Registration Dossier

Administrative data

Description of key information

Seven-day repeat dose toxicity study using the oral route by gavage in the rat, conducted according to a range-finder standard procedure under GLP, and followed by a twenty-eight day repeat oral (gavage) study in the rat. The NOAEL for systemic toxicity was found to be 1000 mg/kg bw/day (OECD 407 and EU Method B.7).

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:
24 September 2014 to 12 March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
higher than planned relative humidity noted on two occasions
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
yes
Remarks:
higher than planned relative humidity noted on two occasions
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Wistar Han
Sex:
male/female
Details on test animals and environmental conditions:
ANIMALS AND ANIMAL HUSBANDRY
- A sufficient number of male and female Wistar Han: RccHan: WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Oxon, UK.
- On receipt the animals were examined for signs of ill-health or injury.
- The animals were acclimatised for nine 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 189 to 225g, the females weighed 164 to 194g, and animals were approximately six to eight weeks old.
- 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).
- Animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK) was used. 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.
- Animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., 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 computerised system, and print-outs of hourly temperatures and humidities were
included in the study records.
- The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20 % respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.
- The animals were randomly allocated to treatment groups using a stratified body weight randomisation 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 randomised.
- The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
TEST ITEM PREPARATION
- The test item was prepared at the appropriate concentrations as a solution in Arachis oil BP.
- Analytical results showed that the formulations were stable for at least twenty days. Formulations were therefore prepared twice during the treatment period and stored at approximately 4 ºC in the dark.

PROCEDURE
- Animals were allocated to treatment groups as shown in the table (below).
- The test item was administered daily, for twenty-eight 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.
- 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 body weight and was adjusted at weekly intervals.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Samples of each test item formulation were taken and analysed for concentration of test material at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services.
- The results indicate that the prepared formulations were within 92 to 105 % of the nominal concentration.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
400 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
- Control (0 mg/kg bw/day): 5 males and 5 females
- Recovery control (0 mg/kg bw/day): 5 males and 5 females
- Low (100 mg/kg bw/day): 5 males and 5 females
- Intermediate: (400 mg/kg bw/day): 5 males and 5 females
- High: (1000 mg/kg bw/day): 5 males and 5 females
- Recovery high: (1000 mg/kg bw/day): 5 males and 5 females
Control animals:
yes, concurrent vehicle
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 during the treatment period.
- During the treatment-free period, animals were observed daily.
- All observations were recorded.

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.

BEHAVIOURAL ASSESSMENTS
- Detailed individual clinical observations were performed for each animal using a purpose built arena. The test was developed from the methods used by Irwin (1968) and Moser et al (1988).
- The parameters observed were gait, tremors, twitches, convulsions, bizzare/abnormal/stereotypic behaviour, salivation, pilo-erection, exophthalmia, lachrymation, hyper/hypothermia, skin colour, respiration, palpebral closure, urination, defaecation, transfer arousal and tail elevation.

FUNCTIONAL PERFORMANCE TESTS
- Motor Activity: Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals were randomly allocated to the activity monitors. The tests were performed at approximately the same time each day, under similar laboratory conditions. The evaluation period was sixteen hours for each animal. The percentage of time each animal was active and mobile was recorded for the overall sixteen hour period and also during the final 20% of the period (considered to be the asymptotic period).
- 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. The animal was pulled by the base of the tail until its grip was broken. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal.

SENSORY REACTIVITY
- Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. The assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).
- The parameters observed were grasp response, vocalisation, toe pinch, tail pinch, finger approach, touch escape, pupil reflex, startle reflex and blink reflex.

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 observed daily, for each cage group, by visual inspection of the water bottles for any overt changes except during Week 3 where water intake was measured gravimetrically.
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 animals were killed by intravenous overdose of sodium pentobarbitone 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 were discarded.

ORGAN WEIGHTS
- Organs, removed ftom animals that were killed at the end of the study, were dissected free from fat and weighed before fixation.
- Organs subjected to fixation were adrenals, brain, epididymides, heart, kidneys, pituitary (post-fixation), prostate and seminal vesicles (with coagulating glands and fluids), liver, ovaries, spleen, testes, thymus, thyroid/parathyroid (post-fixation) and uterus with cervix.

