Registration Dossier

Administrative data

Description of key information

The oral (gavage) administration of the test substance to Wistar Han™:RccHan™:WIST strain rats, at dose levels of 35, 350 or 600 mg/kg bw/day, resulted in premature deaths of three females from the high dose group. These deaths were considered to be related to the irritant properties of the test item and gavage-related reflux rather than an indication of its systemic toxicity.

The surviving animals of either sex from the 600 mg/kg bw/day dose group showed intermittent episodes of respiratory distress which correlated with tracheal lesions in some of the non-recovery animals from this dose group. Animals of either sex from this dose group also showed hyperplasia and hyperkeratosis of the forestomach, an indication of stomach irritation, which was deemed likely to have contributed to lower food intake and body weight gains and possibly higher water consumption.  Animals of either sex previously receiving 600 mg/kg bw/day showed evidence of recovery after a fourteen day treatment-free period and these findings were considered to be due to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity. The findings in male kidneys from the 350 or 600 mg/kg bw/day dose groups were considered to be indicative of alpha-2u-globulin nephropathy, a condition specific to the male rat.

Excluding the above from the overall assessment, 600 mg/kg bw/day could be established as the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity within the confines of this study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental Starting Date: 07 January 2016 Experimental Completion Date: 18 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: The Japanese Ministry of Economy Trade and Industry (METI)
Version / remarks:
Ministry of Health, Labor and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003 for a twenty-eight day repeat dose oral toxicity study as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973 of Ministry of International Trade and Industry (MITI) amended 2004.
Deviations:
no
Guideline:
other: USA Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Wistar Han™:RccHan™:WIST
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 eight days during which time their health status was assessed. A total of sixty animals (thirty males and thirty females) were accepted into the study. At the start of treatment the males weighed 196 to 219g, the females weighed 167 to 189g, and were approximately six to eight weeks old.
Sex:
male/female
Details on test animals and environmental conditions:
The animals were housed in groups of five 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
Details on route of administration:
The test item was administered daily, for up to twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe
Vehicle:
arachis oil
Details on oral exposure:
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 show the formulations to be stable for at least eleven days when stored at approximately 4 ºC, in the dark. Formulations were initially prepared for use over four days; with availability of further stability data, formulations were prepared on an approximately weekly basis.
Formulations were aliquoted for use and stored as above before use.

Samples of each test item formulation were taken and analyzed for concentration of test substance at Envigo Research Limited, Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in the attached Annex 2. The results indicate that the prepared formulations were within 92 to 110% of the nominal concentration.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
The test item was administered daily, for up to twenty-eight consecutive days. Recovery group animals were maintained for a further fourteen days treatment-free period following termination of treatment.
Frequency of treatment:
The test item was administered daily, for up to twenty-eight consecutive days
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control Treatment Group
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Recovery Control Treatment Group
Dose / conc.:
35 mg/kg bw/day (actual dose received)
Remarks:
Low Treatment Group
Dose / conc.:
350 mg/kg bw/day (actual dose received)
Remarks:
Intermediate Treatment Group
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
High Treatment Group
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
Recovery High Treatment Group
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
Animals were allocated to treatment groups as follows:


Treatment Group Dose Level (mg/kg bw/day) Treatment Volume (mL/kg) Concentration (mg/mL) Animal Numbers
Male Female
Control 0 4 0 5 (1-5) 5 (6-10)
Recovery Control 0 4 0 5 (41-45) 5 (46-50)
Low 35 4 8.75 5 (11-15) 5 (16-20)
Intermediate 350 4 87.5 5 (21-25) 5 (26-30)
High 600 4 150 5 (31-35) 5 (36-40)
Recovery High 600 4 150 5 (51-55) 5 (56-60)
The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group.

The test item was administered daily, for up to twenty-eight consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe; see Deviations from Study Plan in OVERALL REMARKS section . Control animals were treated in an identical manner with 4 mL/kg of Arachis oil BP. Recovery group animals were maintained for a further fourteen days treatment-free period following termination of treatment.

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; see Deviations from Study Plan in OVERALL REMARKS section.
Positive control:
No
Observations and examinations performed and frequency:
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; see Deviations from Study Plan. During the treatment-free period, animals were observed daily. All observations were recorded.

Body Weight
Individual body weights were recorded prior to dosing on Day 1 and at weekly intervals thereafter. Body weights were also performed prior to terminal kill and, in the case of recovery group animals, on Days 36 and 43 prior to 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; see Deviations from Study Plan.

Special Evaluations
Functional Observations
Prior to the start of treatment and on Days 7, 14, 21 and 26, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli. Observations were carried out from approximately two hours after dosing on each occasion.

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

Normal ranges for functional performance assessments are given in the attached Annex 7.

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all surviving non- recovery test and control group animals at the end of the treatment period (Day 28) and on all surviving recovery group animals at the end of the treatment-free period (Day 42). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 29 and 43. Animals were not fasted prior to sampling.


Urinalytical investigations were performed on all surviving non-recovery test and control group animals during Week 4 and on all surviving recovery group animals during Week 6. Urine samples were collected overnight by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food.

The methods used for hematological, blood chemical and urinalytical investigations are given in Annex 6 and normal ranges are shown in the attached Annex 8.

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)
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++) Gamma glutamyltranspeptidase
Triglycerides (Tri)


Urinalysis
The following parameters were measured on collected urine:
Volume Ketones
Specific Gravity Bilirubin
pH Urobilinogen
Protein Blood
Glucose Appearance

Sacrifice and pathology:
Necropsy
On completion of the dosing period, or in the case of recovery group animals, at the end of the treatment-free 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.

Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored frozen at approximately -20 °C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples will be discarded.

Organ Weights
The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation:
Adrenals Liver
Brain Ovaries
Epididymides Spleen
Heart Testes
Kidneys Thymus
Pituitary (post-fixation) Thyroid/Parathyroid (post fixation)
Prostate and Seminal Vesicles Uterus with Cervix (with coagulating glands and fluids)

Normal ranges for organ weights are given in the attached Annex 9.


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 Rectum
pons) Salivary glands (submaxillary)
Caecum Sciatic nerve
Colon Seminal vesicles (with coagulating
Duodenum glands and fluids)
Epididymides ♦ Skin
Esophagus Spinal cord (cervical, mid thoracic
Eyes * and lumbar)
Gross lesions Spleen
Heart Stomach
Ileum Testes ♦
Jejunum Thymus
Kidneys Thyroid/Parathyroid
Liver Trachea
Lungs (with bronchi)# Urinary bladder
Lymph nodes (mandibular and mesenteric) Uterus & Cervix
Mammary gland Vagina
Muscle (skeletal)

All tissues were dispatched to the histology processing Test Site (Envigo CRS Limited, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: D Roberts). The tissues shown in bold from all control and 600 mg/kg bw/day dose group animals (including early decedents) were prepared as paraffin blocks, sectioned at a nominal thickness of approximately 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed. In addition, sections of testes from all Control and 600 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain and examined.

Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of the forestomach, liver, thyroid glands and trachea (males and females) and adrenal glands and kidneys (males only) from animals in the low and intermediate groups as well as the surviving recovery animals.




* 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

Pathology
Microscopic examination was conducted by the Study Pathologist (Anna Domenech at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS). A histopathology peer review was conducted at the Test Facility. A complete histopathology phase report is presented in Annex 1 and presents the consensus view of both pathologists.

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, Urinalysis (Volume and Specific Gravity), 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). Urine volume and specific gravity were statistically analyzed using the R Environment for Statistical Computing.
Initially, the distribution of the data was assessed by the Shapiro-Wilk normality test, followed by assessment of the homogeneity of the data using Bartlett’s test. Where considered appropriate, parametric analysis of the data was applied incorporating analysis of variance (ANOVA), which if significant, was followed by pairwise comparisons using Dunnett’s test. Where parametric analysis of the data was considered to be unsuitable, non- parametric analysis of the data was performed incorporating the Kruskal- Wallis test which if significant was followed by the Mann-Whitney "U" test.

Probability values (p) are presented as follows:

p<0.01 **
p<0.05 *
p>0.05 (not significant)


Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
A summary incidence of daily clinical observations is given in the attached Table 1. Individual data are presented in the attached Appendix 1.

At 600 mg/kg bw/day, sporadic episodes of noisy respiration were observed in most males and some surviving females from Days 11 and 18 (relative to the start of dosing), respectively. Male 52 from this dose group also exhibited signs of decreased respiratory rate from Day 27 and was not dosed on the following day for animal welfare reasons.
Intermittent instances of increased post-dose salivation were seen in animals of either sex treated with 600 or 350 mg/kg bw/day from Days 5 and 8, respectively. There were no clinical signs for any of the animals receiving the test item at a dose level of 35 mg/kg bw/day throughout the treatment period.

