p-tert-butylphenyl 1-(2,3-epoxy)propyl ether

Endpoint summary

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

Identification : p-tert-butylphenyl 1-(2,3-epoxy) propyl ether
Physical State/Appearance : Yellow liquid
Chemical Name : Oxirane,[[4-(1,1-dimethylethyl)phenoxy]methyl]-
Purity : 100%
Date Received : 12 February 2016
Storage Conditions : Room temperature in the dark
Expiry Date : 12 October 2017

Species:

rat

Strain:

Wistar

Details on species / strain selection:

A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for nine days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 187 to 223g, the females weighed 125 to 161g, and were approximately six to eight weeks old.

Sex:

male/female

Details on test animals or test system 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; there were no deviations from these targets.

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.

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

Animals were allocated to treatment groups as follows:

Treatment Dose Level Treatment Concentration Animal Numbers
Group (mg/kg bw/day) Volume (mL/kg) (mg/mL) Male Female
Control 0 4 0 10 (1-10) 10 (11-20)
Low 25 4 6.25 10 (21-30) 10 (31-40)
Intermediate 100 4 25 10 (41-50) 10 (51-60)
High 500 4 125 10 (61-70) 10 (71-80)
The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group.

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.

Details on analytical verification of doses or concentrations:

See analytical report

Duration of treatment / exposure:

90 days

Frequency of treatment:

once per day

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

This study was designed to be compatible with the procedures indicated by the following internationally accepted guidelines and recommendations:

 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).

Positive control:

none

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.

Water Consumption
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 animals from Groups 1 to 4 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:
Adrenals Ovaries
Aorta (thoracic) Pancreas
Bone & bone marrow (femur including stifle joint)• Pituitary
Bone & bone marrow (sternum) Prostate
Brain (including cerebrum, cerebellum and pons) Rectum
Caecum Salivary glands (submaxillary)
Colon Sciatic nerve
Duodenum Seminal vesicles
Epididymides ♦ Skin (hind limb)
Esophagus Spinal cord (cervical, mid-thoracic
Eyes* and lumbar)
Gross lesions Spleen
Heart Stomach
Ileum (including Peyer’s patches) Testes ♦
Jejunum Thymus
Kidneys Thyroid/Parathyroid
Liver Tongue•
Lungs (with bronchi) # Trachea
Lymph nodes (mandibular and mesenteric) Urinary bladder
Mammary glands Uterus (with cervix)
Muscle (skeletal)• Vagina

• Retained only and not processed
* Eyes fixed in Davidson’s fluid
♦ Preserved in Modified Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative
• Retained only and not processed

All tissues were dispatched to the Test Site (Propath UK Ltd.,Willow Court, Netherwood Road, Rotherwas, Hereford,HR2 6JU) for processing (Principal Investigator: M Dow). All tissues from control and 500 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 adrenal glands, liver, thyroid glands and thymus from both sexes from animals in the low and intermediate groups.

Pathology
Microscopic examination was conducted by the Study Pathologist (W Henderson). A 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:

Data Evaluation
Data were processed to give summary incidence or group mean and standard deviation values where appropriate. All data were summarized in tabular form.

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, 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 nonhomogeneity 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)

Description (incidence and severity):

A summary incidence of daily clinical observations is given in Table 1. Individual data are presented in Appendix 1.

Animals of either sex treated at 500 or 100 mg/kg bw/day exhibited signs of increased salivation from Day 6 or Day 21 of treatment until the end of study albeit at a lesser incidence in the animals treated at 100 mg/kg bw/day. An isolated instance of increased salivation was also seen in two 25 mg/kg bw/day females on Day 84. Observations 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. Incidental findings included noisy respiration in one 500 mg/kg bw/day female between Days 54 and 86, in isolation this transient condition was considered not to be related to test item toxicity.

Description (incidence):

Two males (Nos, 21 and 26) treated at 25 mg/kg bw/day were killed in extremis on Study Day 52 or 18 respectively and one control male (No.2) was terminated on Study Day 65.

Male No. 26 - was terminated due to the severity of a physical injury to the tail. The following changes were detected at necropsy: dark coloured stomach contents, enlarged spleen, increased pelvic fluid filled space in the kidneys (characteristic of hydronephrosis), and small testes and epididymides.