HISTOPATHOLOGY
- Samples of tissues were removed from all animals and preserved in buffered 10 % formalin.
- - Tissues preserved were adrenals; aorta (thoracic); bone and bone marrow (femur including stifle joint); bone and bone marrow (sternum); brain (including cerebrum, cerebellum and pons); caecum; colon; duodenum; epididymides (preserved in modified Davidson's fluid); eyes (fixed in Davidson's fluid); gross lesions; heart; ileum (including Peyer's patches); jejunum; kidneys; liver; lungs (with bronchi); lymph nodes (mandibular and mesenteric); mammary gland, muscle (skeletal); ovaries; pancreas; pituitary; prostate; rectum; salivary glands (submaxillary); sciatic nerve; seminal vesicles (with coagulating glands and fluids); skin (hind limb); spina cord (cervical, mid thoracic and lumbar); spleen; stomach; testes (preserved in modified Davidson's fluid); thymus; thyroid/parathyroid; trachea; urinary bladder; uterus and cervix; vagina.
- Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative.
- All tissues were dispatched to the histology processing Test Site (Huntingdon Life Sciences Ltd.) for processing (Principal Investigator: J Schofield). - Tissues prepared as paraffin blocks were adrenals; bone and bone marrow (sternum); brain (including cerebrum, cerebellum and pons); caecum; colon; duodenum; epididymides; eyes, gross lesions; heart; ileum (including Peyer's patches); jejunum; kidneys; liver; lungs (with bronchi); lymph nodes (mandibular and mesenteric); ovaries; pituitary; prostate; rectum; sciatic nerve; seminal vesicles (with coagulating glands and fluids); spinal cord (cervical, mid thoracic and lumbar); spleen; stomach; testes; thymus; thyroid/parathyroid; trachea; urinary bladder; uterus and cervix; vagina.
- Microscopic examination was conducted by the Study Pathologist (J Wilson at Propath GmbH, Muttenzerstrasse 30, 4133 Pratteln, Switzerland).
Other examinations:
LABORATORY INVESTIGATIONS
- Hematological and blood chemical investigations were performed on all non-recovery test and control group animals at the end of the treatment period (Day 28) and on all 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 non-recovery test and control group animals during Week 4 and on all 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.

HEMATOLOGY
- Parameters measured were haemoglobin (Hb); erythrocyte count (RBC); haematocrit (Hct); erythrocyte indices (mean corpuscular haemoglobin (MCH), mean corpuscular volume (MCV) and mean corpuscular haemoglobin concentration (MCHC); total leucocyte count (WBC); differential leucocyte count (neutrophils (Neut), lymphocytes (Lymph), monocytes (Mono), eosinophils (Eos) and basophils (Bas)); platelet count (PLT); Reticulocyte count (Retic). Prothrombin time (CT) was assess by 'Innovin' and activated partial prothrombin time (APTT) was assessed by 'Actin FS' using samples collected into sodium citrate solution (0.11 mol/L).

BLOOD CHEMISTRY
- Parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant.
- Parameters measured were urea, glucose, total protein (Tot Prot), albumin, albumin/globulin (A/G) ratio (by calculation), sodium (Na+), potassium (K+), chloride (Cl-), calcium (Ca++), triglycerides, inorganic phosphorus (P), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), alkaline phosphatase (AP), creatinine (Creat), total cholesterol (Chol), total bilirubin (Bili), bile acids and gamma glutamyltranspeptidase.

URINALYS
- Parameters measured on collected urine were volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood and appearance.

Statistics:
- Data were processed to give summary incidence or group mean and standard deviation values where appropriate. All data were summarised in tabular form.
- 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.
- Parameters on which statistical analysis was performed were 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 (see below for further details of statistical methods).
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
no dose relationship (see below)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
no adverse effect (see below)
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
no adverse effect (see below)
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
no toxicological significance (see below)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
no toxicological significance (see below)
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
no toxicological significance (see below)
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
no toxicological significance (see below)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
findings unremarkable and known to occur spontaneously in this strain of rat
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY
- There were no deaths during the study.

CLINICAL OBSERVATIONS
- There were no clinical signs for any of the animals throughout the study.

BEHAVIOURAL ASSESSMENTS
- There were no changes in the behavioral parameters considered to be related to treatment with the test item at any dose level.

