Registration Dossier

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:
2018
Report Date:
2018

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
As per IUCLID Sections 1.1. 1.2. and 4.1.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species: Rat
Strain: Wistar Han ™
Sub-strain: RccHan™:WIST
Source of supply: Envigo RMS (UK) Ltd., Oxon, UK
Number: 80
Sex: Males and females
Age: Approximately 6 to 8 weeks at start of treatment
Other: Weight variation did not exceed ± 20% of the mean weight for either sex at start of treatment
Sex:
male/female
Details on test animals and environmental conditions:
The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Envigo RMS (UK) Limited, Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Annex 5. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.

The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.

The animals were 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. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:
oral: gavage
Details on route of administration:
The test item was administered daily, for up to ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP.

The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.
Vehicle:
polyethylene glycol
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP. The stability and homogeneity of the test item formulations were determined by Envigo Research Limited, Shardlow, UK, Analytical Services. Results from this study showed the formulations to be stable for initially up to at least 4 hours at ambient temperature and also for 14 days. Formulations were initially prepared daily and once further stability was known formulations were prepared weekly and stored at approximately 4°C in the dark.

Samples of test item formulation were taken and analyzed for concentration on six occasions of 2,2’-[(1-Methylethylidene)bis(cyclohexane-4,1-diyloxymethylene)]-bisoxirane (Eponex 1510) at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Annex 2. The results indicate that the prepared formulations were within acceptable ranges for the purpose of this study. The intermediate dose level was below the accepted range on two occasions. However, the animals only received these formulations on one occasion each. Further formulations were within range for the remainder of the dosing period.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Once per day
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Positive control:
NA

Examinations

Observations and examinations performed and frequency:
Serial Observations
General Observations/Measurements
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioral change immediately before dosing, up to thirty minutes post dosing and one hour after dosing. All observations were recorded.

Body Weight
Individual body weights were recorded on Day 1 (prior to dosing) and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency was calculated retrospectively.

Water Consumption
Water intake was measured and recorded daily for each cage group on two occasions at the start of treatment and during the latter stages of treatment; each occasion for fourteen days, with the exception of one day during the first week of treatment; see deviations from study plan. The remaining days water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Specialist Evaluations
Functional Observations
Prior to the start of treatment and at weekly intervals thereafter, all animals were observed for signs of functional/behavioral toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:

Gait Hyper/Hypothermia
Tremors Skin color
Twitches Respiration
Convulsions Palpebral closure
Bizarre/Abnormal/Stereotypic behavior Urination
Salivation Defecation
Pilo-erection Transfer arousal
Exophthalmia Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).

Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

The following parameters were observed:

Grasp response Touch escape
Vocalization Pupil reflex
Toe pinch Blink reflex
Tail pinch Startle reflex
Finger approach

Ophthalmoscopic Examination
The eyes of all Groups 1 to 4 animals were examined pre-treatment. During Week 12, the eyes of all control and high dose animals (Groups 1 and 4, respectively) were examined.

Examinations included observation of the anterior structures of the eye and following pupil dilation with 0.5% Tropicamide solution (Mydriacyl® 0.5%, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using an ophthalmoscope was performed.

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all surviving animals from each test and control group at the end of the study (Day 90). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 91. Animals were not fasted prior to sampling.

The methods used for hematological and blood chemical investigations are given in Annex 6 and normal ranges are shown in Annex 7.

Hematology
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) - Methylene blue stained slides were prepared but reticulocytes were not assessed

Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:

Urea Inorganic phosphorus (P)
Glucose Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.) Alanine aminotransferase (ALAT)
Albumin Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation) Creatinine (Creat)
Sodium (Na+) Total cholesterol (Chol)
Potassium (K+) Total bilirubin (Bili)
Chloride (Cl-) Bile acids
Calcium (Ca++)
Sacrifice and pathology:
Terminal Investigations
Necropsy
On completion of the dosing period all surviving animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:

Adrenals Ovaries
Brain Spleen
Epididymides Testes
Heart Thymus
Kidneys Uterus
Liver

Normal ranges for organ weights are given in Annex 8.

