw/day. The male treated with 200 mg/kg bw/day that was found dead on Day 48 had only previously shown increased salivation and noisy respiration. During the treatment-free, twenty-eight day period, one male and one female that were previously given 200 mg/kg bw/day continued to show episodes of noisy respiration.
At 100 mg/kg bw/day, increased salivation and noisy respiration were evident throughout the treatment period albeit to a lesser extent than at 200 mg/kg bw/day. One male from this treatment group also had a decreased respiratory rate and hunched posture on Day 22 only.
No such effects were detected in males treated with 10 mg/kg bw/day, however, one female from this treatment group had increased salivation on Day 81 only.
One control female showed increased salivation on Day 22 whilst a further one control female showed increased salivation on Day 77. One female treated with 100 mg/kg bw/day had fur loss between Days 44 and 91.
These incidences were considered to be incidental.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male treated with 200 mg/kg bw/day was found dead on Day 48. There were no further unscheduled deaths.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males treated with 200 and 100 mg/kg bw/day showed a statistically significant reduction (p<0.01) in body weight gain during the first week of treatment. Periods of recovery were evident thereafter, however, generally body weight gain in these males was slightly below controls. A further, statistically significant reduction (p<0.01) in body weight gain was evident during Week 12 for males treated with 200 mg/kg bw/day and actual body weight losses were evident in males treated with 200 and 100 mg/kg bw/day during the final week of treatment. Consequently overall body weight gain was lower than controls in these males. Females treated with 200 mg/kg bw/day showed a slight reduction in body weight gain during the first week of treatment, however, statistical significance was not achieved and recovery was evident thereafter. Significant improvement in body weight gain was evident in males previously treated with 200 mg/kg bw/day during the twenty-eight day treatment-free period. Statistically significant increases (p<0.05-0.01) when compared to controls were evident in these males throughout the treatment-free period.
No such effects were detected in females treated with 100 mg/kg bw/day or in animals of both sexes treated with 10 mg/kg bw/day.
A statistically significant increase (p<0.05) in body weight gain during Week 9 and a statistically significant reduction (p<0.05) in body weight gain during Week 10 was evident in females treated with 200 mg/kg bw/day. These intergroup differences were considered to be incidental and not to be of toxicological importance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Animals of both sex treated with 200 mg/kg bw/day and males treated with 100 mg/kg bw/day showed a reduction in overall food consumption. Incidences of reduced food conversion efficiency was also evident in males treated with 200 and 100 mg/kg bw/day during the treatment period and generally followed the fluctuations seen in body weight gain. During the twenty-eight day treatment-free period, recovery in both food consumption and food conversion efficiency were evident in animals of both sexes that were previously given 200 mg/kg bw/day.
No such effects were detected in females treated with 100 mg/kg bw/day or in animals of both sexes treated with 10 mg/kg bw/day.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Animals of both sex treated with 200 mg/kg bw/day and males treated with 100 mg/kg bw/day showed a reduction in overall food consumption. Incidences of reduced food conversion efficiency was also evident in males treated with 200 and 100 mg/kg bw/day during the treatment period and generally followed the fluctuations seen in body weight gain. During the twenty-eight day treatment-free period, recovery in both food consumption and food conversion efficiency were evident in animals of both sexes that were previously given 200 mg/kg bw/day.
No such effects were detected in females treated with 100 mg/kg bw/day or in animals of both sexes treated with 10 mg/kg bw/day.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Visual inspection of water bottles did not reveal any inter-group differences.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmoscopic examination of animals of both sexes from the non-recovery control and 200 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Animals of both sexes treated with 200 and 100 mg/kg bw/day showed a statistically significant increase (p<0.05-0.01) in neutrophils. A statistically significant increase (p<0.01) in neutrophils was also present in females previously treated with 200 mg/kg bw/day at the end of the twenty-eight day treatment-free period. Males treated with 200 mg/kg bw/day also showed a statistically significant increase (p<0.01) in total leukocyte count. The majority of individual values for both parameters at 200 and 100 mg/kg bw/day were outside of historical control ranges.
No toxicologically significant effects were detected in animals of both sexes treated with 10 mg/kg bw/day.
Females treated with 200 mg/kg bw/day showed a statistically significant increase (p<0.05) in platelet count. The majority of individual values were within historical control range and in the absence of any associated histopathological correlates the intergroup difference was considered not to be of toxicological significance.
Males treated with 10 mg/kg bw/day showed statistically significant reductions (p<0.05) in mean corpuscular hemoglobin and mean corpuscular volume. The majority of individual values were within historical control ranges and in the absence of a similar effect in 200 or 100 mg/kg bw/day males or any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.
Following the treatment-free period, females that were previously given 200 mg/kg bw/day showed statistically significant increases (p<0.01) in hemoglobin, erythrocyte count and hematocrit and statistically significant reductions (p<0.05) in mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. In the absence of a similar effect in 200 mg/kg bw/day females at the end of the treatment period or any associated histopathological correlates the intergroup difference was considered not to be of toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no toxicologically significant effects detected in the blood chemical parameters examined.
Males treated with 200 and 100 mg/kg bw/day showed a statistically significant increase (p<0.05) in urea and statistically significant reductions (p<0.05-0.01) in total protein, albumin, alkaline phosphatase, triglycerides and bile acids. Males treated with 200 mg/kg bw/day also showed a statistically significant increase (p<0.01) in albumin/globulin ratio and a statistically significant reduction (p<0.05) in cholesterol. Recovery males that were previously given 200 mg/kg bw/day showed a statistically significant reduction (p<0.05) in total protein and a statistically significant increase (p<0.05) in alkaline phosphatase. Females treated with 200 and 100 mg/kg bw/day showed statistically significant increases (p<0.05-0.01) in urea and albumin/globulin ratio and statistically significant reductions (p<0.05-0.01) in chloride concentration, triglycerides, cholesterol and bile acids. The effect on albumin/globulin ratio also extended to females treated with 10 mg/kg bw/day (p<0.05). Females treated with 200 mg/kg bw/day also showed statistically significant reductions (p<0.05) in sodium concentration and alkaline phosphatase. Recovery females that were previously given 200 mg/kg bw/day showed statistically significant increases (p<0.05) in chloride concentration and bile acids. The majority of individual values for all parameters for both sexes were within historical control ranges and although some of these intergroup differences may indicate minor perturbations in hepatic metabolism, in the absence of any associated microscopic hepatic changes evident these differences were considered not to represent an adverse effect of treatment.
Animals of both sexes treated with 200 and 100 mg/kg bw/day showed statistically significant increases (p<0.05-0.01) in alanine aminotransferase and aspartate aminotransferase. These intergroup differences may again indicate minor perturbations in hepatic metabolism and the majority of individual values did exceed historical control ranges, however, in the absence of any associated microscopic hepatic changes evident, the intergroup differences were considered not to represent an adverse effect of treatment.
Following the treatment-free period, females that were previously given 200 mg/kg bw/day showed statistically significant reductions (p<0.05-0.01) in potassium concentration and inorganic phosphorus. The majority of individual values were within historical control ranges and in the absence of similar effects in 200 mg/kg bw/day females at the end of the treatment period or any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There were no toxicologically significant changes in functional performance.
Males from all treatment groups showed a statistically significant increase (p<0.01) in overall activity. A true dose related response was not evident and in the absence of any clinical signs of neurotoxicity, the intergroup difference was considered not to be of toxicological importance.