After cessation of dosing, respiratory signs persisted in 3/5 recovery males previously treated with 600 mg/kg bw/day, with Male 52 exhibiting signs of noisy respiration up to 11 days into the treatment-free period. In contrast, none of the surviving recovery females from this dose group showed any clinical signs during this phase of the study.

Taking into consideration the overall results from this study, it was considered likely that the premature deaths amongst females from the 600 mg/kg bw/day dose group were related to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity. For the surviving animals, clinical signs of respiratory distress and increased post-dose salivation, reduction in body weight development or associated food intake and slightly higher water consumption were also deemed not related to the systemic toxicity of the test item. The changes identified during hematological or blood chemistry investigations in males and/or females showed reversibility/recovery after the fourteen day dose-free period whilst increases in the liver and kidneys weights in non-recovery males corresponded to the histopathological alterations in these tissues. Excluding the premature deaths, other effects deemed to be related to the irritant properties of the test item and/or gavage-related reflux, and kidney findings indicativeof a male rat-specific condition from the overall assessment, 600 mg/kg bw/day could be established as the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity within the confines of this study.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were three unscheduled deaths during the study, all females treated with the test item at a dose level of 600 mg/kg bw/day.

Female 37 (allocated to non-recovery group) was found dead on Day 14 (before dosing) relative to the start of test item administration. Prior to death, there were no adverse clinical signs for this animal and macroscopic findings at necropsy only included a number of cannibalised tissues. Of the tissues available for histopathological examination, marked tracheal ulceration was evident, which was considered to be the major factor contributing to the death of this animal.

Females 59 and 60 (allocated to recovery group) were killed in extremis on Days 27 and 23 (before dosing), respectively. Before death, clinical signs of respiratory distress were evident in these animals with individuals also showing increased post-dose salivation, lethargy, hunched posture, dehydration and piloerection. Body weight losses of approximately 5% to 16% (relative to body weights on Day 22) were evident in these animals and it is worth noting that both females had shown clinical signs of respiratory distress on previous days. At necropsy, sloughing of the glandular region in the stomach, thickening of the stomach and discoloration of duodenum were noted for Female 59 with both animals also showing gaseous distension of the gastrointestinal tract. Histopathological findings in Female 59 included minimal hyperkeratosis of the forestomach and minimal centrilobular hepatocellular hypertrophy whilst Female 60 showed slight epithelial hyperplasia and hyperkeratosis of the forestomach with a minimal focal ulcer. Additionally, both females showed moderate to marked atrophy of the thymus and slightly increased granulopoiesis in the bone marrow (sternum).

There were no further unscheduled deaths during the study.

DISCUSSION
The oral (gavage) administration of the test substance to rats, at dose levels of 35, 350 or
600 mg/kg bw/day resulted in premature deaths of three females from the high dose group. One female was found dead on Day 14 relative to the start of dosing; no adverse clinical signs were seen in this animal prior todeath whilst histopathological examination identified marked tracheal ulceration, which was considered to be the major factor contributing to the death. A further two females from this dose group were killed in extremis during the final week of dosing due to clinical signs of respiratory distress and general poor clinical condition. At necropsy, gaseous distension of the gastrointestinal tract was noted for both animals with one female also showing thickening of the stomach, sloughing of the glandular region in the stomach and duodenal discoloration. Histopathological findings in these animals included minimal centrilobular hepatocellular hypertrophy and minimal hyperkeratosis of the forestomach in one animal and slight epithelial hyperplasia and hyperkeratosis of the forestomach with a minimal focal ulcer in the other female. Both females also showed moderate to marked atrophy of the thymus and slightly increased granulopoiesis in the bone marrow (sternum). Taking into account the overall findings from this study, whilst it is clear that the treatment with the test item resulted in the premature demise of these animals, these deaths were deemed to be related to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity.
Thymic atrophy and granulopoiesis in the bone marrow (sternum) seen in two females killed in extremis were also considered to be due to stress, secondary to poor clinical condition, and not to represent a direct effect of treatment with the test item (Everds et al. 2013, Gopinath and Mowat, 2014).

Taking into consideration the overall results from this study, it was considered likely that the premature deaths amongst females from the 600 mg/kg bw/day dose group were related to the irritant properties of the test item and/or gavage-related reflux rather than anindication of its systemic toxicity. For the surviving animals, clinical signs of respiratory distress and increased post-dose salivation, reduction in body weight development or associated food intake and slightly higher water consumption were also deemed not related to the systemic toxicity of the test item. The changes identified during hematological or blood chemistry investigations in males and/or females showed reversibility/recovery after the fourteen day dose-free period whilst increases in the liver and kidneys weights in non-recovery males corresponded to the histopathological alterations in these tissues. Excluding the premature deaths, other effects deemed to be related to the irritant properties of the test item and/or gavage-related reflux, and kidney findings indicative of a male rat-specific condition from the overall assessment, 600 mg/kg bw/day could be established as the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity within the confines of this study.
Body weight and weight changes:
effects observed, non-treatment-related
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 the attached Table 7 (statistically significant differences are indicated). Individual data are given in the attached Appendix 7 and Appendix 8.

During the treatment period, group mean body weight gains for males treated with 600 mg/kg bw/day were generally lower than controls achieving statistical significance in Week 1 (p<0.01). At the end of the dosing period, overall body weight gain for these males was approximately 29% lower than controls. Group mean body weight gains for males treated with 350 mg/kg bw/day was also slightly lower than controls over the last week of dosing but without attaining statistical significance; there was no clear dose-relationship and no intergroup difference was apparent when only non-recovery males were compared.

The females treated with test substance at a dose level of 600 mg/kg bw/day showed lower group mean body weight gains in relation to controls from the second week of dosing achieving statistical significance in Week 2 (p<0.01). Overall body weight gain for the surviving females at the end of the treatment period was approximately 34% lower than controls. Females treated with 350 or 35 mg/kg bw/day also showed slightly lower mean body weight gains during Week 4 but without attaining statistical significance or showing dose-dependence.

During the treatment-free period, males previously given 600 mg/kg bw/day showed slightly higher body weight gains than controls. The corresponding females also exhibited slightly higher or comparable body weight gains in relation to controls during this period.

Taking into consideration the overall results from this study, it was considered likely that the premature deaths amongst females from the 600 mg/kg bw/day dose group were related to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity. For the survivinganimals, clinical signs of respiratory distress and increased post-dose salivation, reduction in body weight development or associated food intake and slightly higher water consumption were also deemed not related to the systemic toxicity of the test item. The changes identified during hematological or blood chemistry investigations in males and/or females showed reversibility/recovery after the fourteen day dose-free period whilst increases in the liver and kidneys weights in non-recovery males corresponded to the histopathological alterations in these tissues. Excluding the premature deaths, other effects deemed to be related to the irritant properties of the test item and/or gavage-related reflux, and kidney findings indicative of a male rat-specific condition from the overall assessment, 600 mg/kg bw/day could be established as the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity within the confines of this study.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Group mean weekly food consumptions are given in the attached Table 8 and are presented graphically in the attached Figure 3 and Figure 4. Weekly food efficiencies are given in the attached Table 9.

During the dose administration period, dietary intake for animals of either sex treated with 600 mg/kg bw/day and females receiving 350 mg/kg bw/day was generally marginally lower than controls. Marginally lower food consumption was also observed for males treated with 350 or 35 mg/kg bw/day during the last week of dosing; however, no intergroup differences were apparent in dietary intake when only non-recovery males were compared.

During the two week recovery period, food consumption for animals of either sex previously dosed with 600 mg/kg bw/day generally remained comparable with controls.

Fluctuations in food conversion efficiency were noted at all dose levels for animals of either sex. These were deemed to be reflective of intergroup differences in body weight gains and/or food intake for these animals.


Taking into consideration the overall results from this study, it was considered likely that the premature deaths amongst females from the 600 mg/kg bw/day dose group were related to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity. For the surviving animals, clinical signs of respiratory distress and increased post-dose salivation, reduction in body weight development or associated food intake and slightly higher water consumption were also deemed not related to the systemic toxicity of the test item. The changes identified during hematological or blood chemistry investigations in males and/or females showed reversibility/recovery after the fourteen day dose-free period whilst increases in the liver and kidneys weights in non-recovery males corresponded to the histopathological alterations in these tissues. Excluding the premature deaths, other effects deemed to be related to the irritant properties of the test item and/or gavage-related reflux, and kidney findings indicative of a male rat-specific condition from the overall assessment, 600 mg/kg bw/day could be established as the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity within the confines of this study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, non-treatment-related
Description (incidence and severity):
Group mean daily water consumptions are given in the attached Table 10.