FUNCTIONAL PERFORMANCE TESTS
- There were no treatment-related changes in functional performance at any dose level.
- Any intergroup differences achieving statistical significance were considered to be incidental; there was no dose-relationship and the majority of individual values were within the historical background control data ranges.

SENSORY REACTIVITY ASSESSMENTS
- There were no intergroup differences in sensory reactivity scores for animals given test material when compared with controls.

BODY WEIGHT
- No adverse effect of treatment was noted on body weight development in animals of either sex receiving the test item at any dose level.
- At the start of dosing, group mean body weight gains in males given the test item at 400 or 1000 mg/kg bw/day were slightly lower than controls but without achieving statistical significance or showing any dose-dependence. Subsequent weekly mean body weight gain in test item-treated males from all dose groups were occasionally lower than controls, but statistical significance was only achieved for males receiving 400 mg/kg bw/day during the final week of dosing (p<0.01). At the end of the treatment period, overall weight gains in non-recovery males treated with 100, 400 or 1000 mg/kg bw/day were approximately 10%, 30% and 8% lower than controls; there was no dose-relationship and the majority of individual values were within the control ranges and as such these intergroup differences were considered to be likely due to biological variation. Over the treatment-free period, group mean body weight gains in males previously given the high dose remained slightly lower than controls achieving statistical significance during the second week (p<0.05). It is worth noting, however, that with the exception of one male from this dose group (Animal Number 55) showing a body weight loss of 8g over the recovery period, individual body weight gains for the remaining recovery males were generally within the control ranges throughout the study and as such these intergroup differences were deemed unrelated to treatment with the test item.
- There was no effect of treatment with test item at any dose level on female body weight development throughout the study.

FOOD CONSUMPTION
- There was no adverse effect of treatment on dietary intake in animals of both sexes treated with test material at any dose level.
- Throughout the treatment period, there was no convincing evidence of an effect of treatment with the test item on food intake in males. Marginal reduction in food consumption was observed for males receiving 400 mg/kg bw/day throughout dosing and for males treated with 100 mg/kg bw/day during the final week of dosing; however food intake in high dose males remained similar to controls during the dosing period and as such these intergroup differences were considered unrelated to treatment with the test item. Following the cessation of dosing, males previously given the high dose showed marginally reduced food intake when compared with controls; however the difference was not of sufficient magnitude for it to have been
considered adverse.
- Throughout the treatment and treatment-free period, there was no effect of treatment with test item on food consumption in females at any dose levels.
- Fluctuations in food conversion efficiency were evident in males treated with the test item at all dose levels; however, these followed intergroup differences in body weight gains and/or food intake and were therefore considered not to represent an adverse effect of treatment.

WATER CONSUMPTION
- Gravimetric measurement of water consumption during Week 3 of the study did not reveal any treatment-related differences.

HEMATOLOGY
- There were no hematology findings considered to be of toxicological significance.
- At the end of the dosing period, males given the test item at all dose levels showed marginally but statistically significantly lower hemoglobin levels when compared with controls (p<0.01 at 1000 mg/kg bw/day and p<0.05 for 100 or 400 mg/kg bw/day). The mean cell hemoglobin and the mean cell hemoglobin concentrations in these males were also marginally lower than controls achieving statistical significance in most instances (p<0.01); statistical significance was not achieved for the mean cell hemoglobin in males treated with 100 mg/kg bw/day. Although a dose-relationship was apparent for the mean cell hemoglobin and the mean cell hemoglobin concentration, all individual values were within the historical control background data ranges. Non-recovery males treated with 1000 mg/kg bw/day also showed statistically significantly lower mean neutrophil count when compared w th controls (p<0.05); there was no doserelationship and all individual values were within the background data ranges. At the end of the treatment-free period, group mean hemoglobin level in males previously receiving the high dose was statistically significantly higher than controls (p<0.05) but all individual values were within the background data ranges. The mean cell hemoglobin, the mean cell hemoglobin concentration and neutrophil counts in these recovery males were similar to controls. In the absence of any histopathology correlates from non-recovery animals, these findings were considered to be of no toxicological significance.
- There were no treatment-related hematological observations in females treated with the test item at any dose level.

BLOOD CHEMISTRY
- There were no blood chemistry findings considered to be of toxicological significance.
- At the end of the treatment period, males receiving the test item at 400 or 1000 mg/kg bw/day showed statistically significantly lower group mean total protein levels in plasma when compared with controls (p<0.05). There was no dose-relationship and all individual values were within the historical background data ranges. At the end of the treatment-free period, the corresponding mean value in males previously treated with 1000 mg/kg bw/day was similar to controls indicating reversibility.
- At all dose levels, non-recovery males receiving the test item showed statistically significant lower plasma levels of triglycerides when compared with controls (p<0.01 at 400 or 1000 mg/kg bw/day and p<0.05 at 100 mg/kg bw/day). Non-recovery females from the high dose group also showed slightly lower triglyceride levels than controls but without achieving statistical significance. A dose-relationship was apparent in males but individual values from the test item treated animals of either sex were within the historical control data ranges. Group mean plasma concentration of cholesterol in non-recovery males treated with 1000 mg/kg bw/day also appeared to be slightly lower than control, albeit without attaining statistical significance and individual values were within the background data ranges. At the end of the recovery period, plasma levels of triglycerides and cholesterol in males previously given the high dose remained lower than controls achieving statistical significance in both instances (p<0.05).
- Plasma concentrations of chloride and calcium in males given the test item at all dose levels were statistically significantly higher than controls at the end of the treatment period (p<0.01 for calcium in males treated with 1000 mg/kg bw/day and p<0.05 for the remaining parameters). A dose-relationship was only apparent for calcium, but with the exception of some individual calcium values from the low and intermediate dose groups, the remaining individual values from males treated with the test item were within the background data ranges. The corresponding parameters in non-recovery females treated with the test item at all dose levels were similar to controls. At the end of the treatment-free period, chloride levels in males previously treated with the high dose remained statistically significantly higher than controls (p<0.05) but there were no intergroup differences for calcium levels in these males; group mean plasma concentration of calcium in females previously given the high dose was marginally but statistically significantly higher than controls (p<0.05).
- At 1000 mg/kg bw/day, plasma concentration of gamma glutamyltranspeptidase in non-recovery males was statistically significantly lower than controls (p<0.05) whilst females from this dose group showed statistically significantly lower levels of bilirubin when compared with controls (p<0.05). All individual values were within the historical data ranges with the corresponding values in recovery animals being similar to the respective controls. Group mean aspartate aminotranferase levels in plasma from treated males and females at the end of the dosing period were similar to controls; however, the corresponding value in recovery males previously given the high dose was slightly but statistically significantly higher than controls (p<0.05). The majority of the individual values were within the background data ranges.
- The histopathology examination of the tissues from the high dose animals of either sex at the end of the dosing period did not reveal any treatment-related findings and as such the above observations were deemed unlikely to be of any toxicological significance. Any other intergroup differences achieving statistical significance were considered to be incidental.

URINALYSIS
- Towards the end of the dosing period, urine samples from non-recovery animals receiving the test item at all dose levels appeared to be slightly more acidic when compared with controls; however the remaining urinalysis parameters in non-recovery and recovery animals of either sex were similar to the respective control and in the absence of any associated histopathology correlates, this observation was considered to be of no toxicological significance.

NECROPSY
- The few macroscopic findings were unremarkable and amongst those which occur spontaneously in this strain of rat. These included small/flaccid testes and small epididymides in one nonrecovery male treated with 400 mg/kg bw/day and one recovery male from 1000 mg/kg bw/day group.

ORGAN WEIGHTS
- At the end of the treatment period, group mean absolute and body weight-related brain weights in males given the test item at all dose levels were statistically significantly lower than controls (p<0.01); only exception was body weight-related brain weight in intermediate dose males that
was statistically significantly higher than controls (p<0.01). There was no dose-relationship and with the exception of one animal from the intermediate dose group, all individual values were within the historical background data ranges. The corresponding weights in the recovery males at the end of the treatment-free period were similar to controls. In the absence of any histopathology findings, this observation was deemed not to be toxicologically significant.
- The remaining organ weights from both non-recovery and recovery animals given test material at all dose levels were generally comparable with the respective controls; any intergroup differences achieving statistical significance were deemed incidental.