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:

Tissue/Organ Weigh Retain Process &
Examine
Adrenals X X X
Aorta (thoracic) X X
Bone and bone marrow (femur including stifle joint) X
Bone and Bone marrow (sternum) X X
Brain (including cerebrum, cerebellum and pons) X X X
Caecum X X
Colon X X
Duodenum X X
Esophagus X X
Epididymides (retained in Modified Davidson's X X X
Fluid)
Eyes (retained in Davidson's Fluid) X X
Gross lesions X X
Heart X X X
Ileum (including peyer's patches) X X
Jejunum X X
Kidneys X X X
Liver X X X
Lungs (with bronchi) X X
Lymph nodes (mandibular and mesenteric) X X
Mammary gland X X
Muscle (skeletal) X
Ovaries X X X
Pancreas X X
Pituitary X X
Prostate X X
Rectum X X
Salivary glands (submaxillary) X X
Sciatic nerve X X
Seminal vesicles (including coagulating gland) X X
Skin X X
Spinal cord (cervical, mid-thoracic and lumbar) X X
Spleen X X X
Stomach X X
Testes (retained in Modified Davidson's Fluid) X X X
Thymus X X X
ThyroidlParathyroid X X
Tongue X
Trachea X X
Urinary bladder X X
Uterus (with Cervix) X X X
Vagina X X

All tissues were dispatched to the Test Site (Propath UK Ltd.,Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). All tissues from control and 600 mg/kg bw/day dose group animals 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 treatment-related changes, examination was subsequently extended to include similarly prepared sections of kidneys, bone, adrenal glands, thymus, liver, ovaries, vagina and seminal vesicles from animals in the low and intermediate groups.

Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A peer review of the histopathology results for the study was conducted by an appointed Responsible Scientist (V Mowat) working at a designated Test Site. A complete histopathology phase report is presented in Annex 1 and represents the consensus view of both pathologists.
Statistics:
Statistical Analysis
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, 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).

Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Results and discussion

Results of examinations

Description (incidence and severity):
A summary incidence of daily clinical observations is given in Table 1. Individual data is presented in Appendix 1.

Animals of either sex treated with 600 mg/kg bw/day exhibited increased salivation during the treatment period. This finding was also noted to a lesser extent in animals treated with 100 mg/kg bw/day. One 10 mg/kg bw/day female showed increased salivation on Day 29 only. During the treatment period five males and three females treated with 600 mg/kg bw/day exhibited signs of noisy respiration. Observation of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item and in isolation are considered not to be associated with systemic toxicity.

Isolated incidences of staining around the mouth (one male and one female at 600 mg/kg bw/day) or snout (one male at 100 mg/kg bw/day) were observed among two males and one female at 100 mg/kg bw/day.

One male animal which was killed in extremis on Day 55 exhibited tiptoe gait, hunched posture, dehydration and emaciation. A further male which was killed in extremis on Day 64 exhibited tiptoe gait, increased salivation, pilo-erection and hunched posture. The male animal which was killed in extremis on Day 89 exhibited hunched posture, pilo-erection, lethargy and pallor of the extremities.

No clinical signs were apparent in male animals treated with 10 mg/kg bw/day.
Description (incidence):
Three male animals treated with 600 mg/kg bw/day were killed in extremis on Days 55, 64 or 89 of the study due to the severity of the clinical signs noted.

There were no further unscheduled deaths.
Description (incidence and severity):
Group mean weekly body weights and standard deviations are given in Table 6 and are presented graphically in Figure 1 and Figure 2. Group mean weekly body weight gains and standard deviations are given in Table 7 (statistically significant differences are indicated). Individual data are given in Appendix 7 and Appendix 8.

Animals of either sex treated with 600 mg/kg bw/day generally showed lower body weight gains throughout the treatment period, which resulted in an overall reduction of 29 % and 30 % (for males and females respectively) in body weight gain when compared with controls. There were no effects on body weight development for males treated with 10 or 100 mg/kg bw/day, however, the females from these treatment groups showed fluctuations in body weight gains during the treatment period which resulted in reductions of 16 % and 18 % in overall body weight gains for females treated with 10 or 100 mg/kg bw/day respectively when compared to control.
Description (incidence and severity):
Group mean weekly food consumptions are given in Table 8 and are presented graphically in Figure 3 and Figure 4. Weekly food efficiencies are given in Table 9.