Gravimetric measurements identified sporadic instances of higher water intake for animals of either sex treated with test substance at a dose level of 600 mg/kg bw/day from Week 2 of dosing with subsequent weekly water consumption values for these animals slightly higher than controls. Males treated with 350 mg/kg bw/day also showed intermittent instances of slightly higher water intake from Week 2 resulting in slightly higher weekly water consumption but the effect was not dose-related. Some instances of slightly higher water consumption were also apparent for females receiving 350 mg/kg bw/day during the last week of dosing. At 35 mg/mg kg/day, water consumption generally remained comparable with controls for animals of either sex.

During the dosing-free period, females previously treated with 600 mg/kg bw/day showed a few instances of slightly higher water consumption at the start but the corresponding values in males were generally similar to controls.

Taking into consideration the overall results from this study, it was considered likely that the premature deaths amongst females from the 600 mg/kg bw/day dose group were related to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity. For the surviving animals, clinical signs of respiratory distress and increased post-dose salivation, reduction in body weight development or associated food intake and slightly higher water consumption were also deemed not related to the systemic toxicity of the test item. The changes identified during hematological or blood chemistry investigations in males and/or females showed reversibility/recovery after the fourteen day dose-free period whilst increases in the liver and kidneys weights in non-recovery males corresponded to the histopathological alterations in these tissues. Excluding the premature deaths, other effects deemed to be related to the irritant properties of the test item and/or gavage-related reflux, and kidney findings indicative of a male rat-specific condition from the overall assessment, 600 mg/kg bw/day could be established as the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity within the confines of this study.
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Hematology
Group mean values and standard deviations for test and control group animals are given in the attached Table 11 (statistically significant differences are indicated). Individual data are given in the attached Appendix 9, Appendix 10 and Appendix 11.
No toxicologically significant effects were detected at any dose level in animals of either sex. At the end of the treatment period, group mean red blood cell count in males from the
600 mg/kg bw/day dose group was statistically significantly higher than controls (p<0.05) but
without clear dose-relationship. The mean corpuscular haemoglobin concentrations in males receiving 600 or 350 mg/kg bw/day were also statistically significantly lower than controls (p<0.01 and p<0.05, respectively) with dose-relationship evident for these groups.
Additionally, males treated with 600 mg/kg bw/day showed statistically significantly lower platelet count in comparison with controls (p<0.05). All individual values for the affected parameters remained within the historical control data ranges whilst the corresponding group mean values in females were comparable with controls.

At the end of the recovery period, there were no statistically significant intergroup differences in animals of either sex previously given 600 mg/kg bw/day for any of the examined hematology parameters.



Blood Chemistry

Group mean values and standard deviations for test and control group animals are given in the attached Table 12 (statistically significant differences are indicated). Individual data are given in the attached Appendix 12 and Appendix 13.

No toxicologically significant effects were detected at any dose level in animals of either sex.

At the end of twenty-eight day treatment period, group mean triglyceride levels in animals of either sex dosed with 350 or 600 mg/mg bw/day were lower than controls attaining statistical significance (p<0.05 or p<0.01) in all instances but for females receiving 350 mg/kg bw/day. One control animal of either sex showed individual values that exceeded the historical control data ranges, but the corresponding values for all test item-treated animals were within these ranges. Group mean cholesterol levels for males and females treated with 600 mg/kg bw/day at the end of the dosing period were also lower than controls but statistical significance was only attained in males (p<0.01); all individual values remained within the background data ranges. At the end of the treatment-free period, the mean triglyceride and cholesterol levels in recovery males and females (respectively) were statistically significantly higher than controls (p<0.05) indicating reversibility.

At 600 mg/kg bw/day, animals of either sex showed statistically significantly lower total protein levels when compared with controls at the end of the treatment period (p<0.05 for males and p<0.01 for females) with these females also showing a statistically significant reduction in albumin concentration (p<0.05); most individual values for the females were


lower than the historical data ranges. Males treated with 350 or 600 mg/kg bw/day also showed statistically significantly higher albumin/globulin ratios at the end of dosing (p<0.01) in a dose-dependent manner with all individual values for the latter exceeding the background data ranges. The corresponding values in recovery males and females previously administered with 600 mg/kg bw/day were comparable with the respective controls.

At the end of the treatment period, group mean plasma concentrations of urea in females treated with 350 or 600 mg/kg bw/day were statistically significantly lower than controls (p<0.05) in a dose-related manner. Plasma levels of creatinine in all test item-treated female dose groups were also statistically significantly lower than controls (p<0.01 at 600 mg/kg bw/day and p<0.05 in the remaining instances) with a dose-relationship apparent between the 350 and 600 mg/kg bw/day groups. Whilst most individual urea values were within the historical data ranges, 3/4 females receiving 600 mg/kg bw/day had creatinine values below the low end of these ranges. Such observations were not evident in males from the corresponding dose groups or recovery animals of either sex previously given 600 mg/kg bw/day.

At the end of the dose administration period, males from all dose groups showed an increase in plasma concentration of glucose achieving statistical significance at 350 or 600 mg/kg bw/day (p<0.05) in comparison with controls. The effect in females was in the opposite direction with statistical significance achieved at all doses (p<0.05). It is worth noting that no dose-relationship was evident in either sex and most individual values remained within the historical control data ranges. The corresponding group mean plasma concentrations in recovery males and females previously given 600 mg/kg bw/day were similar to controls.

At the end of the dosing period, males receiving the test substance at all dose levels showed statistically significant decreases in aspartate aminotransferase activities with respect to controls (p<0.01 at 600 mg/kg bw/day and p<0.05 in the remaining instances). A dose- relationship was apparent, but a similar effect was not evident in the corresponding females. Whilst most individual values from test-item treated males were within control data ranges, 2/5 control males showed values that exceeded these ranges and this finding was considered likely to be incidental. In recovery males previously treated with 600 mg/kg bw/day, group mean aspartate aminotransferase activities were similar to controls.

At all dose levels, males showed statistically significantly lower plasma concentrations of inorganic phosphorus relative to controls at the end of the dose administration period (p<0.05) but without any dose-relationship. The corresponding values in females were similar to controls and individual values from all test item-treated males also remained within the historical control data ranges. Females treated with 350 or 600 mg/kg bw/day also showed statistically significantly higher plasma levels of sodium at the end of dosing (p<0.01 and p<0.05 at 600 or 350 mg/kg bw/day, respectively) with some individual values exceeding the background data ranges. Males from these dose groups showed statistically significant increases in calcium concentrations (p<0.05) but the effect in females treated with 600 mg/kg bw/day was in the opposite direction (p<0.05). These parameters in recovery animals of either sex previously administered with 600 mg/kg bw/day were comparable with controls at the end of the fourteen day dosing-free period.



Taking into consideration the overall results from this study, it was considered likely that the premature deaths amongst females from the 600 mg/kg bw/day dose group were related to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity. For the surviving animals, clinical signs of respiratory distress and increased post-dose salivation, reduction in body weight development or associated food intake and slightly higher water consumption were also deemed not related to the systemic toxicity of the test item. The changes identified during hematological or blood chemistry investigations in males and/or females showed reversibility/recovery after the fourteen day dose-free period whilst increases in the liver and kidneys weights in non-recovery males corresponded to the histopathological alterations in these tissues. Excluding the premature deaths, other effects deemed to be related to the irritant properties of the test item and/or gavage-related reflux, and kidney findings indicative of a male rat-specific condition from the overall assessment, 600 mg/kg bw/day could be established as the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity within the confines of this study.
Clinical biochemistry findings:
not examined
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
A summary of urinalytical findings is given in the attached Table 13 (statistically significant differences are indicated where appropriate). Individual data are given in the attached Appendix 14.

During the last week of dosing, the mean urine volume in females appeared to be higher than controls and was associated with lower specific gravity. The intergroup differences were, however, not statistically significant and the corresponding values in non-recovery males from all dose groups and recovery animals of either sex previously receiving 600 mg/kg bw/day were generally similar to controls. Any other parameters examined as part of the urinalysis process, including, presence of proteins, ketones and hemoglobin were similar across all dose groups including controls.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Functional Observations
A summary incidence of behavioral assessment observations is given in the attached Table 2 and group mean behavioral assessment scores are given in the attached Table 3. Individual data are given in the attached Appendix 2 and Appendix 3. Group mean functional performance test values and standard deviations are given in the attached Table 4 (statistically significant differences are indicated). Individual values are given in the attached Appendix 4 and Appendix 5. Group mean sensory reactivity assessments are given in the attached Table 5. Individual responses are given in the attached Appendix 6.

Behavioral Assessments
There were no changes in the assessed behavioral parameters considered to be related to treatment with the test substance at any dose level.

Functional Performance Tests
There were no treatment-related changes in functional performance at any dose level.