HISTOPATHOLOGY
- There were no treatment-related findings. The recorded microscopic findings were within the range of background pathology encountered in Wistar rats of this age in this type of study and occurred at similar incidences and severity in both control and treated rats.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Based on the in-life results and histopathology findings, a dose level of 1000 mg/kg bw/day is considered to be a No Observed Adverse Effect Level (NOAEL) for systemic toxicity.
Remarks on result:
other: Please see remarks section
Remarks:
The oral (gavage) administration of test material to male and female Wistar Han: RccHan: WIST strain rats for four weeks, at dose levels of 100, 400 and 1000 mg/kg bw/day, was well tolerated. Based on the in-life results and histopathology findings, a dose level of 1000 mg/kg bw/day is considered to be a No Observed Adverse Effect Level (NOAEL) for systemic toxicity.
Critical effects observed:
not specified

DISCUSSION

- The oral (gavage) administration of test material to male and female rats of Wistar Han: RccHan: WIST strain at dose levels of 100, 400 or 1000 mg/kg bw/day was well tolerated. There were no clinical signs for any of the animals throughout the dosing or recovery periods. Whilst there was no effect of treatment with the test item on body weight development or food consumption in females throughout the study, group mean body weight gains in males from all dose groups generally remained lower than controls; there was no dose-relationship and food intake in high dose males was unaffected. During the treatment-free period, group mean body weight gains in males previously given the high dose remained slightly lower than controls, which was associated with marginally reduced food intake in these animals. The majority of individual body weight gain values for these animals were, however, within control ranges and any intergroup differences were considered unlikely to be treatment-related.

- Hematological investigations did not reveal any adverse effect of treatment with the test item in animals of either sex. Some statistically significant intergroup differences were evident in males at the end of the treatment and/or treatment-free periods; however, individual values from test item-treated males were within the historical control background data ranges. As there were no associated histopathology findings, these differences were considered to be of no toxicological importance.

- There was no adverse effect of treatment with test material at any dose level on blood chemistry parameters in male and female rats. At the end of the dosing period, plasma levels of triglycerides in males treated with the test item at all dose levels were statistically significant lower than controls in a dose-related manner whilst plasma concentration of cholesterol in high dose males also appeared to be slightly lower than controls albeit without achieving statistical significance; the corresponding values in recovery males remained statistically significantly lower than controls. The majority of individual values were within the control background data ranges and in the absence of any associated histopathology observations, these findings were considered not toxicologically important. Other statistically significant intergroup differences at the end of the treatment period included lower total protein levels in males treated with 400 or 1000 mg/kg bw/day, higher calcium and chloride concentration in males from all dose groups, and lower gamma glutamyltranspeptidase and bilirubin levels in high dose males and females, respectively. At the end of the treatment free period, males previously receiving 1000 mg/kg bw/day showed higher plasma concentrations of chloride and aspartate aminotranferase whilst

the corresponding females showed higher calcium levels. The majority of individual values were, however, within the background data ranges and in the absence of any histopathology correlates, these findings were considered of no toxicological significance.

Conclusions:
The oral (gavage) administration of test material to male and female Wistar Han: RccHan: WIST strain rats for four weeks, at dose levels of 100, 400 and 1000 mg/kg bw/day, was well tolerated. Based on the in-life results and histopathology findings, a dose level of 1000 mg/kg bw/day is considered to be a No Observed Adverse Effect Level (NOAEL) for systemic toxicity.
Executive summary:

GUIDELINE

The study was designed to investigate the systemic toxicity of the test item and is compatible with Commission Directive 96/54/EC (Method B7). Commission Directive 96/54/EC (Method B7) and the OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008). The 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 twenty-eight consecutive days, at dose levels of 100, 400 and 1000 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 (1000 mg/kg bw/day) or the vehicle alone for 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 no deaths during the study.

Clinical observations: There were no clinical signs for any of the animals throughout the study.

Behavioural assessment: 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 scores remained unaffected by treatment with the test item.

Body weight: There was no adverse effect of treatment with the test material on body weight development in animals of either sex at any dose level.

Food consumption: Treatment with the test item had no adverse impact on food consumption in male or female rats at any dose level. Food conversion efficiency in males fluctuated throughout the study reflecting intergroup differences in body weight gains and/or food intake in these males.

Water consumption: Gravimetric measurement of water consumption during Week 3 of the study did not reveal any treatment-related differences.

Hematology: Investigation did not reveal any toxicologically significant findings.

Blood chemistry: No toxicologically adverse effects were detected in blood chemistry investigations.