There were no treatment-related changes in sensory reactivity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No toxicologically significant effects were detected in the organ weights measured.
At the end of the treatment period, males from all treatment groups showed statistically significant reductions (p<0.05-0.01) in kidney and thymus weights both absolute and relative to terminal body weight and absolute brain weight. Males treated with 10 mg/kg bw/day also showed a statistically significant reduction in relative brain weight, however, males treated with 200 and 100 mg/kg bw/day showed a statistically significant increase (p<0.05-0.01) in relative brain weight. Males treated with 200 mg/kg bw/day also showed a statistically significant increase (p<0.05) in spleen weight both absolute and relative to terminal body weight. The majority of individual values for both absolute and relative weights were within historical control ranges and no associated micropscopic changes were evident in these organs, therefore, the intergroup differences were considered not to be of toxicological significance.
Following the treatment-free period, females that were previously given 200 mg/kg bw/day showed a statistically significant increase (p<0.05) in brain weight both absolute and relative to terminal body weight. All of the individual values were within historical control ranges and in the absence of similar effects in 200 mg/kg bw/day females at the end of the treatment period or any associated histopathological correlates the intergroup differences were considered not to be of toxicological significance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No toxicologically significant macroscopic abnormalities were detected in surviving animals.
The male treated with 200 mg/kg bw/day that was found dead on Day 48 had a dark liver and dark lungs. Histopathological examination revealed moderate prostatic inflammation, unilateral lymphoid aggregates and urothelial hyperplasia in the kidneys and adrenal cortical hypertrophy. The adrenal change was likely to indicate stress and was probably secondary to the prostatic inflammation. The cause of death in this animal was undetermined, and none of the microscopic or macroscopic changes were considered to be treatment-related.
A number of animals across most dose groups including controls showed reddened lungs. Such findings are common in this type of study and were considered unrelated to treatment with the test item. One male treated with 200 mg/kg bw/day had an enlarged spleen. One female treated with 10 mg/kg bw/day had increased renal pelvic space. In the absence of any associated treatment-related microscopic changes, these findings were considered to be incidental.
One female that was previously given 200 mg/kg bw/day had a fluid filled (dark) right uterine horn at the end of the treatment-free period. In the absence of a similar effect at the end of the treatment period, the intergroup difference was considered to be incidental.
One non-recovery control male had enlarged fluid filled kidneys and the left kidney was also pale and malformed. A further non-recovery control male had a hard and pale left seminal vesicle. One recovery control female had increased renal pelvic space in both kidneys. In the absence of treatment, these were considered to be incidental findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following treatment-related microscopic abnormalities were detected:
Mesenteric Lymph Nodes: Histiocytosis with granulomas was evident in animals of both sexes from all treatment groups, varying from minimal to moderate in severity and following a dose-dependent response. At 100 and 200 mg/kg bw/day the more severe grades of histiocytosis were often accompanied by minimal abscesses. Following the twenty-eight day recovery period, histiocytosis with granulomas and abscesses were evident in both sexes previously treated with 200 mg/kg bw/day. When compared with the non-recovery animals, the incidence of abscesses was lower, whereas histiocytosis persisted at much the same incidence and severity.
The following microscopic abnormalities were evident, however, these were considered to reflect individual variation rather than an effect of treatment.
Adrenals: A slightly increased incidence of cortical vacuolation was evident in males treated with 200 mg/kg bw/day when compared with controls. The change was minimal in all cases, and was not apparent in females. As cortical vacuolation is a background finding with a variable incidence, this intergroup difference is considered to be chance and to represent normal variation rather than an effect of treatment.