Males treated with the test substance at 600 mg/kg bw/day showed statistically significant intergroup differences for hindlimb grip strength during tests 2 and 3, but the corresponding values were in the opposite directions and these differences were considered to be incidental.

Sensory Reactivity Assessments
There were no treatment-related changes detected at any dose level.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Group mean absolute and relative organ weights and standard deviations for test and control group animals are presented in the attached Table 15 (statistically significant differences are indicated). Individual data are given in the attached Appendix 16 and Appendix 17.

At the end of the dosing period, group mean absolute and body weight-related liver weights in males treated with 350 or 600 were statistically significantly higher than controls (p<0.01). A dose-relationship was evident and 3/5 males from the high dose group showed body weight-related liver weights exceeding the background data ranges. Males from these groups also showed dose-related statistically significant increases in absolute and body weight- related kidneys weights (p<0.01) with most individual body weight-related values for either group exceeding the historical control data ranges. The corresponding liver and kidneys weights in female dose groups at the end of the treatment period and recovery animals of


either sex previously given 600 mg/kg bw/day at the end of the treatment-free period were comparable with controls.

At the end of the treatment period, females receiving 600 mg/kg bw/day showed statistically significant increases in absolute and body weight-related brain weights in relation to controls (p<0.05). The absolute and body weight-related pituitary weights in these females were also statistically significantly lower than controls (p<0.01). Whilst some individual control values for either parameter were outside the historical control data ranges, the corresponding values from all test item-treated females were within these ranges. At the end of recovery period, these parameters in females previously receiving 600 mg/kg bw/day were similar to controls. In the absence of any histopathology correlates, the above findings were deemed unlikely to be treatment-related.

At the end of the dosing period, males from the 35 mg/kg bw/day dose group showed statistically significantly higher thyroid/parathyroid weights when compared with controls. The corresponding values in males receiving 350 or 600 mg/kg bw/day were similar to controls and this observation was considered likely to be incidental. Recovery males previously treated with 600 mg/kg bw/day also showed statistically significant increases in these weights at the end of the treatment-free period but in the absence of any histopathology correlates in these recovery animals, this finding was considered to be of no toxicological significance.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
A summary incidence of necropsy findings is given in the attached Table 14. Individual data are given in the attached Appendix 15.

At the end of the twenty-eight day treatment period, all males and 3/4 surviving females treated with 600 mg/kg bw/day showed sloughing of the non-glandular region of the stomach. The remaining surviving female showed sloughing of the glandular region of the stomach.

At 350 mg/kg bw/day, Male 25 was observed with reddened lungs which microscopically corresponded to multifocal alveolar hemorrhages, a change considered to be agonal in nature. Small testes and epididymides noted in Male 13 from the 35 mg/kg bw/day dose group corresponded to bilateral testicular tubular degeneration/atrophy and reduced luminal sperm in the epididymides. These observations were considered to be unrelated to treatment with the test item.

At the end of the two week recovery period, there were no macroscopic observations at terminal necropsy for any of the surviving animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
A complete histopathology phase report is presented in the attached Annex 1.

Findings considered to be related to treatment with the test substance were seen in the forestomach, trachea, liver and thyroid glands of males and surviving females given
600 mg/kg bw/day; the findings in the liver and thyroid glands were also observed in males receiving 350 mg/kg bw/day. Males receiving 600 mg/kg bw/day also showed treatment- related findings in the kidneys and adrenal glands, with the kidney findings extending to males from the 350 mg/kg bw/day dose group. The details are given below:

Fore stomach: Minimal to slight, focally extensive to diffuse epithelial hyperplasia and hyperkeratosis of the fore stomach was detected in most surviving animals receiving the test substance at 600 mg/kg bw/day. Similar findings were not observed in recovery animals of either sex previously treated with 600 mg/kg bw/day after a fourteen day treatment-free period.

Trachea: Moderate ulceration of the respiratory epithelium, with underlying inflammation, was observed in a male given 600 mg/kg bw/day. Evidence of epithelial degeneration/ regeneration with or without associated inflammation was also seen in other males and females receiving the test substance at 600 mg/kg bw. These findings were no longer present in the recovery animals of either sex previously treated with 600 mg/kg bw/day.

Liver: Minimal to slight centrilobular hepatocellular hypertrophy was observed in most males receiving the test substance at 350 or 600 mg/kg bw/day, with an increased severity in males given 600 mg/kg bw/day. Minimal centrilobular hepatocellular hypertrophy was also


observed in some females treated at 600 mg/kg bw/day. Following the treatment-free period, minimal centrilobular hepatocellular hypertrophy was observed in 1/5 male previously treated with the test substance at 600 mg/kg bw/day whilst the surviving females showed complete recovery.

Thyroid glands: Minimal hypertrophy of the follicular epithelium of the thyroid glands was seen in some male rats treated with the test substance at 350 or 600 mg/kg bw/day, with a similar incidence, and in some female rats given 600 mg/kg bw/day. At the end of the two week dose-free period, animals of either sex previously receiving 600 mg/kg bw/day showed complete recovery.

Kidneys: Males receivingthe test item at 350 or 600 mg/kg bw/day showed increased incidence and severity of accumulation of hyaline droplets in cortical tubules, in association with tubular basophilia and a few granular casts. These findings increased in severity in males given the high dose. Partial recovery from the changes seen in males at the end of the treatment with the test substance at 600 mg/kg bw/day was observed in recovery males.
Accumulation of hyaline droplets in cortical tubules was no longer present, but minimal tubular basophilia and a few granular casts could be observed.

Adrenal glands: Minimal hypertrophy of the zona glomerulosa was seen in some males treated with the test substance at 600 mg/kg bw/day. These changes were no longer evident in males previously dosed with 600 mg/kg bw/day at the end of the recovery period.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
A complete histopathology phase report is presented in the attached Annex 1.

Findings considered to be related to treatment with the test substance were seen in the forestomach, trachea, liver and thyroid glands of males and surviving females given
600 mg/kg bw/day; the findings in the liver and thyroid glands were also observed in males receiving 350 mg/kg bw/day. Males receiving 600 mg/kg bw/day also showed treatment- related findings in the kidneys and adrenal glands, with the kidney findings extending to males from the 350 mg/kg bw/day dose group. The details are given below:

Forestomach: Minimal to slight, focally extensive to diffuse epithelial hyperplasia and hyperkeratosis of the forestomach was detected in most surviving animals receiving the test substance at 600 mg/kg bw/day. Similar findings were not observed in recovery animals of either sex previously treated with 600 mg/kg bw/day after a fourteen day treatment-free period.

Trachea: Moderate ulceration of the respiratory epithelium, with underlying inflammation, was observed in a male given 600 mg/kg bw/day. Evidence of epithelial degeneration/ regeneration with or without associated inflammation was also seen in other males and females receiving the test substance at 600 mg/kg bw. These findings were no longer present in the recovery animals of either sex previously treated with 600 mg/kg bw/day.

Liver: Minimal to slight centrilobular hepatocellular hypertrophy was observed in most males receiving the test substance at 350 or 600 mg/kg bw/day, with an increased severity in males given 600 mg/kg bw/day. Minimal centrilobular hepatocellular hypertrophy was also


observed in some females treated at 600 mg/kg bw/day. Following the treatment-free period, minimal centrilobular hepatocellular hypertrophy was observed in 1/5 male previously treated with the test substance at 600 mg/kg bw/day whilst the surviving females showed complete recovery.

Thyroid glands: Minimal hypertrophy of the follicular epithelium of the thyroid glands was seen in some male rats treated with the test substance at 350 or 600 mg/kg bw/day, with a similar incidence, and in some female rats given 600 mg/kg bw/day. At the end of the two week dose-free period, animals of either sex previously receiving 600 mg/kg bw/day showed complete recovery.

Kidneys: Males receiving the test substance at 350 or 600 mg/kg bw/day showed increased incidence and severity of accumulation of hyaline droplets in cortical tubules, in association with tubular basophilia and a few granular casts. These findings increased in severity in males given the high dose. Partial recovery from the changes seen in males at the end of the treatment with the test substance at 600 mg/kg bw/day was observed in recovery males.
Accumulation of hyaline droplets in cortical tubules was no longer present, but minimal tubular basophilia and a few granular casts could be observed.