Male No. 21 - was terminated on Day 52 of treatment due to clinical observations of red staining around the snout, decreased respiratory rate, piloerection, hunched posture, lethargy, pallor of the extremities and tip toe gait. Macroscopic findings included pallid kidneys, liver and spleen. Dark coloured contents were present in the stomach and intestines.

With exception of the physical injury to the tail there were no notable histopathological changes to account for the demise of either low dose male.

Male No. 2 - was killed in extremis due to body weight loss and labored respiratory observations, other clinical signs included hunched posture, staining around the snout, and light brown fluid was observed coming out of the mouth and nostrils. Macroscopic examination revealed a fluid filled cavity in the right side of the thoracic chamber, a fluid filled mass located externally on the thoracic cavity and discoloured lungs. The histopathology examination indicated these macroscopic findings were the result of dosing trauma.

There were no further unscheduled deaths on the study.

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.

The body weight development of males treated at 500 mg/kg bw/day throughout the study remained consistently lower than that of the controls often attaining statistical significance (p<0.05 or p<0.01) and culminating in lower than control overall weight gain (ca. 31%). Males treated at 100 or 25 mg/kg bw/day sporadically exhibited lower than control weight gains (with or without attaining statistical significance) which also resulted in lower than control overall weight gains (ca. 13% or 7% respectively) these latter changes may have some association with the palatability of the test item formulations. Females from all test groups also showed slightly lower than control weight gains during the course of treatment resulting in approximately 11% (500 mg/kg bw/day), 13% (100 mg/kg bw/day) or 15% (25 mg/kg bw/day) overall lower gains than the concurrent control. However, in the absence of a true dose relationship these female mean weight values were considered to be of no toxicological importance.

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.

Dietary intake in males receiving 500 mg/kg bw/day remained slightly below that of the control throughout the treatment period with an overall lower (11%) than control dietary intake by the end of treatment.

The magnitude of variance in food consumptions between males treated at 25 or 100 mg/kg bw/day and the concurrent control remained marginal throughout the study. A similar trend was apparent in all female test groups indicating these minor fluctuations were of no toxicological importance.

Animals of either sex from each dose group showed fluctuations in food conversion efficiency when compared to controls. However, these intergroup differences were of no particular consequence and were considered to correlate directly to the body weight and food intake fluctuations seen during the study.

Description (incidence and severity):

Visual inspection of water residues did not indicate any effect of treatment on water intake throughout the study.

Description (incidence and severity):

Individual ophthalmoscopic examination findings are given in Appendix 9.

No adverse ocular changes were detected in any animal prior to start of treatment or in control and 500 mg/kg bw/day (high dose) animals during Week 12 of the study.

Description (incidence and severity):

Group mean values and standard deviations for test and control group animals are given in Table 10 (statistically significant differences are indicated). Individual data are given in Appendix 10 and Appendix 11.

There were no toxicologically significant findings in the haematological parameters examined.

Incidental changes detected included reductions (p<0.05) in both mean corpuscular hemoglobin concentration (MCHC) and platelet count (Plt) in males treated at 500 mg/kg bw/day when compared to controls. However, without any supporting histopathological correlates these isolated changes are considered to be of no toxicological significance.

Females from all treatment groups showed a statistically significant reduction (p<0.05) in hemoglobin (Hb), without a dose related response implying in the absence of supporting changes to be due to natural biological variation. Reductions (p<0.05) in MCHC, mean corpuscular hemoglobin (MCH) and platelet counts were apparent in high dose females. However, as the majority of individual values for these parameters were within the background control ranges it is considered unlikely these findings are of any toxicological consequence.

At 500 mg/kg bw/day (high dose) females also exhibited a statistically significant reduction in total leucocyte count with an associated reduction in circulating lymphocyte levels when compared to controls. While the precise aetiology of this condition is uncertain the absence of any associated histopathological changes and as the majority of individual values for both parameters were within the background control ranges it is reasonable to conclude these changes are of no toxicological consequence.

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 12 and Appendix 13.

Treatment-related changes were identified in each sex of high dose (500 mg/kg bw/day) animals with statistically significant variations (p<0.05 or p<0.01) from control in either sex for alkaline phosphatase, cholesterol, bile acids and albumin/globulin ratio together with changes in total protein (males only) and alanine aminotransferase and bilirubin (females only). High dose females also showed a reduction in plasma chloride concentration, however, as only a few individual values were below normal historical ranges and there was no histopathological evidence of renal impairment it is reasonable to associate this electrolyte change with natural biological variation rather than test item toxicity.