Histopathological findings: neoplastic:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

haematology

histopathology: non-neoplastic

mortality

2

Dose descriptor:

LOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical signs

haematology

histopathology: non-neoplastic

mortality

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

100 mg/kg bw/day (actual dose received)

System:

immune system

Organ:

mesenteric lymph node

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

*         Significantly different from control group p<0.05

**        Significantly different from control group p<0.01

n        Data not appropriate for statistical analysis

.         not examined

Group Mean Body Weight Values 

Group (sex)		Day Numbers Relative to Start Date
		1	8	15	22	29	36	43	50	57
1(M)	Mean SD N	248 11.3 20	278.6 17.2 20	302.8 23 20	325.4 26.8 20	345.6 29.8 20	357.7 31.9 20	374.1 32.2 20	384.4 33.3 20	394.7 34.4 20
2(M)	Mean SD N	243.3 11.1 10	272.4 15.4 10	297.1 21 10	316.3 23.9 10	338 26.9 10	351.4 29.2 10	366.3 33.1 10	376.8 32.8 10	387.8 32.7 10
3(M)	Mean SD N	239.7 11 10	260.9 15.3 10	283.5 18.7 10	300.6 24.3 10	321.1 27.2 10	335.6 28.3 10	349.7 28.9 10	361.9 30 10	368.5 31.5 10
4(M)	Mean SD N	241.5   11.6 20	254.5  14.3    20	277.0   22.1 20	295.1 26.6 20	318.7 32.1    20	327.6   35.8    20	341.8   37.6    20	353.5   41.4    19	362.8 44.0                       19

 

Group (sex)		Day Numbers Relative to Start Date
		64	71	78	85	91	98	105	112	119
1(M)	Mean SD N	407.9 36.6 20	416.9 39.3 20	425.6 38.9 20	434.1 39.7 20	437.1 40.2 20	434.5 44.4 10	445.1 47.4 10	447.3 48 10	453.7 50 10
2(M)	Mean SD N	396.9 33.3 10	404.3 35.2 10	415 37.8 10	424.7 39.1 10	424.8 39.2 10	. . 0	. . 0	. . 0	. . 0
3(M)	Mean SD N	379.8 29.8 10	387.7 28.5 10	397.6 30.4 10	404.1 32 10	398.9 31.1 10	. . 0	. . 0	. . 0	. . 0
4(M)	Mean SD N	372.8 43.4 19	378.5 45.5 19	387 47.7 19	390.5 48.7 19	383.6 48 19	398.3 55.4 9	412.6 58.8 9	417.6 59.3 9	431.9 61.3 9

 

  

Group (sex)		Day Numbers Relative to Start Date
		1	8	15	22	29	36	43	50	57
1(F)	Mean SD N	173.8 8.5 20	186.9 10.6 20	199.1 12.4 20	212 12.9 20	225.7 17.5 20	231.8 18.8 20	238.4 19.9 20	242.4 18.8 20	248.9 20 20
2(F)	Mean SD N	169.8 7.7 10	181.5 10.1 10	193 10.9 10	204.1 14.5 10	215.1 14.4 10	221.3 13 10	228.2 15 10	227.5 15.9 10	235.9 14.8 10
3(F)	Mean SD N	179 7.4 10	191.7 10.6 10	200.4 13.2 10	217.1 15.4 10	231.4 15.1 10	236.6 17.1 10	242.5 15.9 10	247.3 18.9 10	255.5 18 10
4(F)	Mean SD N	174.5 7.4 20	184.2 10.4 20	195.6 13.6 20	206 13 20	219.1 14.8 20	226.4 16.8 20	232 17.3 20	236.4 17.5 20	240.3 20.8 20

 

Group (sex)		Day Numbers Relative to Start Date
		64	71	78	85	91	98	105	112	119
1(F)	Mean SD N	251.6 18.8 20	257.2 20.3 20	261.4 18.9 20	262 19.3 20	266.2 20.5 20	261.1 20.4 10	261.9 20.6 10	261.9 21 10	264.1 23 10
2(F)	Mean SD N	238.8 13.1 10	243 14.3 10	243.7 15.8 10	246.9 15.9 10	248 16.7 10	. . 0	. . 0	. . 0	. . 0
3(F)	Mean SD N	259.3 19 10	261.2 17.8 10	263.4 18.2 10	267.3 18.5 10	269.5 20.4 10	. . 0	. . 0	. . 0	. . 0
4(F)	Mean SD N	247.3 18.1 20	248 18.3 20	250.9 21.8 20	253.6 19.9 20	254.5 19.1 20	262.9 24.4 10	265.9 25 10	265 25.7 10	265 28.2 10

 

Group Mean Body Weight Gains 

Group (sex)		Day Numbers Relative to Start Date
		1 – 8	8-15	15-22	22-29	29-36	36-43	43-50	50-57	57-64
1(M)	Mean SD N	30.6 6.6 20	24.3 7.1 20	22.6 4.8 20	20.2 5.1 20	12.1 3.3 20	16.5 5.4 20	10.3 3.4 20	10.4 3.4 20	13.2 4.5 20
2(M)	Mean SD N	29.1 5.2 10	24.7 6 10	19.2 4.2 10	21.7 5 10	13.4 3.6 10	14.9 4.4 10	10.5 4.4 10	11 3.4 10	9.1 4.2 10
3(M)	Mean SD N	21.2** 7.8 10	22.6 8.2 10	17.1 8.7 10	20.5 4.7 10	14.5 4.6 10	14.1 4.3 10	12.2 4.7 10	6.6 6.7 10	11.3 5.7 10
4(M)	Mean SD N	13.0** 9.4 20	22.6 12.3 20	18.1 8 20	23.6 6.6 20	9 8.6 20	14.2 5.4 20	12.4 6.6 19	9.3 4.7 19	10.1 4.6 19

 

Group (sex)		Day Numbers Relative to Start Date
		64-71	71-78	78-85	85-91	91-98	98-105	105-112	112-119
1(M)	Mean SD N	9 4.4 20	8.7 3.6 20	8.6 4.8 20	3 5.3 20	5.5 4.1 10	10.6 4.3 10	2.2 2.7 10	6.4 4.6 10
2(M)	Mean SD N	7.4 4.7 10	10.7 3.9 10	9.7 4.9 10	0.1 3.2 10	. . .	. . .	. . .	. . .
3(M)	Mean SD N	7.9 8 10	9.9 3.5 10	6.5 6.5 10	5.2 8.3 10	. . .	. . .	. . .	. . .
4(M)	Mean SD N	5.6 7.6 19	8.5 4.5 19	3.5** 6 19	6.9 9.6 19	12.6* 7.4 9	14.2 4.7 9	5 6.3 9	14.3** 6.2 9