Adrenal glands: Minimal hypertrophy of the zona glomerulosa was seen in some males treated with the test substance at 600 mg/kg bw/day. These changes were no longer evident in males previously dosed with 600 mg/kg bw/day at the end of the recovery period.
Details on results:
The oral (gavage) administration of the test substance to rats, at dose levels of 35, 350 or
600 mg/kg bw/day resulted in premature deaths of three females from the high dose group. One female was found dead on Day 14 relative to the start of dosing; no adverse clinical signs were seen in this animal prior to death whilst histopathological examination identified marked tracheal ulceration, which was considered to be the major factor contributing to the death. A further two females from this dose group were killed in extremis during the final week of dosing due to clinical signs of respiratory distress and general poor clinical condition. At necropsy, gaseous distension of the gastrointestinal tract was noted for both animals with one female also showing thickening of the stomach, sloughing of the glandular region in the stomach and duodenal discoloration. Histopathological findings in these animals included minimal centrilobular hepatocellular hypertrophy and minimal hyperkeratosis of the forestomach in one animal and slight epithelial hyperplasia and hyperkeratosis of the forestomach with a minimal focal ulcer in the other female. Both females also showed moderate to marked atrophy of the thymus and slightly increased granulopoiesis in the bone marrow (sternum). Taking into account the overall findings from this study, whilst it is clear that the treatment with the test item resulted in the premature demise of these animals, these deaths were deemed to be related to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity.
Thymic atrophy and granulopoiesis in the bone marrow (sternum) seen in two females killed in extremis were also considered to be due to stress, secondary to poor clinical condition, and not to represent a direct effect of treatment with the test item (Everds et al. 2013, Gopinath and Mowat, 2014).

The surviving males and females receiving the test sustance at a dose level of 600 mg/kg bw/day exhibited sporadic episodes of respiratory distress from Weeks 2 or 3 of dosing, respectively, which extended into the recovery period for individual males previously treated with 600 mg/kg bw/day. It is worth noting, however, that no clinical signs were observed for any of the recovery animals towards the end of the fourteen day treatment-free period, indicating recovery. Findings in the trachea which included moderate ulceration and/or areas of epithelial degeneration/regeneration in some animals along with changes in the forestomach of high dose males and females after the administration period were suggestive of gavage- related reflux due to an irritant effect of treatment on the forestomach. Sloughing of the forestomach reported at necropsy correlated histopathologically with hyperkeratosis. The lack of tracheal lesions in the remaining animals from the 600 mg/kg bw/day dose group, however, does not rule out the possibility that similar changes might be present in other sites or other regions in the upper respiratory tract, which were not evaluated in this study, which could explain the audible respiration (Damsch et al, 2011). Such histopathological lesions were no longer apparent in any of the animals previously given 600 mg/kg bw/day toward the end of the treatment-free period indicating recovery. Intermittent instances of increased post- dose salivation observed in males and females administered with 350 or 600 mg/kg bw/day during the treatment period appeared to correspond to increased water consumption for these animals. Such observations are common in this type of study and usually reflect unpalatability and/or an irritant nature of the test item formulation and are generally deemed to be of no toxicological importance. The surviving males and females given 600 mg/kg bw/day also showed lower overall body weight gains at the end of the twenty-eight dose administration period. This finding was associated with lower dietary intake for these animals with females from the 350 mg/kg bw/day dose group also showing lower food consumption. Taking into account the macroscopic observations and histopathological findings in the forestomach and trachea from animals of either sex treated with 600 mg/kg bw/day, these observations were also considered to be related to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity.

Hematological investigations at the end of twenty-eight day dosing period identified statistically significant intergroup differences in some red blood cell parameters and platelet counts in males but all individual values remained within the historical control data ranges. Whilst these findings may be suggestive of minor variations in blood cell parameters, it is worth noting that similar differences were not evident in females. Recovery was apparent in males previously treated with 600 mg/kg bw/day following the fourteen day treatment-free period and, in the absence of any associated histopathological findings in the spleen or bone marrow, these observations were considered not to be of any toxicological significance.

The centrilobular hepatocellular hypertrophy seen in the liver at the end of the treatment period in 350 mg/kg bw/day males and 600 mg/kg bw/day males and females correlated with the liver weight increases reported in males at necropsy. Hepatocyte enlargement is commonly observed in the rodent liver following the administration of xenobiotics and in the absence of any degenerative or inflammatory changes, is generally considered to be an adaptive change or reflect a metabolic disturbance that is a result of hepatic enzyme induction. Blood chemistry evaluations in the surviving males and/or females treated with 600 mg/kg bw/day at the end of the treatment period showed various other changes including lower plasma concentrations of triglycerides, cholesterol, total protein, albumin (females only), urea (females only), higher creatinine (females only) and albumin/globulin ratio (males only) with respect to controls. Some of these changes were also apparent in males and/or females treated with 350 mg/kg/day but by the end of the fourteen day recovery period, reversibility/recovery was evident. When compared with controls, males receiving the test item at all dose levels showed an increase in glucose concentration at the end of the 28 day dose period, but the corresponding values in test item-treated females were in the opposite direction; the corresponding group mean values in recovery animals of either sex previously receiving 600 mg/kg bw/day were similar to controls. Variations in these plasma markers may well be indicative of fluctuations in metabolic processes associated with test item detoxification and taken together these observations were deemed not to represent an adverse effect of treatment. Only 1/5 males previously treated with 600 mg/kg bw/day still showed centrilobular hypertrophy in the liver at a minimal level after the recovery period but there was no morphological evidence to assume that this would not reverse completely over time. Thyroid follicular hypertrophy, seen in animals at the end of the dose administration period was also considered to be adaptive and secondary to centrilobular hypertrophy. The most likely explanation for this alteration is a disruption in thyroid hormone synthesis, transport or metabolism probably associated with liver enzyme induction. Since the rat thyroid gland is markedly more sensitive than humans in its response to xenobiotics (Chandra et al., 2013), it is reasonable to conclude that the minor difference seen in this study would not carry


significant risk to humans. These observations were no longer evident in animals of either sex previously treated with 600 mg/kg bw/day following the treatment-free period.

Males receiving 350 or 600 mg/kg bw/day showed higher kidney weights in a dose- dependent manner at the end of the dosing period; these intergroup differences were no longer evident in recovery males previously given 600 mg/kg bw/day after the fourteen day treatment-free period. Histopathological examination of the kidneys from non-recovery males revealed accumulation of hyaline droplets at 350 or 600 mg/kg bw/day, which correlated with the organ weight increases. This change was associated with other histologic features of pathology (tubular basophilia and granular casts), which were altogether suggestive of male rat-specific alpha-2u-globulin nephropathy (Frazier et al., 2012). Given that this is a species-specific condition to the male rat, it was considered to be of no relevance to human exposure. After the fourteen day recovery period, none of the males previously given 600 mg/kg bw/day showed accumulation of hyaline droplets, indicating reversibility of the primary cause of the condition. A few granular casts and minimal tubular basophilia were still present, but in the absence of hyaline droplet accumulation, it was thought likely that given sufficient recovery time these too would resolve. Statistically significant variations in urea and creatinine seen in test item-treated females at the end of the dose administration period were not associated with any treatment-related kidney findings in females from the 600 mg/kg bw/day dose group and were deemed to be of no toxicological bearing.

In the adrenal glands, the minimal hypertrophy of the zona glomerulosa observed in some males given 600 mg/kg bw/day at the end of the treatment period suggested changes in aldosterone production. This was unassociated with histologic features of toxicity and was therefore considered a non-adverse functional change within the confines of this study. This may also correlate with increased water consumption and electrolyte imbalance identified in non-recovery males at blood chemistry evaluations but these findings were considered to be of no toxicological significance.

Taking into consideration the overall results from this study, it was considered likely that the premature deaths amongst females from the 600 mg/kg bw/day dose group were related to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity. For the surviving animals, clinical signs of respiratory distress and increased post-dose salivation, reduction in body weight development or associated food intake and slightly higher water consumption were also deemed not related to the systemic toxicity of the test item. The changes identified during hematological or blood chemistry investigations in males and/or females showed reversibility/recovery after the fourteen day dose-free period whilst increases in the liver and kidneys weights in non-recovery males corresponded to the histopathological alterations in these tissues. Excluding the premature deaths, other effects deemed to be related to the irritant properties of the test item and/or gavage-related reflux, and kidney findings indicative of a male rat-specific condition from the overall assessment, 600 mg/kg bw/day could be established as the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity within the confines of this study.
Key result
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Please see remarks section
Critical effects observed:
no
Lowest effective dose / conc.:
600 mg/kg bw/day (nominal)
System:
other:
Organ:
other: increases in liver kidneys weights non recovery males corresponded to histopathological alterations in these tissues other effects related to irritant prop of TI andor gavage related reflux & kidney findings indicative of a male rat-specific condition
Treatment related:
no
Dose response relationship:
no
Relevant for humans:
no
Conclusions:
The oral (gavage) administration of the test substance to Wistar Han™:RccHan™:WIST strain rats, at dose levels of 35, 350 or 600 mg/kg bw/day, resulted in premature deaths of three females from the high dose group. These deaths were considered to be related to the irritant properties of the test item and gavage-related reflux rather than an indication of its systemic toxicity.