Male animals treated with 600 mg/kg bw/day showed marginally lower food intake than the controls from week 8 of treatment, whereas the females from this test group showed no effect on dietary intake when compared to controls. There were no convincing effects detected in animals of either sex that received 10 or 100 mg/kg bw/day.

Slight reductions in food conversion efficiency were evident in male animals treated at 600 mg/kg bw/day, however, these generally followed the fluctuations in body weight gain seen in these animals.
Description (incidence and severity):
Group mean daily water consumptions are given in Table 10.

Animals of either sex treated with 600 mg/kg bw/day were shown to be consuming larger volumes of water than the controls when water intake was measured gravimetrically. Overall group mean water consumptions were 29% and 39% higher than controls (male and females respectively). The increase in water consumptions also correlated to the histopathological changes to the kidneys.

No such effect was observed in animals treated with 100 or 10 mg/kg bw/day.
Description (incidence and severity):
Group mean values and standard deviations for test and control group animals are given in Table 11 (statistically significant differences are indicated). Individual data are given in Appendix 10 and Appendix 11.

Males treated with 100 or 600 mg/kg bw/day showed a statistically significant increase (p<0.05) in neutrophil count compared to controls. The majority of individual values were within the background control ranges with the exception of two animals from each dose group. Females at 600 mg/kg bw/day also showed an increase (p<0.05) in neutrophils compared to controls. The majority of individual values were above the background control ranges and correlate with the histopathological kidney changes. However the elevated levels of neutrophils did not impact on the overall white blood cell count for animals at 600 mg/kg bw/day.

At 600 mg/kg bw/day males showed statistically significant reductions in hemoglobin (p<0.01), hematocrit (p<0.01) and erythrocytes (p<0.05) compared with the controls, this resulted in associated reductions (p<0.01) in mean corpuscular hemoglobin and mean corpuscular volume, with no dose related response. The majority of individual values for these parameters were below the background control ranges. All female dose groups showed statistically significant reductions in mean corpuscular volume (MCV) (p<0.05 - p<0.01) and mean corpuscular hemoglobin (MCH) (p<0.01) compared to controls. The majority of individual values for MCH were within the background control ranges, whereas, the majority of the MCV values were below the background control ranges. Females treated with 600 mg/kg bw/day showed a statistically significant reduction (p<0.01) in mean corpuscular hemoglobin concentration in relation to controls, however, all individual values were within the background control ranges. These findings are of unknown etiology, but cannot be dismissed.

All treated male dose groups showed a statistically significant reduction (p<0.01) in activated partial thromboplastin times (but no disruption in prothrombin time or platelet counts) in relation to controls. However, all individual values were within the background control ranges without a dose related response. This finding may be associated with the kidney and adaptive liver changes.
Description (incidence and severity):
Group mean values and standard deviations for test and control group animals are given in Table 12 (statistically significant differences are indicated). Individual data are given in Appendix 12 and Appendix 13.

All treated males showed a statistically significant increase (p<0.05 - p<0.01) in phosphorous compared to controls, with the majority of the individual values within the background control ranges. This can be associated with the changes seen in the kidneys at histopathology.

Males at 600 mg/kg bw/day showed statistically significant reductions in total protein (p<0.05), alanine aminotransferase (p<0.01) and alkaline phosphatase (p<0.01). The majority of the individual values were within the background control ranges; albeit within the lower end of the range. These changes can be linked to the histopathological changes in the liver. These males also showed statistically significant increases (p<0.01) in both calcium and albumin/globulin ratio in relation to controls, the individual values were within the background control ranges, however, the findings correlate with the microscopic findings of the kidneys.

All treated females showed statistically significant increases (p<0.05 - p<0.01) in calcium compared to controls, all individual values within the background control ranges. Females treated with 100 or 600 mg/kg bw/day showed elevated levels (p<0.05 and p<0.01, respectively) of phosphorous in relation to the controls. Individual values of three and seven females respectively were above the background control ranges, and can be associated with the kidney changes.