 

Group (sex)		Day Numbers Relative to Start Date
		1 – 8	8-15	15-22	22-29	29-36	36-43	43-50	50-57	57-64
1(F)	Mean SD N	13.1 4.9 20	12.3 7 20	12.9 5.6 20	13.7 7.8 20	6.1 5.9 20	6.6 7 20	4.1 4.9 20	6.5 4.2 20	2.7 5.5 20
2(F)	Mean SD N	11.7 4.8 10	11.5 4.6 10	11.1 5.6 10	11 5.8 10	6.2 5 10	6.9 4.2 10	0.7 6.3 10	8.4 6.4 10	2.9 5.7 10
3(F)	Mean SD N	12.7 6.5 10	8.7 9.3 10	16.7 11.4 10	14.3 5.6 10	5.2 4.6 10	5.9 5.3 10	4.8 6.8 10	8.2 6.2 10	3.8 5.4 10
4(F)	Mean SD N	9.8 5.2 20	11.4 8.7 20	10.4 7.4 20	13.1 6.1 20	7.4 5.9 20	5.6 6.3 20	4.4 6.9 20	3.9 8.1 20	7.1* 8 20

 

Group (sex)		Day Numbers Relative to Start Date
		64-71	71-78	78-85	85-91	91-98	98-105	105-112	112-119
1(F)	Mean SD N	5.6 8.3 20	4.2 5.9 20	0.6 3.7 20	4.2 6.4 20	1.3 7.4 10	0.8 3.5 10	0 3.8 10	2.2 4.4 10
2(F)	Mean SD N	4.2 5 10	0.7 5.7 10	3.2 4.9 10	1.1 4.5 10	. . .	. . .	. . .	. . .
3(F)	Mean SD N	1.9 5.4 10	2.2 6.7 10	3.9 4.7 10	2.2 3.8 10	. . .	. . .	. . .	. . .
4(F)	Mean SD N	0.7* 6.8 20	2.9 7.1 20	2.7 6.1 20	0.9 5.7 20	0.6 7.2 10	3 5.7 10	0.9 4 10	0 5.2 10

 

Group Mean Hematological Values

Non-recovery animals:       

		Hb	RBC	Hct	MCH	MCV	MCHC	WBC	Neut
Group (sex)		g/dl	10^12/l	%	pg	fl	g/dl	10^9/l	10^9/l
1(M)	Mean SD N	15.9 0.4 10	9.037 0.326 10	48.01 1.52 10	17.63 0.64 10	53.16 1.68 10	33.15 0.42 10	7.67 1.68 10	1.17 0.553 10
2(M)	Mean SD N	15.38 0.34 10	9.136 0.371 10	46.63 1.01 10	16.85* 0.69 10	51.06* 1.57 10	33 0.45 10	7.35 1.53 10	1.31 0.571 10
3(M)	Mean SD N	16.07 0.5 10	9.328 0.391 10	48.44 1.68 10	17.23 0.67 10	51.97 1.59 10	33.15 0.37 10	7.36 1.28 10	1.985** 0.64 10
4(M)	Mean SD N	16.05 0.62 10	9.149 0.344 10	48.66 1.75 10	17.54 0.54 10	53.21 1.36 10	33 0.32 10	10.02** 2.15 10	3.751** 1.793 10

       

		Lymph	Mono	Eos	Bas	CT	PLT	APTT
Group (sex)		10^9/l	10^9/l	10^9/l	10^9/l	Seconds	10^9/l	Seconds
1(M)	Mean SD N	6.435 1.485 10	0.011n 0.023 10	0.052 0.049 10	0.000n 0 10	9.16 0.43 10	589.2 53.3 10	13.9 0.86 10
2(M)	Mean SD N	5.955 1.399 10	0.000n 0 10	0.088 0.053 10	0.000n 0 10	8.74 0.46 10	565.9 90.6 10	13.11 0.66 10
3(M)	Mean SD N	5.253 1.136 10	0.000n 0 10	0.122 0.074 10	0.000n 0 10	9.02 0.38 10	558.1 58.6 10	13.98 2.02 10
4(M)	Mean SD N	6.212 1.813 10	0.000n 0 10	0.058 0.07 10	0.000n 0 10	9.14 0.43 10	653.9 108.7 10	13.03 0.95 10

 

		Hb	RBC	Hct	MCH	MCV	MCHC	WBC	Neut
Group (sex)		g/dl	10^12/l	%	pg	fl	g/dl	10^9/l	10^9/l
1(F)	Mean SD N	14.93 0.39 10	8.059 0.395 10	42.95 1.34 10	18.56 0.7 10	53.38 1.71 10	34.75 0.3 10	4.83 0.75 10	0.894 0.547 10
2(F)	Mean SD N	15.16 0.33 10	8.129 0.265 10	43.45 1.06 10	18.67 0.59 10	53.49 1.63 10	34.92 0.31 10	4.68 0.92 10	0.514 0.337 10
3(F)	Mean SD N	15 0.67 10	8.059 0.377 10	43.05 1.96 10	18.63 0.57 10	53.44 1.34 10	34.87 0.35 10	5.52 1.7 10	1.569* 1.002 10
4(F)	Mean SD N	14.75 1.23 10	8.042 0.765 10	42.71 3.96 10	18.35 0.52 10	53.14 1.1 10	34.54 0.44 10	6.32 2.6 10	1.644* 0.648 10

       

		Lymph	Mono	Eos	Bas	CT	PLT	APTT
Group (sex)		10^9/l	10^9/l	10^9/l	10^9/l	Seconds	10^9/l	Seconds
1(F)	Mean SD N	3.887 0.614 10	0.000n 0 10	0.048 0.031 10	0.000n 0 10	8.81 0.35 10	571.6 53 10	15.18 1.98 10
2(F)	Mean SD N	4.107 0.751 10	0.000n 0 10	0.059 0.059 10	0.000n 0 10	8.93 0.59 10	550.1 88.1 10	15.39 1.78 10
3(F)	Mean SD N	3.908 1.071 10	0.000n 0 10	0.043 0.056 10	0.000n 0 10	8.79 0.64 10	626.1 76.4 10	14.44 1.36 10
4(F)	Mean SD N	4.607 2.235 10	0.000n 0 10	0.069 0.066 10	0.000n 0 10	9.03 0.75 10	655.4* 104.8 10	14.61 1.83 10