Males treated at 100 mg/kg bw/day showed increases (p<0.05) in plasma levels of alkaline phosphatase and cholesterol. An increase in A/G ratio (p<0.05) and reduction in cholesterol levels (p<0.01) were detected in females from this test group. However, in the absence of supporting treatment-related histological changes in animals at this dose level these changes were considered to be of no toxicological importance.

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. 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
There were no treatment-related changes in the behavioral parameters measured. Incidental findings involved one instance of tip toe gait that was detected in one control animal.

Functional Performance Tests
There were no treatment-related changes in functional performance.

A statistically significant reduction in one of three trials for forelimb and hind limb grip strengths were detected in males treated at 500 mg/kg bw/day together with a reduction (p<0.01) in overall activity. In addition males treated with 100 mg/kg bw/day also showed a statistically significant reduction (p<0.05) in one of three hind limb grip strength measurements. In addition, females treated with 25 or 100 mg/kg bw/day exhibited an increase (p<0.01) in overall activity. In relation to these findings, in the absence of any supporting credible evidence of neurotoxicity these changes were considered more plausibly due to natural biological variation rather than through exposure to the test item.

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 13 (statistically significant differences are indicated). Individual data are given in Appendix 15 and Appendix 16.

At 500 mg/kg bw/day animals of either sex showed a statistically significant increase in liver weights in both absolute and relative to terminal body weight.

Females from this test group (500 mg/kg bw/day) showed a reduction in thymus weights. The majority of individual thymic values were within the normal background control ranges but would correlate with the histological identification in these animals of thymic atrophy. These females also showed a reduction in spleen weights which in the absence of a histopathological correlate was considered to be of no toxicological significance.

Description (incidence and severity):

A summary incidence of necropsy findings is given in Table 12. Individual data are given in Appendix 14.

Notable macroscopic findings identified in animals at study termination included liver enlargement in one 500 mg/kg bw/day male with sloughing in the glandular region of the stomach detected in two further 500 mg/kg bw/day males.

Incidental findings included the following:

A number of animals across all groups including controls showed either red discolouration or red patches on the lungs. It is reasonable to assume this was associated with the terminal exsanguination procedure and not to exposure to the test item.

Increased pelvic space was detected in one or both kidneys of two control males and in one male treated at 100 and in one male at 500 mg/kg bw/day. In addition, one 100 mg/kg bw/day female showed a malformation of the uterus. Findings of this nature are consistent with normally expected low incidence findings in laboratory maintained rats and are therefore considered to have no direct relationship to treatment with the test item.

Description (incidence and severity):

A complete histopathology phase report is presented in Annex 1.

Premature Decedents
One Control male (No. 2) was killed prematurely due to clinical symptoms. The changes were as a result of a mal-dose.

At 100 mg/kg bw/day one male (No. 21) was killed prematurely due to clinical symptoms. There were no notable changes noted at histopathology to account for its early death. One further male (No. 26) was killed prematurely due to a tail injury.

Terminal Kill

Adrenal Glands
Hypertrophy of the zona glomerulosa was present at a mild level in 1/9 control and 7/10 Group 4 males along with 2/10 Group 3 males, minimal hypertrophy was noted in another Group 3 male. In females hypertrophy of the zona glomerulosa was present in 3/10 control, 1/10 Group 2, 3/10 Group 3 and 6/10 Group 4 females at a minimal level and in 1/10 Group 3 and 4/10 Group 4 females at a mild level.

Liver
There was an increase in cell turnover (increased single cell degeneration/necrosis and increased mitoses) in one male and 4/10 females in Group 4.

Diffuse hepatocellular hypertrophy was present in 8/10 males and 9/10 females in Group 4. Centriloblular hypertrophy was present in another one male and female in Group 4.

No such change was apparent in Group 2 and 3 animals.

Thyroid Gland
Follicular cell hypertrophy, minimal or mild, was present in 8/10 males and females in Group 4. It was present at a minimal level in one Group 3 male but this is considered to be incidental at low incidence and severity.