At 600 mg/kg bw/day, females showed an increase (p<0.05) in glucose and a reduction in urea (p<0.05), total protein (p<0.01) and albumin (p<0.05) compared to controls. The majority of individual values for urea and glucose were within the background control ranges, whereas, only half of the females for total protein and albumin were within the background control ranges. These findings can be linked to the changes seen in the liver and kidneys.
Description (incidence and severity):
Functional Observations
A summary incidence of behavioral assessment observations is given in Table 2 and group mean behavioral assessment scores are given in Table 3. Individual values are given in Appendix 2 and Appendix 3. Group mean functional performance test values and standard deviations are given in Table 4 (statistically significant differences are indicated). Individual values are given in Appendix 4 and Appendix 5. Group mean sensory reactivity assessments are given in Table 5. Individual responses are given in Appendix 6.

Behavioral Assessments
One male animal treated with 600 mg/kg bw/day exhibited hunched posture during Weeks 11, 12 and 13. Another male from this treatment group exhibited hunched posture and pilo-erection during Week 12. Tiptoe gait was also noted in one male during Week 8, however, this animal was killed in extremis the next day. One male and one female treated with 600 mg/kg bw/day exhibited increased salivation during Week 4.

One female treated with 100 mg/kg bw/day exhibited generalized fur loss during the final two weeks of treatment, however, due to the isolated nature of this finding it was considered not to be related to treatment with the test item.

No such effects were detected in male animals treated with 100 mg/kg bw/day or in animals of either sex treated with 10 mg/kg bw/day.

Functional Performance Tests
There were no toxicologically significant changes in functional performance.

Males treated with 600 mg/kg bw/day showed a statistically significant increase (p<0.01) in overall motor activity. In the absence of any clinical signs of neurotoxicity, the intergroup difference was considered not to be of toxicological importance.

Females treated with 600 mg/kg bw/day showed a statistically significant increase (p<0.01) in forelimb grip strength. The intergroup difference was confined to one out of the three tests and in the absence of any clinical signs of neurotoxicity, the intergroup difference was considered not to be of toxicological importance.

Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.
Description (incidence and severity):
Group mean absolute and relative organ weights and standard deviations for test and control group animals are presented in Table 14 (statistically significant differences are indicated). Individual data are given in Appendix 15 and Appendix 16.

At 600 mg/kg bw/day there were statistically significant increases in kidney and liver weights; both absolute and relative to terminal body weights; males and females in relation to controls. The majority of the individual values were above the background control ranges. This would correlate to the microscopic findings of the kidneys and liver. The males also showed an increase in adrenal weights; both absolute and relative to terminal body weights. This would be indicative of a stress response.

At 100 mg/kg bw/day, animals of either sex showed a statistically significant increase (p<0.05) in liver weights for both absolute and relative to terminal body weight. The majority of individual values were within background control ranges and correlated with the histopathological findings.

Females treated with 600 or 100 mg/kg bw/day also showed a statistically significant decrease (p<0.05) in brain weights both absolute and relative to terminal body weight compared to controls. The majority of individual values were within the background control range, however, control values were mainly at the upper end of the range. Without any histopathological correlates this was considered to be of no toxicological significance.

All female treatment groups showed statistically significantly lower (p<0.05) thymus weights in relation to controls for both absolute and relative to terminal body weight values, all individual values were within the background control ranges and would correlate to the microscopic findings of the thymus and a potential stress response.
Description (incidence and severity):
A summary incidence of necropsy findings is given in Table 13. Individual data are given in Appendix 14.

The following findings were observed in animals treated with 600 mg/kg bw/day.

Six males and seven females showed one or both kidneys to be pale, with four males and five females having enlarged kidneys including one male with increased pelvic space in the right kidney. These findings would correlate to the histopathological changes to the kidneys. Five males and five females had pale adrenals.

Four males and one female had a pale pituitary and one male had a pale glandular region in the stomach. With no histopathological correlates these findings were considered to be of no toxicological significance.

Two males were observed to have small seminal vesicles which would correlate to the histopathological findings and one female had a mass on the right ovary which was considered incidental and unrelated to treatment.

Incidental findings included one 100 mg/kg bw/day male with a mass on the right epididymis and one control male had an enlarged spleen. There were a number of animals across the treatment groups (five intermediate females and one high dose female) with red discolored lungs including controls (one male and one female). With no histopathological correlates, these findings were considered to be of no toxicological significance.

Premature decedents from 600 mg/kg bw/day dose groups:

One male (No.61) killed in extremis on Day 55, microscopic findings included pale and enlarged kidneys, thickening of the stomach wall in the glandular region. Also the seminal vesicles were small.

One male (No.63) killed in extremis on Day 64, the necropsy findings included pale and enlarged kidneys, reddening of the lungs, pale coloration of the glandular region of the stomach and small seminal vesicles.

One male (No.68) killed in extremis on Day 89, showed pale and enlarged kidney with increased pelvic space (right kidney), pallor and thickening of the glandular stomach region with a raise limiting ridge. In addition, the thoracic aorta was enlarged, pale colon, and small seminal vesicles.
Description (incidence and severity):
A complete histopathology phase report is presented in Annex 1.

Premature Decedents at 600 mg/kg bw/day

Male 61 had marked nephropathy in the kidneys. Hyperostosis was present in the sternum with mineralisation in the kidneys and stomach. Zona glomerulosa hypertrophy and general cortical hyperplasia was present in the adrenal glands, ulceration in the stomach and atrophy in the thymus. There was also hypertrophy in the liver and decreased cytoplasmic rarefaction. There was decreased secretion in the seminal vesicles and diffuse tubular degeneration in the testes.

Male 63 had marked nephropathy as well as papillary inflammation and urothelial hyperplasia in the kidneys. Hyperostosis was present in the sternum with mineralisation was present in the kidneys, heart, aorta and stomach. There was also atrophy in the thymus and hypertrophy and decreased cytoplasmic rarefaction in the liver. There was decreased secretion in the seminal vesicles.

Male 68 had marked nephropathy in the kidneys. Hyperostosis was present in the sternum with moderate or marked mineralisation in the kidneys, aorta, heart and stomach. Zona glomerulosa hypertrophy was present in the adrenal glands and atrophy in the thymus. There was also hypertrophy in the liver and decreased cytoplasmic rarefaction. There was decreased secretion in the seminal vesicles as well as other minor changes.

Terminal Kill
Adrenal Glands
Mild hypertrophy of the zona glomerulosa was present in two controls, three 10 mg/kg bw/day, five 100 mg/kg bw/day and four 600 mg/kg bw/day males. In females this finding was present in one control, five 100 mg/kg bw/day and eight 600 mg/kg bw/day females. Cortical hypertrophy was present in one male at 600 mg/kg bw/day and cortical vacuolation was increased in males treated with 100 mg/kg bw/day.

Bone
At 600 mg/kg bw/day, hyperostosis was present in the sternal bone in six males (minimal to marked) and all the females (minimal to moderate). This did not occur in animals treated with 10 or 100 mg/kg bw/day.

Mineralisation
Mineralisation was present in the kidneys in one or more areas of most animals treated with 600 mg/kg bw/day. In addition mineralisation was also present occasionally in the aorta, heart and gastrointestinal tract at 600 mg/kg bw/day.

Kidneys
Nephropathy, defined as two or more of tubular degeneration/regeneration, tubular dilation, inflammatory cell infiltration, fibrosis, was present in all males (moderate or marked) and seven females (mild or moderate) at 600 mg/kg bw/day. This did not occur at 10 or 100 mg/kg bw/day.

Liver
Minimal centrilobular hepatocyte hypertrophy was present in three and five males (100 and 600 mg/kg bw/day, respectively) and six females at 600 mg/kg bw/day. Decreased rarefaction was present in five 600 mg/kg bw/day males.

Thymus
Atrophy was present in one male (minimal) and two females (minimal or mild) at 600 mg/kg bw/day.

Ovaries
Interstitial cell hypertrophy, minimal or mild was present in the ovaries of all 600 mg/kg bw/day females. This did not occur in 10 or 100 mg/kg bw/day females.

Seminal Vesicles
Decreased secretion was noted in two 600 mg/kg bw/day males only.

Vagina
At 600 mg/kg bw/day, mucification was present in two females and atrophy of the epithelium present in another one female. No abnormalities were present in 10 or 100 mg/kg bw/day females.

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:
mortality
body weight and weight gain
food consumption and compound intake
water consumption and compound intake
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
600 mg/kg bw/day (actual dose received)
System:
other: multiple
Organ:
liver
kidney
thymus
bone
adrenal glands
seminal vesicle
ovary
vagina
Treatment related:
yes
Dose response relationship:
not specified
Relevant for humans:
not specified

Applicant's summary and conclusion

Conclusions:
The oral administration of 2,2'-[(1-Methylethylidene)bis(cyclohexane-4,1-diyloxymethylene)]-bisoxirane (Eponex 1510) to rats by gavage, at dose level of 600 mg/kg bw/day, resulted in the early deaths of three males. Treatment-related changes in this treatment group included reductions in body weight development and dietary intake (males only), increased water consumption and histopathological changes in the kidneys, bone, adrenal glands, thymus, liver, ovaries, vagina, seminal vesicles. Mineralisation was also present in occasional tissues.

The findings detected at 100 mg/kg bw/day were considered to be more adaptive in nature and under the conditions of this study a No Observed Adverse Effect Level (NOAEL) for either sex can be considered to be 100 mg/kg bw/day for systemic toxicity.
Executive summary:

Introduction

The study was designed to investigate the systemic toxicity of the test item and is compatible with the following regulatory guideline:

The OECD Guidelines for Testing of Chemicals No. 408 "Subchronic Oral Toxicity - Rodent: 90 Day Study” (Adopted 21 September 1998).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

Methods

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 600 mg/kg bw/day.  A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

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 animals at the end of the study.  Ophthalmoscopic examination was also performed on all study animals before the start of treatment and on control and high dose animals during Week 12 of treatment.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from all control and high dose animals (Groups 1 and 4) as well as any gross lesions from Groups 1 to 4 was performed in the first instance.  As there were treatment-related findings in the kidneys, bone, adrenal glands, thymus, liver, ovaries, vagina and seminal vesicles, examination of these tissues was subsequently extended to include relevant animals from the low and intermediate dose groups.

Results

Mortality

Three male animals treated with 600 mg/kg bw/day were killed in extremis during the study due to the severity of the clinical signs.  All three had marked nephropathy in the kidneys and this is considered to be the primary cause of death.

There were no further unscheduled deaths.

Clinical Observations

Animals of either sex treated with 600 mg/kg bw/day exhibited increased salivation during the treatment period.  This finding was also noted to a lesser extent in animals treated with 100 mg/kg bw/day.  One 10 mg/kg bw/day female showed increased salivation on Day 29 only.  During the treatment period five males and three females treated with 600 mg/kg bw/day exhibited signs of noisy respiration.

Isolated incidences of staining around the mouth were seen in one male and one female at 600 mg/kg bw/day and staining around the snout in one male at 100 mg/kg bw/day.  

One male animal which was killed in extremis on Day 55 exhibited tiptoe gait, hunched posture, dehydration and emaciation.  A further male which was killed in extremis on Day 64 exhibited tiptoe gait, increased salivation, pilo-erection and hunched posture.  The male animal which was killed in extremis on Day 89 exhibited hunched posture, pilo-erection, lethargy and pallor of the extremities.  

No clinical signs were apparent in male animals treated with 10 mg/kg bw/day.

Behavioral Assessment

Male animals treated with 600 mg/kg bw/day exhibited instances of hunched posture towards the end of the treatment period.  Isolated occurrences of tiptoe gait, increased salivation and pilo-erection were also noted.  Female animals from this treatment group exhibited an isolated occurrence of increased salivation.  Female animals treated with 100 mg/kg bw/day exhibited isolated instances of generalized fur loss towards the end of the treatment period.  