            

Recovery animals:

		Hb	RBC	Hct	MCH	MCV	MCHC	WBC	Neut
Group (sex)		g/dl	10^12/l	%	pg	fl	g/dl	10^9/l	10^9/l
1(M)	Mean SD N	16.24 0.48 10	9.428 0.377 10	48.86 1.39 10	17.25 0.56 10	51.86 1.25 10	33.23 0.32 10	7.34 1.26 10	1.359 0.708 10
4(M)	Mean SD N	15.88 1.57 9	9.019 0.726 9	47.31 4.73 9	17.61 1.14 9	52.39 3.04 9	33.57 0.58 9	7.09 1.99 9	1.593 0.818 9

       

		Lymph	Mono	Eos	Bas	CT	PLT	APTT
Group (sex)		10^9/l	10^9/l	10^9/l	10^9/l	Seconds	10^9/l	Seconds
1(M)	Mean SD N	5.907 1.019 10	0.000n 0 10	0.077 0.064 10	0.000n 0 10	10.03 0.83 10	610.4 104.8 10	15.18 1.4 10
4(M)	Mean SD N	5.432 1.433 9	0.008n 0.023 9	0.056 0.057 9	0.000n 0 9	9.84 0.55 9	536.4 89.8 9	14.94 1.82 9

       

		Hb	RBC	Hct	MCH	MCV	MCHC	WBC	Neut
Group (sex)		g/dl	10^12/l	%	pg	fl	g/dl	10^9/l	10^9/l
1(F)	Mean SD N	15.42 0.59 10	8.145 0.522 10	44.86 2.32 10	18.97 0.6 10	55.14 1.32 10	34.39 0.59 10	3.87 0.69 10	0.604 0.18 10
4(F)	Mean SD N	16.37** 0.51 10	8.866** 0.251 10	48.41** 1.32 10	18.35* 0.53 10	54.31 1.26 10	33.81* 0.28 10	4.32 1.02 10	1.046** 0.402 10

       

		Lymph	Mono	Eos	Bas	CT	PLT	APTT
Group (sex)		10^9/l	10^9/l	10^9/l	10^9/l	Seconds	10^9/l	Seconds
1(F)	Mean SD N	3.225 0.557 10	0.000n 0 10	0.133 0.306 10	0.000n 0 10	8.98 0.38 10	588.1 54.8 10	15.28 1.79 10
4(F)	Mean SD N	3.235 0.975 10	0.000n 0 10	0.043 0.042 10	0.000n 0 10	8.81 0.56 10	545.2 115.5 10	15.49 1.45 10

 

Group Mean Blood Chemical Values

Non-recovery animals:       

		Urea	Glucose	total Prot.	Albumin	A/G	Na+	K+	Cl	Ca++	P
Group (sex)		mg/dl	mg/dl	g/dl	g/dl	Ratio	mmol/l	mmol/l	mmol/l	mmol/l	mmol/l
1(M)	Mean SD N	37.5 4.6 10	174.2 19.3 10	6.965 0.271 10	3.85 0.14 10	1.241 0.063 10	148.1 2.5 10	4.541 0.15 10	104.8 1.8 10	2.57 0.33 10	1.4 0.21 10
2(M)	Mean SD N	38 7.8 10	187.5 22.4 10	6.891 0.308 10	3.79 0.1 10	1.243 0.074 10	147.4 2.3 10	4.592 0.735 10	105.3 1.8 10	2.557 0.192 10	1.5 0.26 10
3(M)	Mean SD N	45.8* 9.3 10	194.8 38.6 10	6.711* 0.411 10	3.69* 0.16 10	1.235 0.099 10	147.1 1.7 10	4.585 0.767 10	104.8 1.4 10	2.66 0.108 10	1.42 0.32 10
4(M)	Mean SD N	44.6* 7.4 10	193.7 26 10	6.535** 0.245 10	3.75* 0.08 10	1.366** 0.085 10	146.4 1.9 10	4.918 0.732 10	103.5 1.6 10	2.714 0.095 10	1.48 0.29 10

       

		yGT	ASAT	ALAT	AP	Creat	Tri	Chol	Bili	Bile acids
Group (sex)		IU/l	IU/l	IU/l	IU/l	mg/dl	mg/dl	mg/dl	mg/dl	µmol/l
1(M)	Mean SD N	1.4 0.7 10	97.1 20.1 10	69 17.8 10	115 20.9 10	0.71 0.05 10	230.3 81.9 10	84.2 10.8 10	0.114 0.023 10	9.76 7.23 10
2(M)	Mean SD N	0.9 0.57 10	89.2 20.3 10	67.6 11.4 10	114.7 24.5 10	0.762 0.17 10	278.5 81.2 10	97.4 20.4 10	0.121 0.01 10	9.25 6.13 10
3(M)	Mean SD N	1.6 1.17 10	128.2* 43.3 10	108.5** 31.5 10	88.8* 15.7 10	0.776 0.237 10	127.8** 44.6 10	74.5 13.6 10	0.116 0.02 10	3.73* 1 10
4(M)	Mean SD N	0.9 0.57 10	169.7** 54 10	176.7** 39.8 10	93.9* 22.2 10	0.831 0.227 10	126.3** 47.2 10	68.6* 12.5 10	0.122 0.018 10	4.22* 1.11 10

       