Urinalysis: No toxicologically significant effects were detected in the urinalysis parameters examined at dose levels up to 1000 mg/kg bw/day.

Necropsy: Macroscopic examination did not reveal any treatment-related findings.

Organ weights: Analysis of organ weight did not indicate any toxicologically significant findings at the end of the treatment or treatment-free periods.

Histopathology: Examination did not reveal any treatment-related findings.

CONCLUSION

The oral (gavage) administration of test material to male and female Wistar Han: RccHan: WIST strain rats for four weeks, at dose levels of 100, 400 and 1000 mg/kg bw/day, was well tolerated. Based on the in-life results and histopathology findings, a dose level of 1000 mg/kg bw/day is considered to be a No Observed Adverse Effect Level (NOAEL) for systemic toxicity.

Endpoint:
repeated dose toxicity: oral
Remarks:
other: range-finding study
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
28 August 2014 to 22 January 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed under GLP conditions.
Qualifier:
no guideline required
Principles of method if other than guideline:
The study was designed to provide information for further repeated dose toxicity studies.
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Wistar Han
Sex:
male/female
Details on test animals and environmental conditions:
ANIMALS AND ANIMAL HUSBANDRY
- A sufficient number of male and female Wistar Han: RccHan: WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Oxon, UK.
- On receipt the animals were examined for signs of ill-health or injury.
- The animals were acclimatized for six days during which time their health status was assessed.
- A total of twenty four animals (twelve males and twelve females) were accepted into the study.
- At the start of treatment the males weighed 207 to 224g, the females weighed 159 to 180g, and animals were approximately six to eight weeks old.
- 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, Harlan Laboratories U.K. Ltd., Oxon, UK) was used. 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.
- Animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., 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 computerised system, and print-outs of hourly temperatures and humidities were included in the study records.
- The Study Plan target ranges for temperature and relative humidity were 22 ± 3 ºC and 50 ± 20 % respectively; short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.
- The animals were allocated to dose groups using a randomisation 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
- 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. This is an exception with regard to GLP and was reflected in the GLP compliance statement.

PROCEDURE
- Animals were allocated to treatment groups as shown in the table below.
- The test material 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/day of dried Arachis oil BP.
- The volume of test and control item administered to each animal was based on the most recently scheduled body weight and was adjusted on Days 3 and 5.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Seven days
Frequency of treatment:
Daily
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
other: nominal in arachis oil
Remarks:
Doses / Concentrations:
250 mg/kg bw/day
Basis:
other: nominal in arachis oil
Remarks:
Doses / Concentrations:
500 mg/kg bw/day
Basis:
other: nominal in arachis oil
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
other: nominal in arachis oil
No. of animals per sex per dose:
- Control: 3 male and 3 female
- Low (250 mg/kg/day): 3 males and 3 females
- Intermediate (500 mg/kg/day): 3 males and 3 females
- High (1000 mg/kg/day): 3 males and 3 females
Control animals:
yes, concurrent vehicle
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.
- Animals were also examined four hours after dosing on week days.
- 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.
- No tissues were retained.
Statistics:
EVALUATION OF DATA
- Data were processed to give summary incidence or group mean and standard deviation values where appropriate.
- All data were summarised in tabular form.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
no detrimental effect (see below)
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
see below
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
not related to treatment (see below)
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY
- There were no deaths during the study.

CLINICAL OBSERVATIONS
- There were no clinical signs for any of the animals throughout the dosing period.

BODY WEIGHT
- There was no detrimental effect of treatment with the test item at any dose level on body weight development in animals of either sex.
- The degree of individual variation was high amongst the female groups with occasional body weight losses noted for individual animals.
- Overall group mean body weight gains for animals of either sex at all dose levels were slightly higher than controls, albeit without a dose relationship.

FOOD CONSUMPTION
- There was no detrimental effect of treatment with test material at any dose level on food consumption or food conversion efficiency in animals of either sex.
- Males receiving the test item at all dose levels and females from the high dose group showed slightly higher food intake over Days 3 to 8 of dosing when compared with the respective controls.

FOOD CONVERSION EFFICIENCY
- Food conversion efficiency in animals from all test item-treated groups was occasionally higher than controls reflecting differences in body weight gain and/or food consumption for these animals.