No such effects were detected in male animals treated with 100 mg/kg bw/day or in animals of either sex treated with 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

Animals of either sex treated with 600 mg/kg bw/day generally showed lower body weight gains throughout the treatment period, which resulted in an overall reduction in body weight gain when compared with controls.  There were no effects on body weight development for males treated with 100 or 10 mg/kg bw/day, however, the females from these treatment groups showed fluctuations in body weight gains during the treatment period which resulted in reductions in overall body weight gains when compared to control.

Food Consumption

Male animals treated with 600 mg/kg bw/day exhibited marginally lower food intake than the controls from week 8 of treatment.

No such effects were noted in female animals treated with 600 mg/kg bw/day or in animals of either sex treated with 10 or 100 mg/kg bw/day.

At 600 mg/kg bw/day food conversion efficiency for males was generally lower compared to controls.  These reductions generally followed similar trend of the body weight changes.

Water Consumption

Animals of either sex treated with 600 mg/kg bw/day were shown to be consuming larger

volumes of water than the controls when water intake was measured gravimetrically.  

No such effects were noted in animals of either sex treated with 10 or 100 mg/kg bw/day.

Ophthalmoscopy

Ophthalmoscopic examination of animals of both sexes from the control and surviving animals from the 600 mg/kg bw/day dose group during Week 12 of the treatment period did not indicate any treatment-related differences.

Hematology

Animals of either sex at 600 mg/kg bw/day and males treated with 100 mg/kg bw/day showed a statistically significant increase (p<0.05) in neutrophil count compared to controls.  

At 600 mg/kg bw/day, males showed statistically significant reductions in hemoglobin, hematocrit and erythrocytes, which resulted in associated reductions in the red blood cell indices.  All female dose groups showed significant reductions in mean corpuscular volume and mean corpuscular hemoglobin, and 600 mg/kg bw/day females had reduced mean corpuscular hemoglobin concentrations compared to controls.  

All treated male dose groups showed a significant reduction in activated partial thromboplastin times in relation to controls.

Blood Chemistry

At 600 mg/kg bw/day both sexes showed a reduction in plasma levels of total protein and increases in electrolyte levels of calcium and phosphorus with the males also exhibiting an increase in albumin/globulin ratio and reductions in levels of alanine aminotransferase and alkaline phosphatase in comparison with controls.  In addition, females from this dose group showed an increase in glucose and a reduction in albumin levels and plasma urea.

Males treated at 10 and both sexes at 100 mg/kg bw/day also showed slightly elevated levels of phosphorus and females from both these test groups showed a slight increase in levels of calcium.

Necropsy

Premature decedents from 600 mg/kg bw/day dose groups:  

Male 61 was killed in extremis on Day 55, microscopic findings included pale and enlarged kidneys, thickening of the stomach wall in the glandular region.  Also the seminal vesicles were small.  

Male 63 was killed in extremis on Day 64, the necropsy findings included pale and enlarged kidneys, reddening of the lungs, pale coloration of the glandular region of the stomach and small seminal vesicles.  

Male 68 was killed in extremis on Day 89, showed pale and enlarged kidneys with increased pelvic space (right kidney), pallor and thickening of the glandular stomach region with a raised limiting ridge.  In addition, the thoracic aorta was enlarged, pale colon, and small seminal vesicles.  

Teminal Kill

At 600 mg/kg bw/day six males and seven females showed pallor in one or both kidneys, four males and five females also exhibited enlarged kidneys including one male with increased pelvic space in the right kidney.  Five males and five females had pale adrenals.  

Organ Weights

At 600 mg/kg bw/day there were statistically significant increases in male and female absolute and relative kidney and liver weights.  The males also showed an increase in adrenal weights.  At 100 mg/kg bw/day, animals of either sex showed statistically significant increase in liver weights (p<0.05) for both absolute and relative to terminal body weights.  All female treatment groups showed statistically significant lower thymus weights in relation to controls.