		Urea	Glucose	total Prot.	Albumin	A/G	Na+	K+	Cl	Ca++	P
Group (sex)		mg/dl	mg/dl	g/dl	g/dl	Ratio	mmol/l	mmol/l	mmol/l	mmol/l	mmol/l
1(F)	Mean SD N	41.4 12.8 10	160.8 14.7 10	7.141 0.359 10	4.26 0.24 10	1.483 0.069 10	148.8 2.1 10	4.776 1.435 10	105.1 1.3 10	2.735 0.161 10	1.44 0.24 10
2(F)	Mean SD N	39 4.2 10	165.9 18.4 10	7.245 0.338 10	4.45 0.25 10	1.585* 0.08 10	148.1 2.7 10	4.116 0.442 10	105.3 2.1 10	2.781 0.092 10	1.39 0.26 10
3(F)	Mean SD N	49.2* 8.8 10	160.1 24.7 10	7.415 0.382 10	4.51 0.19 10	1.562* 0.116 10	147 2.6 10	4.34 0.858 10	103.5* 1.8 10	2.863 0.086 10	1.39 0.56 10
4(F)	Mean SD N	55.8** 8 10	158.5 18.9 10	6.763 0.786 10	4.14 0.39 10	1.601* 0.135 10	146.3* 2.1 10	4.914 0.722 10	103.0* 1.9 10	2.788 0.117 10	1.24 0.22 10

 

		yGT	ASAT	ALAT	AP	Creat	Tri	Chol	Bili	Bile acids
Group (sex)		IU/l	IU/l	IU/l	IU/l	mg/dl	mg/dl	mg/dl	mg/dl	µmol/l
1(F)	Mean SD N	0.22 0.44 9	92.9 45.5 10	65.4 16.9 10	48.2 10.9 10	0.815 0.197 10	164.9 44 10	69.2 9 10	0.074 0.034 10	16.51 8.93 10
2(F)	Mean SD N	0.1 0.32 10	79.1 12.3 10	58.2 9.4 10	57.9 9.9 10	0.821 0.063 10	137.6 32.3 10	65.6 5.5 10	0.09 0.019 10	20.33 13.11 10
3(F)	Mean SD N	0.3 0.48 10	117.0* 19.4 10	124.9** 30.5 10	43.3 10 10	0.865 0.17 10	103.6** 47.2 10	58.9** 7.9 10	0.095 0.013 10	7.98** 4.02 10
4(F)	Mean SD N	0.4 0.52 10	109.3* 26.6 10	112.2** 46.7 10	37.4* 9.1 10	0.824 0.138 10	80.9** 33.1 10	50.1** 7.8 10	0.088 0.023 10	6.75** 2.27 10

 

Recovery animals:

		Urea	Glucose	total Prot.	Albumin	A/G	Na+	K+	Cl	Ca++	P
Group (sex)		mg/dl	mg/dl	g/dl	g/dl	Ratio	mmol/l	mmol/l	mmol/l	mmol/l	mmol/l
1(M)	Mean SD N	46.1 5.3 10	159.2 15.7 10	7.16 0.219 10	3.95 0.14 10	1.232 0.076 10	145.7 1.3 10	6.662 4.874 10	104.8 1.9 10	2.742 0.232 10	1.82 0.29 10
4(M)	Mean SD N	52.2 8.9 9	159.7 12.7 9	6.923* 0.26 9	3.89 0.17 9	1.282 0.11 9	146.7 2.3 9	5.191 1.111 9	105.3 1.4 9	2.782 0.093 9	1.86 0.27 9

       

 

		yGT	ASAT	ALAT	AP	Creat	Tri	Chol	Bili	Bile acids
Group (sex)		IU/l	IU/l	IU/l	IU/l	mg/dl	mg/dl	mg/dl	mg/dl	µmol/l
1(M)	Mean SD N	0.00n 0 10	149 187.6 10	102.2 120.4 10	103 23.5 10	0.656 0.051 10	232.9 89.9 10	95.1 21.6 10	0.092 0.049 10	8.53 5.21 10
4(M)	Mean SD N	0.00n 0 9	88.7 15.7 9	71.1 10.4 9	121.4* 11.5 9	0.737 0.187 9	178 68.8 9	82 11.7 9	0.106 0.027 9	11.57 5.84 9

 

 

		Urea	Glucose	total Prot.	Albumin	A/G	Na+	K+	Cl	Ca++	P
Group (sex)		mg/dl	mg/dl	g/dl	g/dl	Ratio	mmol/l	mmol/l	mmol/l	mmol/l	mmol/l
1(F)	Mean SD N	55.1 9.9 10	168.2 46.7 10	7.979 0.797 10	4.59 0.42 10	1.364 0.15 10	147.9 1.5 10	7.097 5.293 10	103 2.3 10	2.599 0.184 10	1.71 0.47 10
4(F)	Mean SD N	52.7 4.5 10	152.4 28.3 10	7.871 0.382 10	4.62 0.25 10	1.43 0.063 10	148.6 3 10	4.029** 0.349 10	105.5* 2.7 10	2.735 0.118 10	1.10* 0.52 10

       

 

		yGT	ASAT	ALAT	AP	Creat	Tri	Chol	Bili	Bile acids
Group (sex)		IU/l	IU/l	IU/l	IU/l	mg/dl	mg/dl	mg/dl	mg/dl	µmol/l
1(F)	Mean SD N	0.00n 0 10	112.9 84.3 10	60.9 14.9 10	39.5 23.5 10	0.719 0.09 10	167.7 87.6 10	85.2 16 10	0.079 0.051 10	9.95 8.97 10
4(F)	Mean SD N	0.10n 0.32 10	85 22.7 10	61.9 13.4 10	47.2 9.7 10	0.72 0.095 10	121.1 27.3 10	73.9 15.2 10	0.105 0.02 10	19.80* 9.65 10

 