WATER CONSUMPTION
- Gravimetric measurement of water intake in males did not indicate any treatment-related differences.
- In females, instances of higher water intake were noted at all dose levels, in particular for the intermediate dose group, when compared with controls. As there was no dose-relationship and water intake for the high dose females was generally similar to controls, this finding was considered not to be treatment-related.

NECROPSY
- No macroscopic abnormalities were detected at any dose level.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified
Conclusions:
Administration of test material to Wistar Han: RccHan: WIST strain rats for seven consecutive days at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated. Based on these results and after consultation with the Sponsor, dose levels of 100, 400 and 1000 mg/kg bw/day were considered suitable for use in a 28 day repeated dose toxicity study in the rat.
Executive summary:

METHOD

The test item was administered by gavage to three groups of male and female Wistar Han RccHan strain rats for seven consecutive days. Dose levels were 250, 500 or 1000 mg/kg bw/day. A control group of three males and three females was dosed with vehicle alone (arachis oil).

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

RESULTS

Mortality: There were no deaths during the study.

Clinical observations: There were no clinical signs for any of the animals during the treatment period.

Body weight: There was no detrimental effect of treatment with test material at any dose level on body weight development in animals of either sex.

Food consumption: There was no detrimental effect of treatment with the test item at any dose level on food consumption or food conversion efficiency in animals of either sex.

Water consumption: Gravimetric measurement of water intake did not reveal any treatment-related effects on water consumption in animals of both sexes.

Necropsy: There were no macroscopic findings for animals of both sexes at any dose level.

CONCLUSION

Administration of test material to Wistar Han: RccHan: WIST strain rats for seven consecutive days at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated. Based on these results and after consultation with the Sponsor, dose levels of 100, 400 and 1000 mg/kg bw/day were considered suitable for use in a 28 day repeated dose toxicity study in the rat.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

GUIDELINES

The study was designed to investigate the systemic toxicity of the test item and was compatible with Commission Directive 96/54/EC (Method B.7) and the OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008). The 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

Administration of test material to Wistar Han: RccHan: WIST strain rats for seven consecutive days at dose levels of 250, 500 or 1000 mg/kg bw/day was well tolerated and no treatment-related effects were seen. Based on these results and after consultation with the Sponsor, dose levels of 100, 400 and 1000 mg/kg bw/day were considered suitable for use in a 28 day repeated dose toxicity study in the rat.

In a key study, the test item was subsequently administered by gavage to three groups, each of five male and five female Wistar Han: RccHan: WIST strain rats, for twenty-eight consecutive days, at dose levels of 100, 400 and 1000 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 (1000 mg/kg bw/day) or the vehicle alone for 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 OF KEY STUDY

Mortality: There were no deaths during the study.

Clinical observations: There were no clinical signs for any of the animals throughout the study.

Behavioural assessment: 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 scores remained unaffected by treatment with the test item.

Body weight: There was no adverse effect of treatment with the test material on body weight development in animals of either sex at any dose level.

Food consumption: Treatment with the test item had no adverse impact on food consumption in male or female rats at any dose level. Food conversion efficiency in males fluctuated throughout the study reflecting intergroup differences in body weight gains and/or food intake in these males.

Water consumption: Gravimetric measurement of water consumption during Week 3 of the study did not reveal any treatment-related differences.

Hematology: Investigation did not reveal any toxicologically significant findings.

Blood chemistry: No toxicologically adverse effects were detected in blood chemistry investigations.

Urinalysis: No toxicologically significant effects were detected in the urinalysis parameters examined at dose levels up to 1000 mg/kg bw/day.

Necropsy: Macroscopic examination did not reveal any treatment-related findings.

Organ weights: Analysis of organ weight did not indicate any toxicologically significant findngs at the end of the treatment or treatment-free periods.

Histopathology: Examination did not reveal any treatment-related findings.

CONCLUSION

The oral (gavage) administration of test material to male and female Wistar Han: RccHan: WIST strain rats for four weeks, at dose levels of 100, 400 and 1000 mg/kg bw/day, was well tolerated. Based on the in-life results and histopathology findings, a dose level of 1000 mg/kg bw/day is considered to be a No Observed Adverse Effect Level (NOAEL) for systemic toxicity.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:

GLP guideline study.

Justification for classification or non-classification

No classification for repeat dose toxicity is required as there were no adverse effects at up to and including the maximum recommended dose level of 1000 mg/kg bw/day.