The surviving animals of either sex from the 600 mg/kg bw/day dose group showed intermittent episodes of respiratory distress which correlated with tracheal lesions in some of the non-recovery animals from this dose group. Animals of either sex from this dose group also showed hyperplasia and hyperkeratosis of the forestomach, an indication of stomach irritation, which was deemed likely to have contributed to lower food intake and body weight gains and possibly higher water consumption. Animals of either sex previously receiving 600 mg/kg bw/day showed evidence of recovery after a fourteen day treatment-free period and these findings were considered to be due to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity. The findings in male kidneys from the 350 or 600 mg/kg bw/day dose groups were considered to be indicative of alpha-2u-globulin nephropathy, a condition specific to the male rat.

Excluding the above from the overall assessment, 600 mg/kg bw/day could be established as the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity within the confines of this study.
Executive summary:

Introduction

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

            Commission Directive 96/54/EC (Method B7).

            The Japanese Ministry of Economy Trade and Industry (METI), Ministry of Health, Labor and Welfare (MHLW) and Ministry of the Environment (MOE) Guidelines of 21 November 2003 for a twenty-eight day repeat dose oral toxicity study as required by the Law Concerning the Evaluation of Chemical Substances and Regulation of their Manufacture, etc (Chemical Substance Control Law) 1973 of Ministry of International Trade and Industry (MITI) amended 2004.

            The OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).

            USA Environmental Protection Agency (EPA) Health Effects Test Guidelines, OPPTS 870.3050 Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.

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 five male and five female Wistar Han™:RccHan™:WIST strain rats, for up to twenty-eight consecutive days, at dose levels of 35, 350 or 600 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (600 mg/kg bw/day) or the vehicle alone for up to twenty-eight consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, body weight change, food and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals towards the end of the treatment period and for all recovery group animals towards the end of the treatment-free period.

 

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

Results Mortality

There were three unscheduled deaths during the study, all belonging to the 600 mg/kg bw/day dose group. Female 37 was found dead on Day 14 with no previous adverse clinical signs.

The major factor contributing to death was marked tracheal ulceration. Females 59 and 60 showed signs of respiratory distress along with poor clinical condition and actual body weight losses, and were killed for welfare reasons on Days 27 and 23, respectively. At necropsy, macroscopic findings for these animals were confined to gastrointestinal tract whilst histopathological observations consisted of minimal centrilobular hepatocellular hypertrophy and minimal hyperkeratosis of the forestomach in Female 59 and slight epithelial hyperplasia and hyperkeratosis of the forestomach with a minimal, focal ulcer in Female 60. These deaths were considered to be related to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity. Both females also showed moderate to marked atrophy of the thymus and slightly increased granulopoiesis in the bone marrow (sternum), observations deemed to be stress-related and secondary to poor clinical condition, and not to represent a direct systemic effect of treatment with the test item.

Clinical Observations

At 600 mg/kg bw/day, sporadic episodes of respiratory signs were seen in surviving animals of either sex during the treatment period. These observations persisted in some recovery males previously treated at this dose level for a few days into the fourteen day treatment-free period. At the end of the dosing period, these findings corresponded to microscopic lesions in the trachea, which included moderate ulceration and/or areas of epithelial degeneration/ regeneration in some animals. These observations were deemed to reflect gavage-related reflux due to the irritant properties of the test item and not an indication of its systemic toxicity. Instances of increased post-dose salivation seen in animals of either sex treated with 600 or 350 mg/kg bw/day were also considered to be due to an irritant nature of the test item formulations and of no toxicological significance.

Behavioral Assessment

Behavioral assessment scores across the treated animals of either sex remained similar to the respective controls.

Functional Performance Tests

There were no treatment-related changes in functional performance at any dose level.

Sensory Reactivity Assessments

Sensory reactivity evaluation did not identify any effect of treatment with the test item.

 Body Weight

 

During the dosing period, weekly body weight gains in animals of either sex treated with 600 mg/kg bw/day were generally lower than controls (from Week 2 in females). This resulted in lower overall body weight gains in these animals at the end of the treatment period; approximately 29% lower in males and 34% lower in females with respect to controls. Taking into view the histopathological indications of the irritation in the forestomach and trachea, these findings were considered likely to to be due to the irritant properties of the test item rather than a direct effect of treatment on body weight development. During the fourteen day dosing-free phase of the study, recovery was evident with both males and females generally showing comparable or slightly higher body weight gains than controls.

 

Food Consumption

During the twenty-eight day dose administration phase of the study, weekly dietary intake in animals of either sex given 600 mg/kg bw/day and females receiving 350 mg/kg bw/day was generally marginally lower than controls. Histopathological lesions in the forestomach from animals treated with 600 mg/kg bw/day showed signs of irritation and these observations were considered to be linked to the irritant properties of the test item rather than a direct effect of treatment with the test item. During the fourteen day treatment-free phase of the study, recovery was evident with both males and females generally generally showing similar food consumption to controls.

At all dose levels, fluctuations in food conversion efficiency were evident for animals of either sex and were considered to reflect intergroup differences in body weight gains and/or food intake for these animals.

Water Consumption

When compared with controls, sporadic instances of slightly higher water intake were evident for animals of either sex treated with 600 mg/kg bw/day and males given 350 mg/kg bw/day from Week 2 of dosing. There was also an indication of slightly higher water consumption in females treated with 350 mg/kg bw/day during the last week of dosing. Taken together with signs of increased post-dose salivation, these findings may be due to an irritant nature of the test item formulations rather than an indication of its systemic toxicity.

Hematology

No toxicologically significant effects were detected at any dose level in animals of either sex.

Blood Chemistry

No toxicologically significant effects were detected at any dose level in animals of either sex.

 

Urinalysis

No toxicologically significant alterations were detected in the urinalysis parameters examined for animals of either sex at dose levels up to 600 mg/kg bw/day.

Necropsy

At the end of the dose administration period, treatment-related macroscopic findings at necropsy were confined to the 600 mg/kg bw/day dose group and included sloughing of the non-glandular region of the stomach with one female also showing sloughing of the glandular region of the stomach. These findings were considered to be related to the irritant properties of the test item rather than an indication of its systemic toxicity.

Organ Weights

No toxicologically significant changes were detected at any dose level in animals of either sex.

Histopathology

At the end of the twenty-eight days of dosing, microscopic examination of the selected tissues from animals of either sex revealed the following treatment-related findings:

Forestomach: At 600 mg/kg bw/day, minimal to slight, focally extensive to diffuse epithelial hyperplasia and hyperkeratosis of the forestomach was observed in most surviving animals.

Trachea: At 600 mg/kg bw/day, moderate ulceration of the respiratory epithelium, with underlying inflammation, was seen in one male. Evidence of epithelial degeneration/ regeneration with or without associated inflammation was also seen in other males and females from this dose group.

Liver: Animals of either sex receiving 600 mg/kg bw/day and males from the 350 mg/kg bw/day dose group showed minimal to slight centrilobular hepatocellular, with an increased severity in males given 600 mg/kg bw/day.

Thyroid glands: Minimal hypertrophy of the follicular epithelium of the thyroid glands was observed in some male rats treated with the test item at 350 or 600 mg/kg bw/day, with a similar incidence, and in some female rats given 600 mg/kg bw/day.

Kidneys: At 350 or 600 mg/kg bw/day, males exhibited an increased incidence and severity of accumulation of hyaline droplets in cortical tubules, in association with tubular basophilia and a few granular casts. These findings increased in severity in males from the 600 mg/kg bw/day dose group.

Adrenal glands: At 600 mg/kg bw/day, minimal hypertrophy of the zona glomerulosa was seen in some males.

The findings in the forestomach and trachea were suggestive of irritation caused by the test item which was deemed likely to have resulted in gavage-related reflux. The liver and

thyroid glands alterations were considered to be of an adaptive nature whilst those in the adrenal glands were also deemed not to represent an adverse effect of treatment.

Histopathological observations noted in male kidneys were deemed to be indicative of alpha- 2u-globulin nephropathy, a condition specific to the male rat, and of no relevance to human risk assessment.

At the end of the fourteen day treatment-free period, complete recovery was evident for microscopic alterations in the forestomach, trachea, thyroid glands and adrenal glands seen at the end of the dosing period. Whilst females previously treated with 600 mg/kg bw/day also no longer showed any evidence of liver changes, 1/5 males exhibited signs of minimal centrilobular hepatocellular hypertrophy, but there was no morphological evidence to assume that this would not reverse completely over time. Partial recovery for microscopic findings in the male kidneys was also apparent with accumulation of hyaline droplets no longer evident. A few granular casts and minimal tubular basophilia were still present, but with reversibility of the primary cause of the condition, i.e. hyaline droplet accumulation, it was deemed likely that given sufficient recovery time these too would resolve.

Conclusion

The oral (gavage) administration of the test substance to Wistar Han™:RccHan™:WIST strain rats, at dose levels of 35, 350 or 600 mg/kg bw/day, resulted in premature deaths of three females from the high dose group. These deaths were considered to be related to the irritant properties of the test item and gavage-related reflux rather than an indication of its systemic toxicity.

The surviving animals of either sex from the 600 mg/kg bw/day dose group showed intermittent episodes of respiratory distress which correlated with tracheal lesions in some of the non-recovery animals from this dose group. Animals of either sex from this dose group also showed hyperplasia and hyperkeratosis of the forestomach, an indication of stomach irritation, which was deemed likely to have contributed to lower food intake and body weight gains and possibly higher water consumption. Animals of either sex previously receiving 600 mg/kg bw/day showed evidence of recovery after a fourteen day treatment-free period and these findings were considered to be due to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity. The findings in male kidneys from the 350 or 600 mg/kg bw/day dose groups were considered to be indicative of alpha-2u-globulin nephropathy, a condition specific to the male rat.

Excluding the above from the overall assessment, 600 mg/kg bw/day could be established as the ‘No Observed Adverse Effect Level’ (NOAEL) for systemic toxicity within the confines of this study.

Endpoint:
repeated dose toxicity: oral, other
Remarks:
Seven Day Repeated Dose Oral (Gavage) Range-Finding Toxicity Study in the Rat
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Experimental Starting Date: 09 December 2015 Experimental Completion Date: 06 January 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: The study was designed to provide information for further repeated dose toxicity studies.
Version / remarks:
The study was designed and conducted to cause the minimum suffering or distress to the animals consistent with the scientific objectives
The study was conducted in accordance with the UK Home Office Guidance document on Regulatory Toxicology and Safety Evaluation Studies and the OECD guidance document on recognition, assessment and use of clinical signs as humane endpoints for experimental animals used in safety evaluation.
Deviations:
no
Principles of method if other than guideline:
The study was performed according to the Study Plan and was designed to provide information for further repeated dose toxicity studies. Following the preliminary sighting work, the test item was administered to the Wistar Han™:RccHan™:WIST strain rat for a period of seven consecutive days at dose levels of 300, 600 or 1000 mg/kg bw/day. A control group was dosed with vehicle alone (Arachis oil BP).

The dose levels were chosen in consultation with the Sponsor and were based on available toxicity information including preliminary toxicity work performed as part of this study (see Section 3.3.3). The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item. The results of the study will aid dose level selection for subsequent studies.

The in-life phase of the seven day dose-range finding phase of the study was performed between 30 December 2015 (start of treatment) and 06 January 2016 (final necropsy).
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Wistar Han™:RccHan™:WIST strain rat
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

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 for the main phase of the study were acclimatized for fourteen days during which time their health status was assessed. A total of twenty-four animals (twelve males and twelve females) were accepted into the main study. At the start of treatment the main studymales weighed 196 to 235g, the females weighed 153 to 178g, and were approximately seven to nine weeks old.
Sex:
male/female
Details on test animals and environmental conditions:
Animal Care and Husbandry

The main study animals were housed in groups of three 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, (formerly known as Harlan Laboratories Ltd.) Oxon, UK) was used. A certificate of analysis of the batch of diet used is given in Annex 1. 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 was 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 main study animals were allocated to dose groups using a randomization procedure based on stratified body weights and the group mean body weights were then determined to ensure similarity between the dose groups. The animals were uniquely identified within the study, by an ear punch system routinely used in these laboratories.

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
Test Item Preparation and Analysis
For the purpose of this study the test item was prepared at the appropriate concentrations as a solution in Arachis oil BP.

The test item was administered within two hours of it being formulated. It is assumed that the formulation was stable for this duration.

No analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation as part of this study. This is an exception with regard to GLP and has been reflected in the GLP compliance statement.

Dose Administration

In order to increase the toxicity information, preliminary toxicity work was undertaken, during which the test item was administered by gavage to one male and one female Wistar Han™:RccHan™:WIST strain rats for one day, at a dose level of 500 mg/kg bw/day, using a dose volume of 4 mL/kg (vehicle: Arachis oil BP). As there were no clinical signs or any detrimental effect on body weight development at this dose level, a further one male and one female rat were dosed, at a dose level of 1000 mg/kg bw/day for up to three consecutive days. The female showed increased salivation and respiratory signs soon after dosing on Day 1 and was killed in extremis on the following day (prior to dosing) due to a further decline in clinical condition and marked body weight loss. At necropsy, macroscopic findings for this female were confined to reddened lungs and gaseous distention of the stomach and small/large intestine. As this premature death was was considered likely to be due to dosing trauma on the first day of treatment, the female previously treated with 500 mg/kg bw/day was dosed with 1000 mg/kg bw/day for three consecutive days. Clinical signs for the surviving animals from this dose level were confined to noisy respiration observed in the male on Day 4 and whilst the female showed a body weight gain of 7g over Days 1 to 4, the corresponding male showed a body weight loss of 12g over this period. At necropsy, macroscopic findings for the male included reddened lungs and gaseous distension of the small/large intestine whilst the female showed patchy reddened lungs and a thickened non- glandular region in the stomach. Based on these observations, a dose sequence of 300, 600 and 1000 mg/kg bw/day was considered suitable for use in the main phase of this study. All data from this preliminary work will be retained in the study records but will not be discussed further in this report.




Following preliminary work, animals were allocated to treatment groups for the seven day range-finding part of the study as follows:



Treatment Group Dose Level (mg/kg bw/day) Treatment Volume (mL/kg) Concentration (mg/mL) Animal Numbers
Male Female
Control 0 4 0 3 (1-3) 3 (4-6)
Low 300 4 75 3 (7-9) 3 (10-12)
Intermediate 600 4 150 3 (13-15) 3 (16-18)
High 1000 4 250 3 (19-21) 3 (22-24)
The numbers in parentheses ( ) show the individual animal numbers allocated to each treatment group.

The test item was administered daily, for seven 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 body weight and was adjusted on Days 3 and 5.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Seven day range-finding study
Frequency of treatment:
The test item was administered daily, for seven consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe.
The volume of test and control item administered to each animal was based on the most recent body weight and was adjusted on Days 3 and 5.
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Low Treatment Group
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Remarks:
Intermediate Treatment Group
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High Treatment Group
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Details on study design:
Following preliminary sighting work, the test item was administered by gavage to three groups, each of three male and three female Wistar Han™:RccHan™:WIST strain rats, for seven consecutive days, at dose levels of 300, 600 or 1000 mg/kg bw/day. A control group of three males and three females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, body weight change, dietary intake and water consumption were monitored during the main phase of the study. All animals were subjected to gross necropsy examination.
Positive control:
No
Observations and examinations performed and frequency:
Clinical Observations
All animals were examined for overt signs of toxicity, ill health or behavioral change immediately before dosing, up to thirty minutes after dosing and one hour after dosing. Additional observations were also made four hours following dosing (not at weekends or public holidays). All observations were recorded.

Body Weight
Individual body weights were recorded on Days 1, 3, 5 and 8.

Food Consumption
Food consumption was recorded for each cage group for Days 1 to 3, 3 to 5 and 5 to 8. Food conversion efficiency was calculated retrospectively.

Water Consumption
Water intake was measured and recorded daily for each cage group.
Sacrifice and pathology:
Necropsy
On completion of the dosing period, all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination and subjected to an internal and external macroscopic examination; see deviation from Study Plan. No tissues were retained.
Other examinations:
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.

Major Computerized Systems
The following computerized systems were used on the study: Provantis Version 6.4
Delta Controls - ORCAview
Clinical signs:
no effects observed
Description (incidence and severity):
Clinical Observations

A summary incidence of daily clinical observations is given in Table 1 please see the attached “tables & figures” . Individual data are presented in Appendix 1please see the attached “tables & figures” .

Throughout the seven day treatment period, there were no clinical signs considered to be related to the toxicity of the test item at any dose level.

At 600 or 1000 mg/kg bw/day, increased post-dose salivation was seen in animals of either sex from Days 4 and 1 of dosing, respectively. Observations of this nature are commonly observed following the oral administration of an unpalatable or slightly irritant test item formulation and, were therefore, considered to be of no toxicological importance.

There were no clinical signs for any of the remaining animals throughout the study.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Group mean body weights and standard deviations are given in Table 2 and are presented graphically in Figure 1 and Figure 2. Group mean body weight gains and standard deviations are given in Table 3. Individual data are given in Appendix 2 and Appendix 3 please see the attached “tables & figures” .

At 600 or 1000 mg/kg bw/day, males showed a dose-related reduction in group mean body weight gains throughout the treatment period with the high dose animals showing mean body weight losses from Day 3 which resulted in an overall group mean body weight loss of 5.7g for this group of males. For males receiving 600 mg/kg bw/day, the reductions in periodic body weight gains relative to controls were minimal over Days 1 to 5; although these males showed an actual mean weight loss of 2.3g over Days 5 to 8 which resulted in reduced overall body weight gain, this was mainly due to Male 15 from this dose group showing a body weight loss of 21g over this period whilst the corresponding body weight gains for the remaining 2/3 males from this dose group were similar to controls. There was no effect of treatment with the test item at a dose level of 300 mg/kg bw/day on body weight development in males.

Females treated with 1000 mg/kg bw/day showed a group mean body weight loss of 1.3g at the start of dosing. Subsequent improvement was apparent and the mean body weight gain for these females over Days 3 to 5 was slightly higher than controls. Fluctuations in body weight gains were apparent for females treated with 600 or 300 mg/kg bw/day over Days 3 to 8 and although overall weight gains for these females were slightly lower than controls, there was no dose-relationship and the overall body weight gain for the high dose females was similar to controls.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Group mean food consumptions are given in Table 4 and are presented graphically in Figure 3 and Figure 4. Food efficiencies are given in Table 5 please see the attached “tables & figures” .

Throughout the treatment period, males given 600 or 1000 mg/kg bw/day showed a dose- related reduction in food consumption with the effect being particularly marked for the high dose animals. Females treated with 1000 mg/kg bw/day also showed a marginal reduction in food consumption at the start of dosing, but subsequent dietary intake values for these animals were comparable with controls. Females treated with 600 mg/kg bw/day also showed a slight decrease in food intake towards the end of the treatment period (Day 5 to 8), but the corresponding value for the high dose females was similar to controls. There was no effect of treatment with the test item at a dose level of 300 mg/kg bw/day on food intake for animals of either sex.

Throughout the dosing period, a dose-related reduction in food conversion efficiency was generally evident for males treated with 600 or 1000 mg/kg bw/day. Fluctuation in food conversion efficiency were also apparent for females from all test item-treated dose groups and were deemed to be reflective of minor intergroup differences in body weight gains and/or food intake.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
Group mean daily water consumptions are given in Table 6 please see the attached “tables & figures”.

Gravimetric measurement of water intake for animals of either sex indicated an increase in water consumption for females given 1000 mg/kg bw/day from Day 2. Any other fluctuations in water intake were generally not dose-related and were deemed unlikely to be related to treatment with the test item.
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
A summary incidence of necropsy findings is given in Table 7. Individual data are given in Appendix 4 please see the attached “tables & figures”.

No macroscopic findings were identified for animals of either sex at necropsy.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Details on results:
DISCUSSION
Administration ofthe test item to Wistar Han™:RccHan™:WIST strain rats for seven consecutive days at a dose level of 1000 mg/kg bw/day resulted in marked effects on body weight and food intake in males which persisted throughout the dosing period; these observations were considered to be of an adverse nature. Females from the corresponding dose group also showed an initial reduction in body weight gain and food consumption, but subsequent improvement was apparent from Day 3 and overall body weight gain for these females was similar to controls. These females also showed an increase in water intake from Day 2. Taking into consideration the overall results at this dose level, particularly for the males, this dose level was deemed to be too high for subsequent repeated dose oral gavage toxicity studies.

Males treated with 600 mg/kg bw/day also showed a slight reduction in body weight gain and food intake throughout the dosing period. Although 1/3 males from this dose group showed a body weight loss of 21g over Days 5 to 8, there were no adverse clinical signs or any macroscopic necropsy findings for this male and the remaining 2/3 males from this dose group showed comparable body weight gains to controls over this period. Females from this dose group also showed a slight reduction in the mean weight gain over Days 3 to 5 but the corresponding value for the females treated with 1000 mg/kg bw/day was similar to controls. Clinical signs at this dose level were confined to some instances of increased post-dose salivation and there were no macroscopic findings at necropsy. Taking into consideration the overall results, these effects were considered insufficient to exclude this dose level from investigations in a subsequent twenty-eight repeated dose oral gavage toxicity study in the rat with recovery groups.
Conclusions:
Administration of the test item to Wistar Han™:RccHan™:WIST strain rats for seven consecutive days at a dose level of 1000 mg/kg bw/day resulted in adverse effects on body weight development and associated food intake in males. Taking into account the overall results and in consultation with the Sponsor, a high dose level of 600 mg/kg bw/day is considered suitable for use in a subsequent twenty-eight day repeated dose oral gavage toxicity study. Furthermore, dose levels of 35 and 350 mg/kg bw/day are considered suitable as low and intermediate doses, respectively.
Executive summary:

Introduction

 The study was designed to provide information for further repeated dose toxicity studies.

 

Methods

 Following preliminary sighting work, the test item was administered by gavage to three groups, each of three male and three female Wistar Han™:RccHan™:WIST strain rats, for seven consecutive days, at dose levels of 300, 600 or 1000 mg/ kg bw/day. A control group of three males and three females was dosed with vehicle alone (Arachis oil BP).

 

Clinical signs,body weight change, dietary intake and water consumption were monitored during the main phase of the study. All animals were subjected to gross necropsy examination.

 

ResultsMortality

There were no unscheduled deaths during the study.

 

Clinical Observations

 There were no clinical signs for animals of either sex considered to be related to the toxicity of the test item at any dose level.

 

Body Weight

Treatment at 1000 mg/kg bw/day resulted in a marked effect on body weight development with a number of males showing body weight losses during the dosing period. Group mean body weight gains for males treated with 600 mg/kg bw/day also remained slightly lower than controls throughout the dosing period with 1/3 males showing a body weight loss of 21g towards the end of the dosing period.At 300 mg/kg bw/day, group mean body weight gains in males were comparable with controls throughout the treatment period.

Females treated with 1000 mg / kg bw/ day also showed a slight reduction in bodyweight gain at the start of dosing. Subsequent recovery was, however, evident and overall body weight gains for these females was comparable with controls. No adverse effect on body weight development was evident in females treated with 600 or 300 mg/kg bw/day.

 

Food Consumption

At 600 or1000 mg/kgbw/ day,there was a dose-related reduction in dietary intake for males from the start of dosing with the effect being particularly marked for the latter dose group. Food conversion efficiency for the high dose males also remained lower than controls throughout the dosing period. No adverse effect on food consumption or food conversion efficiency was evident for males receiving 300 mg/kg bw/day.


 There was a marginal reduction in food consumption at the start of dosing for females receiving 1000 mg/kg bw/day resulting in reduced food conversion efficiency for these animals during this period. Thereafter, dietary intake for these animals was similar to controls.No adverse effect on food consumption or food efficiency was evident in females treated with 600 or 300 mg/kg bw/day.

 

Water Consumption

At 1000 mg/kg bw/day, females showed a marked increase in water intake from Day 2 of dosing. No such effects were detected in males treated with 1000 mg/kg bw/day or in animals of either sex treated with 600 or 300 mg/kg bw/day.

 

Necropsy

 No macroscopic findings were detected for animals at terminal necropsy.

 

Conclusion

Administration of the test item to Wistar Han™:RccHan™:WIST strain rats for seven consecutive days at a dose level of 1000 mg/kg bw/day resulted in adverse effects on body weight development and associated food intake in males. Taking into account the overall results and in consultation with the Sponsor, a high dose level of 600 mg/kg bw/day is considered suitable for use in a subsequent twenty-eight day repeated dose oral gavage toxicity study. Furthermore, dose levels of 35 and 350 mg/kg bw/day are considered suitable as low and intermediate doses, respectively.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
600 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Klimisch grade 1, GLP study conducted according to OECD407 test guideline.Klimisch grade 2, GLP study conducted according to OECD407 test guideline.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: dermal
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

In accordance with the CLP Regulation, No 1272/2008, the test material is not classified for repeat dose toxicity via the oral route. The NOAEL from the key study is 600 mg/kg bw/day, the highest dose tolerated by rats (adverse effects on body weight development and associated food intake). although the surviving animals of either sex from the 600 mg/kg bw/day dose group showed intermittent episodes of respiratory distress which correlated with tracheal lesions in some of the non-recovery animals from this dose group. Animals of either sex from this dose group also showed hyperplasia and hyperkeratosis of the forestomach, an indication of stomach irritation, which was deemed likely to have contributed to lower food intake and body weight gains and possibly higher water consumption. Animals of either sex previously receiving 600 mg/kg bw/day showed evidence of recovery after a fourteen day treatment-free period and these findings were considered to be due to the irritant properties of the test item and/or gavage-related reflux rather than an indication of its systemic toxicity. The findings in male kidneys from the 350 or 600 mg/kg bw/day dose groups were considered to be indicative of alpha-2u-globulin nephropathy, a condition specific to the male rat and to have no relevance to humans. The test material is therefore not considered to have an adverse effect that would require classification for repeat oral exposure.