Histopathology

Non-recovery animals

	1M	2M	3M	4M	1F	2F	3F	4F
Dose (mg/kg bw/d)	0	10	100	200	0	10	100	200
Mesenteric lymph node Histiocytosis/Granulomas
Minimal	0	4	1	0	1	5	0	0
Slight	0	1	5	3	1	1	4	2
Moderate	0	0	3	4	0	0	6	4
Marked	0	0	1	3	0	0	0	4
Total	0	5	10	10	2	6	10	10
Abscess
Minimal	0	0	2	5	0	0	1	4
Total	0	0	2	5	0	0	1	4
N	10	10	10	10	10	10	10	10
Adrenals Vacuolation, cortical, diffuse
Minimal	1	0	2	4
total	1	0	2	4
N	10	10	10	10

 Recovery animals:                

	1M	4M	1F	4F
Dose (mg/kg bw/d)
Mesenteric lymph node Histiocytosis/Granulomas
Slight	0	1	0	3
Moderate	0	4	0	4
Marked	0	4	0	3
Total	0	9	0	10
Abscess
Minimal	0	1	0	2
Total	0	1	0	2
N	10	10	10	10

 

Summary Incidence of Necropsy Findings – fertility related parameters

 

Males

	0 (control)	10 mg/kg bw/d	100 mg/kg bw/d	200 mg/kg bw/d
Group:	1	2	3	4
Seminal Vesicles (Including Coagulating Gland)
Submitted	20	10	10	19
No Visible Lesions	19	10	10	19
Hard; left	1	0	0	0
Pale; left	1	0	0	0

 

Females

	0 (control)	10 mg/kg bw/d	100 mg/kg bw/d	200 mg/kg bw/d
Group:	1	2	3	4
Uterus And Cervix
Submitted	20	10	10	20
No Visible Lesions	20	10	9	19
Damaged On Removal	0	0	1	0
Fluid Filled; dark; right horn	0	0	0	1

 

 

Group Mean Organ Weights with Corresponding Relative (% of Bodyweight) Organ Weights – fertility related parameters

Non-recovery animals

		0 (control)	10 mg/kg bw/d	100 mg/kg bw/d	200 mg/kg bw/d	0 (control)	10 mg/kg bw/d	100 mg/kg bw/d	200 mg/kg bw/d
Group:		1	2	3	4	1	2	3	4
Sex		M	M	M	M	F	F	F	F
Epididymides	Mean (g) S.D.     N	1.73247  0.19320  10	3.12171 4.72007    10	1.53095 0.17581    10	1.65394 0.25675                                                                            10	-	-	-	-
	Mean (%) S.D.     N	0.390    0.036      10	0.711      1.018      10	0.384      0.031      10	0.436 0.061 10	-	-	-	-
Testes	Mean (g) S.D.     N	3.90426    0.37256 10	3.79877 0.31706 10	3.82483 0.46413 10	3.75560 0.43943 10	-	-	-	-
	Mean (%) S.D.     N	0.883 0.110      10	0.898  0.078      10	0.959      0.091      10	0.988                                                                           0.081                                                                           10	-	-	-	-
Ovaries	Mean (g) S.D.     N	-	-	-	-	0.10444 0.02286    10	0.10428    0.01783    10	0.12207    0.02816    10	0.10916                                0.02254                                10
	Mean (%) S.D.     N	-	-	-	-	0.039      0.009      10	0.042      0.007      10	0.046 0.012 10	0.044 0.009                               10
Uterus And Cervix	Mean (g) S.D.     N	-	-	-	-	0.83856  0.21425    10	0.78873    0.31097    10	0.79504    0.25003    10	0.69453                                0.23548                                10
	Mean (%) S.D.     N	-	-	-	-	0.311      0.076      10	0.318  0.122      10	0.298 0.103      10	0.279 0.086                               10

 

Recovery animals

		0 (control)	200 mg/kg bw/d	0 (control)	200 mg/kg bw/d
Group:		1	4	1	4
Sex		M	M	F	F
Epididymides	Mean (g) S.D.     N	1.68946 0.21805   10	1.73983 0.21805                                                                                                   9	-	-
	Mean (%) S.D.     N	0.375   0.052     10	0.409       0.069                                                                                                  9	-	-
Testes	Mean (g) S.D.     N	3.85441 0.48796   10	3.76723     0.24391                                                                                                   9	-	-
	Mean (%) S.D.     N	0.852   0.095     10	0.889    0.141                                                                                                  9	-	-
Ovaries	Mean (g) S.D.     N	-	-	0.09953  0.01985   10	0.11018 0.02005                                                                               10
	Mean (%) S.D.     N	-	-	0.038     0.007     10	0.042                                                                              0.009                                                                              10
Uterus And Cervix	Mean (g) S.D.     N	-	-	0.94423  0.40225   10	0.98089                                                                               0.49808                                                                               10
	Mean (%) S.D.     N	-	-	0.360  0.151     10	0.374  0.196                                                                              10

 

  

 

 

                                                                            

Applicant's summary and conclusion

Conclusions:

The oral (gavage) administration of BADGE-IPD for up to ninety consecutive days, to Wistar rats of both sexes at dose levels of 10, 100 or 200 mg/kg bw/day resulted in adverse treatment-related effects in animals of both sexes treated with 200 and 100 mg/kg bw/day. These findings included the death of one male treated with 200 mg/kg bw/day, clinical signs of toxicity, reduced body weight development and food consumption, changes in the hematology parameters measured and microscopic changes in the mesenteric lymph nodes. These changes were considered to represent an adverse effect of treatment at these levels. The effects evident in both sexes at 10 mg/kg bw/day were confined to minimal histiocytosis with granulomas in the mesenteric lymph nodes. Based on the level of severity of these changes evident in both sexes treated with 10 mg/kg bw/day, the 'No Observed Adverse Effect Level (NOAEL) was considered to be 10 mg/kg bw/day.

Executive summary:

The present study was designed to investigate the systemic toxicity of the test item BADGE-IPD in accordance with OECD Guideline 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for up to ninety consecutive days, at dose levels of 10, 100 and 200 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Propylene Glycol). Two recovery groups, each of ten males and ten females, were treated with the high dose (200 mg/kg bw/day) or the vehicle alone for up to ninety consecutive days and then maintained without treatment for a further twenty-eight days.

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all non-recovery group animals at the end of the treatment period and for all surviving recovery group animals at the end of the treatment-free period. Ophthalmoscopic examination was also performed on all animals prior to the start of treatment and on all non-recovery control and high dose animals during Week 12 of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues was performed.

 

Mortality

One male treated with 200 mg/kg bw/day was found dead on Day 48. There were no further unscheduled deaths.

 

Clinical Observations

Increased salivation and noisy respiration was evident in animals of either sex treated with 200 mg/kg bw/day and to a lesser extent in animals of either sex treated with 100 mg/kg bw/day throughout the treatment period. Isolated incidences of a stained snout, labored respiration, decreased respiratory rate, hunched posture and fur loss were also evident in females treated with 200 mg/kg bw/day, a stained snout and sneezing was also evident in some males treated with 200 mg/kg bw/day and a decreased respiratory rate and hunched posture was also evident in some males treated with 100 mg/kg bw/day. During the treatment-free period, one male and one female that were previously given 200 mg/kg bw/day showed episodes of noisy respiration. No such effects were detected in males treated with 10 mg/kg bw/day however one female from this treatment group had increased salivation on Day 81 only.

 

Behavioral Assessment

Weekly behavioral assessments revealed instances of noisy respiration in some animals of both sexes treated with 200 mg/kg bw/day. No such effects were detected in animals of both sexes treated with 100 or 10 mg/kg bw/day.

Functional Performance Tests

There were no toxicologically significant changes in functional performance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

 

Body Weight

Males treated with 200 and 100 mg/kg bw/day showed a reduction in body weight gain during the first week of treatment. Periods of recovery were evident thereafter, however, generally body weight gain in these males was slightly below controls. Consequently overall body weight gain was lower than controls in these males. Significant improvement in body weight gain was evident in males previously treated with 200 mg/kg bw/day during the twenty-eight day treatment-free period. Females treated with 200 mg/kg bw/day showed a slight reduction in body weight gain during the first week of treatment, however, recovery was evident thereafter. No such effects were detected in females treated with 100 mg/kg bw/day or in animals of both sexes treated with 10 mg/kg bw/day.

 

Food Consumption

Animals of both sex treated with 200 mg/kg bw/day and males treated with 100 mg/kg bw/day showed a reduction in overall food consumption. Incidences of reduced food conversion efficiency were also evident in males treated with 200 and 100 mg/kg bw/day during the treatment period. Improvement in food consumption was evident during the twenty-eight day treatment-free period. No such effects were detected in females treated with 100 mg/kg bw/day or in animals of both sexes treated with 10 mg/kg bw/day.

 

Water Consumption

Visual inspection of water bottles did not reveal any inter-group differences.

 

Ophthalmoscopy

Ophthalmoscopic examination of animals of both sexes from the non-recovery control and 200 mg/kg bw/day dose groups during Week 12 of the treatment period did not indicate any treatment-related differences.

 

Hematology

Animals of both sexes treated with 200 and 100 mg/kg bw/day showed an increase in neutrophils. Males treated with 200 mg/kg bw/day also showed an increase in total leukocyte count. The effect on neutrophils was also present in females previously treated with 200 mg/kg bw/day at the end of the twenty-eight day treatment -free period. No such effects were detected in both sexes treated with 10 mg/kg bw/day.

 

Blood Chemistry

There were no toxicologically significant effects detected in the blood chemical parameters examined.

 

Necropsy

No toxicologically significant macroscopic abnormalities were detected in surviving animals.

The male treated with 200 mg/kg bw/day that was found dead on Day 48 had a dark liver and lungs.

 

Organ Weights

No toxicologically significant effects were detected in the organ weights measured.

 

Histopathology

The following treatment-related microscopic abnormalities were detected:

Mesenteric Lymph Nodes: Histiocytosis with granulomas was evident in animals of both sexes from all treatment groups, varying from minimal to moderate in severity and following a dose-dependent response. At 100 and 200 mg/kg bw/day the more severe grades of histiocytosis were often accompanied by minimal abscesses. Following the twenty-eight day recovery period, histiocytosis with granulomas and abscesses were evident in both sexes previously treated with 200 mg/kg bw/day. When compared with the main study, the incidence of abscesses was lower, whereas histiocytosis persisted at much the same incidence and severity.

Histiocytosis in the mesenteric lymph nodes is known to occur in response to the oral administration of some test items and is likely to represent accumulation of material, indicating delayed clearance of an exogenous or endogenous material.

The following microscopic abnormalities were evident, however, these were considered to reflect individual variation rather than an effect of treatment.

Adrenals: A slightly increased incidence of cortical vacuolation was evident in males treated with 200 mg/kg bw/day when compared with controls. The change was minimal in all cases, and was not apparent in females. As cortical vacuolation is a background finding with a variable incidence, this intergroup difference is considered to be by chance and to represent normal variation rather than an effect of treatment.

 

Conclusion

The oral (gavage) administration of BADGE-IPD for up to ninety consecutive days, to Wistar rats of both sexes at dose levels of 10, 100 or 200 mg/kg bw/day resulted in adverse treatment-related effects in animals of both sexes treated with 200 and 100 mg/kg bw/day. These findings included the death of one male treated with 200 mg/kg bw/day, clinical signs of toxicity, reduced body weight development and food consumption, changes in the hematology parameters measured and microscopic changes in the mesenteric lymph nodes. These changes were considered to represent an adverse effect of treatment at these levels. The effects evident in both sexes at 10 mg/kg bw/day were confined to minimal histiocytosis with granulomas in the mesenteric lymph nodes. Based on the level of severity of these changes evident in both sexes treated with 10 mg/kg bw/day, the 'No Observed Adverse Effect Level (NOAEL) was considered to be 10 mg/kg bw/day.