Histopathology

Histopathological examination revealed the following treatment-related abnormalities:

Premature Decedents at 600 mg/kg bw/day

Male 61 had marked nephropathy in the kidneys. Hyperostosis was present in the sternum with mineralisation in the kidneys and stomach.  Zona glomerulosa hypertrophy and general cortical hyperplasia was present in the adrenal glands, ulceration in the stomach and atrophy in the thymus.  There was also hypertrophy in the liver and decreased cytoplasmic rarefaction.  There was decreased secretion in the seminal vesicles and diffuse tubular degeneration in the testes.

Male 63 had marked nephropathy as well as papillary inflammation and urothelial hyperplasia in the kidneys.  Hyperostosis was present in the sternum with mineralisation present in the kidneys, heart, aorta and stomach.  There was also atrophy in the thymus and hypertrophy and decreased cytoplasmic rarefaction in the liver.  There was decreased secretion in the seminal vesicles.

Male 68 had marked nephropathy in the kidneys.  Hyperostosis was present in the sternum with moderate or marked mineralisation in the kidneys, aorta, heart and stomach.  Zona glomerulosa hypertrophy was present in the adrenal glands and atrophy in the thymus.  There was also hypertrophy in the liver and decreased cytoplasmic rarefaction.  There was decreased secretion in the seminal vesicles as well as other minor changes.  

Terminal Kill

Kidneys - Nephropathy, defined as two or more of tubular degeneration/regeneration, tubular dilation, inflammatory cell infiltration, fibrosis, was present in all males (moderate or marked) and seven females (mild or moderate) treated with 600 mg/kg bw/day.  No abnormalities were detected at 10 or 100 mg/kg bw/day.

Bone - Hyperostosis was present in the sternal bone in six males (minimal to marked) and all females (minimal to moderate) treated with 600 mg/kg bw/day.  No abnormalities were detected at 10 or 100 mg/kg bw/day.

Mineralisation - Mineralisation was present in the kidneys in one or more areas of most   600 mg/kg bw/day animals.  In addition mineralisation was occasionally present in the aorta, heart and gastrointestinal tract for animals of either sex at 600 mg/kg bw/day.

Ovaries - Interstitial cell hypertrophy, minimal or mild was present in the ovaries of all females at 600 mg/kg bw/day.  No abnormalities were detected at 10 or 100 mg/kg bw/day.

Seminal Vesicles - Decreased secretion was noted in 2/7 Group 4 males only.

Vagina - Mucification was present in two females with atrophy of the epithelium present in another one female at 600 mg/kg bw/day.  No abnormalities were present at 10 or 100 mg/kg bw/day.

Adrenal Glands - Mild hypertrophy of the zona glomerulosa was present in two, three, five and four males from the control, 10, 100 and 600 mg/kg bw/day dose groups respectively.  Also this finding was present in one, five and eight females from control, 100 and 600 mg/kg bw/day respectively.  Cortical hypertrophy was present in one 600 mg/kg bw/day  male. Cortical vacuolation was increased in 100 mg/kg bw/day males.

Liver - Minimal centrilobular hepatocyte hypertrophy was present in three and five males treated with 100 and 600 mg/kg bw/day respectively and six females treated with 600 mg/kg bw/day.  Decreased rarefaction was present in five 600 mg/kg bw/day males.  

Thymus - Atrophy was present in one male (minimal) and 2 females (minimal or mild) from 600 mg/kg bw/day.

Conclusion

The oral administration of 2,2'-[(1-Methylethylidene)bis(cyclohexane-4,1 -diyloxymethylene)]-bisoxirane (Eponex 1510) to rats by gavage, at dose level of 600 mg/kg bw/day, resulted in the early deaths of three males.  Treatment-related changes in this treatment group included reductions in body weight development and dietary intake (males only), increased water consumption and histopathological changes in the kidneys, bone, adrenal glands, thymus, liver, ovaries, vagina, seminal vesicles.  Mineralisation was also present in occasional tissues.

The findings detected at 100 mg/kg bw/day were considered to be more adaptive in nature and under the conditions of this study a No Observed Adverse Effect Level (NOAEL) for either sex can be considered to be 100 mg/kg bw/day for systemic toxicity.