Thymus
Minimal thymic atrophy was present in 7/10 females in Group 4 only. After examination of the thymus from all males it was decided that all males were within expected limits.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

histopathology: non-neoplastic

Critical effects observed:

not specified

Conclusions:

Oral administration of p-tert-butylphenyl 1-(2,3-epoxy) propyl ether to rats by gavage to male and female Wistar Han™:RccHan™:WIST strain rats for ninety days at dose levels of 25, 100 and 500 mg/kg bw/day resulted in a significant level of adverse treatment-related changes in either sex dosed at 500 mg/kg bw/day precluding identification of a No Observed Adverse Effect Level (NOAEL) in animals at this dosage. However, as response to treatment in both sexes at 100 mg/kg bw/day and below were interpreted as adaptive in nature with no evidence of degenerative changes, under the conditions of this study the NOAEL for either sex was considered to be 100 mg/kg bw/day.

Executive summary:

Introduction

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

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 25, 100 and 500 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 animals prior to 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 was performed.

Results

Mortality

Two low dose (25 mg/kg bw/day) group males were killed in extremis on Study Day 18 or 52 respectively and one control male was terminated on humane grounds on Study Day 65.

The premature termination of the control was due to a suspected mal-dose while the termination of one low dose group male resulted from an unrelated to treatment injury to the tail. Furthermore, while the cause of the deterioration in health of the remaining low dose group male was undetermined it is reasonable to assume in the absence of further deaths from this dose group or from animals of either sex from the higher dose groups that none of these early terminations had a true relationship to treatment.

There were no further unscheduled deaths.

Clinical Observations

Post-dose increased salivation was initially observed in both sexes treated at 500 mg/kg bw/day during the first week of treatment persisting thereafter through to study termination. Increased salivation was also observed later in the study in each sex that received 100 mg/kg bw/day and in females treated at 25 mg/kg bw/day.

Behavioral Assessment

There were no treatment-related changes in the behavioural parameters measured.

Functional Performance Tests

There were no treatment-related changes in functional performance.

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

Body Weight

Slightly impaired weight development was evident in all male test groups during the course of the study but most pronounced at the high dose (500 mg/kg bw/day) in comparison with the concurrent controls. Minor body weight fluctuations were also occasionally apparent in all female test groups.

Food Consumption

Dietary intake and food conversion efficiency in high dose (500 mg/kg bw/day) males remained slightly below that of the control throughout the treatment period. Minor fluctuations in food consumption and utilization were also apparent in high dose females and in both sexes of animals treated at 25 and 100 mg/kg bw/day. However, as the variance from control was more marginal in these animals the changes were considered to be of no toxicological importance.

Water Consumption

Visual inspection of water residues did not indicate any effect of treatment on water intake throughout the study.

Ophthalmoscopy

There were no treatment related ocular effects detected.

Hematology

There were no toxicologically significant findings in the hematological parameters examined.

Blood Chemistry

Treatment associated changes in alkaline phosphatase, cholesterol, bile acids, total protein, albumin/globulin ratio, bilirubin and alanine aminotransferase were identified males and or females treated at 500 mg/kg bw/day.

Necropsy

Notable macroscopic findings were confined to males treated at 500 mg/kg bw/day and involved one instance of liver enlargement and two instances of sloughing in the glandular region of the stomach.

Organ Weights

Notable changes were confined to an increase in liver weights in animals of either sex at 500 mg/kg bw/day when compared to control values.

Histopathology

Treatment in both sexes at 500 mg/kg bw/day resulted in an adverse change in the liver involving an increase in cell turnover (increased mitoses and single cell degeneration/necrosis). There were also adaptive changes in the liver, thyroid glands, adrenal glands of both sexes and secondary changes in the thymus of females.

Conclusion

Oral administration of p-tert-butylphenyl 1-(2,3-epoxy) propyl ether to rats by gavage to male and female Wistar Han™:RccHan™:WIST strain rats for ninety days at dose levels of 25, 100 and 500 mg/kg bw/day resulted in a significant level of adverse treatment-related changes in either sex dosed at 500 mg/kg bw/day precluding identification of a No Observed Adverse Effect Level (NOAEL) in animals at this dosage. However, as response to treatment in both sexes at 100 mg/kg bw/day and below were interpreted as adaptive in nature with no evidence of degenerative changes under the conditions of this study the NOAEL for either sex was considered to be 100 mg/kg bw/day.

Endpoint conclusion

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat