Registration Dossier

Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27th December 2012 (dosing) to 27th March 2013 (post-mortem)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no/minor deviations from test guidelines and/or minor methodological deficiencues, which do not affect the quality of relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
Sponsor's identification :2,2’-(octadec-9-enylimino)bisethanol CAS No 25307-17-9
Description : pale brown liquid
Chemical name :2,2’-(octadec-9-enylimino)isethanol
Purity : >97%
Batch number : S-001018
CAS Number : 25307-17-9
REACH Registration Number :01-2119510876-35-0000
Date received : 07 May 2013
Storage conditions :ambient temperature, under nitrogen, (used/ formulated in light) in the dark
Expiry date : 05 June 2016

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
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 eight days during which time their health status was assessed. A total of eighty animals (forty males and forty females) were accepted into the study. At the start of treatment the males weighed 155 to 194g (See Deviations from Study Plan), the females weighed 138 to 160g, and were approximately six to eight weeks old.
The animals were housed in groups of three or four by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used. Certificates of analysis of the batches of diet used are given in Addendum 1. Mains drinking water was supplied from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).

The 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 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 the target ranges.

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.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
As Arachis Oil was successfully used on a previous twenty-eight day toxicity study, for 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 previously determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services (Harlan Laboratories Project Number 0142-0418) at concentrations of 3.75 and 250 mg/kg bw/day. Results from the previous study show the formulations to be stable for at least twenty one days at 4 °C. An additional investigation of the stability of the Test Item at a concentration of 1.25 mg/ml for one day was performed as part of this study. Bulk formulations for the high and intermediate dosage groups were generally prepared in batches of approximately two weeks duration and divided into daily aliquots. These formulations were stored at approximately 4 °C in the dark prior to use. The low dose formulations were made on a daily basis using the formulation prepared for the high dosage group.

Represenative samples of test item formulations were taken and analysed for concentration of 2,2’-(octadec-9-enylimino)bisethanol CAS No 25307-17-9 at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Appendix 17. The results indicate that the prepared formulations were within 86-115% of the nominal concentration confirming the accuracy of the formulation procedure and were considered acceptable for the study.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of 2,2 (octadec-9 enylimino) bisthanol CAS No: 25307-17-9 in the test item formulations was determined by gas chromatography (GC) using an external standard technique.

Samples
The test item formulations were extracted with methanol to give a final, theoretical test item concentration of approximately 0.1 mg/ml.

Standards
Standard solutions of test item were prepared in methanol at a nominal concentration of 0.1 mg/ml.

Procedure
The standard and sample solutions were analysed by GC using the following conditions:

GC system : Agilent Technologies 5890, incorporating autosampler and workstation
Column : DB-1 (15 m x 0.53 mm id x 1.5 µm film)
Oven temperature program :initial 200 ºC for 0 mins, rate 10 ºC/min, final 300 ºC for 12 mins
Injection temperature :300 ºC
Flame ionisation detector temperature :300 ºC
Injection volume: : 1 µl
Retention time : ~ 5.5 mins

Homogeneity Determinations
The 1.25 mg/ml test item formulations were assessed by visual inspection. The homogeneity for the other dose levels was performed under Harlan Laboratories Ltd., Project Number 0142-0418.

Stability Determinations
The 1.25 mg/ml test item formulations were sampled and analysed initially and then after storage at approximately +4ºC in the dark for 1 days. The stability for the other dose levels was performed under Harlan Laboratories Ltd., Project Number 0142-0418.

Verification of Test Item Formulation Concentrations
The test item formulations were sampled and analysed within 1 day of preparation.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
5 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
30 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
150 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 maes
10 females
Control animals:
yes, concurrent vehicle
Details on study design:
Dosages were selected, in collaboration with the sponsor based on available toxicity data including a twenty-eight day toxicity study performed at these laboratories (Harlan Laboratories Project Number 0142-0419). In the twenty-eight day toxicity study, adverse toxicity was observed at 250 mg/kg bw/day that precluded this dosage from use on this study. However the dosage of 250 mg/kg bw/day was subsequently lowered to 150 mg/kg bw/day and this dosage was well tolerated, although histopathological changes were apparent for the stomach and small intestines. It was anticipated that the effects observed for the stomach and small intestines would not increase significantly with the longer duration of treatment utilised in this 90 day study therefore 150 mg/kg bw/day was chosen for the high dosage in this study. Lower dosages of 5 and 30 mg/kg bw/day were also selected.

The test item was administered daily, for ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg of Arachis oil BP.
The volume of test and control item administered to each animal was based on the most recent body weight and was adjusted at weekly intervals.
Positive control:
None

Examinations

Observations and examinations performed and frequency:
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends and public holidays. All observations were recorded.

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

Behavioural Assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin colour, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behaviour, 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 Behavioural 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 Behavioural Assessments and Sensory Reactivity Tests.

The following parameters were observed: Grasp response, Touch escape, Vocalisation, Pupil reflex, Toe pinch, Blink reflex, Tail pinch, Startle reflex,
Finger approach

Body Weight
Individual body weights were recorded on Day 1 and at weekly intervals thereafter. Body weights were also recorded at terminal kill.

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

Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Ophthalmoscopic Examination
The eyes of all control and high dose animals were examined pre-treatment and before termination of treatment (during Week 12). Examinations included observation of the anterior structures of the eye, pupillary and corneal blink reflex. Following pupil dilation with 0.5 % Tropicamide solution (Mydriacyl® 0.5 %, Alcon Laboratories (UK) Ltd., Pentagon Park, Boundary Way, Hemel Hampstead, Hertfordshire), detailed examination of the internal structure of the eye using a direct ophthalmoscope was performed.


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

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


Blood Chemistry

The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea, Inorganic phosphorus (P), Glucose, Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin, Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+), Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Bile acids, Calcium (Ca++)
Sacrifice and pathology:
Pathology
On completion of the dosing 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.

Organ Weights
The following organs, removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation:
Adrenals, Ovaries, Brain, Spleen, Epididymides, Testes, Heart, Thymus, Kidneys, Uterus, Liver

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

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

All tissues from control and 150 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Haematoxylin and Eosin for subsequent microscopic examination.

Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of the stomach, duodenum, jejunum, ileum and mesenteric lymph nodes from animals in the low and intermediate groups.

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. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Haematology, Blood Chemistry, Urinalysis (Volume and Specific Gravity), Absolute Organ Weights, Body Weight-Relative Organ Weights.
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed below:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. Any transformed data were analysed 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 analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p>0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Males only. Please refer to the "Details on Results" section below
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Males only. Please refer to the "Details on Results" section below
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Males only. Please refer to the "Details on Results" section below
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Please refer to the "Details on Results" section below
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Please refer to the "Details on Results" section below
Histopathological findings: neoplastic:
not examined

Effect levels

Dose descriptor:
NOEL
Effect level:
5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Mortality

There were no unscheduled deaths on the study.

Clinical observations

Neither the type, incidence nor distribution of clinical signs observed during the study indicated any adverse effect of treatment at dosages up to 150 mg/kg bw/day.

At 150 mg/kg bw/day all animals showed increased salivation post dosing during the study. Similar increased salivation post dosing was also observed at 30 mg/kg bw/day but the incidence and number of animals affected was much lower. At 5 mg/kg bw/day only one male showed increased post-dosing salivation during the study. Increased salivation is frequently observed when animals are dosed via the oral gavage route and this is generally considered to reflect distaste/irritancy of the test item formulation rather than any systemic effect of treatment.

Additionally at 150 mg/kg bw/day, two males and one female showed noisy respiration and one male also showed staining around the snout during the study. These findings are probably associated with the increased salivation observed at this dosage.

Functional observations

Behavioural Assessments

 Assessment of the animals in a standard arena did not reveal any effect of treatment at 5, 30 or 150 mg/kg bw/day.

 

Functional Performance Tests

Assessment of functional performance using grip strength and measurement of motor activity did not indicate any obvious effects of treatment at dosages of 5, 30 or 150 mg/kg bw/day.

Sensory Reactivity Assessments

Sensory reactivity to different stimuli (auditory, visual and proprioceptive) also appeared unaffected by treatment at 5, 30 or 150 mg/kg bw/day.

       

Body Weight

At 150 mg/kg bw/day body weight gain of males was lower than control throughout the treatment period, with differences frequency attaining statistical significance. By the end of the fourth week of treatment differences in absolute body weights attained statistical significance when compared with control and overall body weight gain of these males at the end of treatment was approximately 71% of their control counterparts. There was considered to be no effect of treatment on body weight or body weight gain of females at this dosage.

At 5 and 30 mg/kg bw/day there was no effect of treatment on body weight or body weight gain for either sex.

           

Food Consumption

At 150 mg/kg bw/day food consumption of males tended to be lower than control throughout the treatment period. This finding was consistent with the lower body weight gains observed at this dosage and was considered to be related to treatment. For females at this dosage, food intake also tended to be lower than control throughout treatment; however on most occasions, these differences were unremarkable and overall these differences in food intake were considered to reflect normal biological variation rather than any effect of treatment.

At 5 and 30 mg/kg bw/day was no obvious effect of treatment on food consumption for either sex.

    

Food Conversion Efficiency

There were no clear effects of treatment on food conversion efficiency for either sex at 5, 30 or 150 mg/kg bw/day.

For males at 150 mg/kg bw/day food efficiency was often lower than control, but these differences were generally not marked and did not indicate any consistent adverse effect of treatment.

Water Consumption

Visual assessment of water intake did not indicate any effect of treatment at 5, 30 or 150 mg/kg bw/day.

   

Opthalmoscopic Examination

Ophthalmic examination during the last week of treatment did not indicate any effect of treatment for either sex at 150 mg/kg bw/day.

              

Oestrus Cycle Assessments

Assessment of female oestrus cycles did not indicate any effect of treatment at 5, 30 or 150 mg/kg bw/day.

    

       

Laboratory Investigations

Haematology


Males

There was no clear effect of treatment on haematology parameters at 5, 30 or 150 mg/kg bw/day.

For males at 150 mg/kg bw/day lower mean cell haemoglobin and mean cell volume attained statistical significance when compared with control. The majority of values for these treated animals were within the historical control range for these parameters and, in the absence of any histopathological correlates, these differences were considered incidental and unrelated to treatment.   

For males at 30 mg/kg bw/day, lower platelet count attained statistical significance when compared with control but all values for treated animals were within the historical control range. In the absence of any similar effect at 150 mg/kg bw/day, this finding was considered incidental and unrelated to treatment.  

Females

At all dosages, lower mean cell haemoglobin and mean cell haemoglobin concentration attained statistical significance when compared with control. For mean cell haemoglobin concentration, the majority of individual values for all dosage groups were outside the historical control. For mean cell haemoglobin, four of the ten individual values at 150 mg/kg bw/day were outside the historical control range; however, at lower dosages only one value at 5 mg/kg bw/day was below this historical range. 

At 150 mg/kg bw/day these findings were accompanied by lower mean haemoglobin and mean cell volume, with differences from control again attaining statistical significance compared with control. While the majority of values for mean haemoglobin at 150 mg/kg bw/day exceeded the historical control range, all values for mean cell volume were within this historical range.

Additionally at 150 mg/kg bw/day the number of neutrophils was statistically significantly higher than control, with the majority of individual values exceeding the historical control range. There was no accompanying statistically significant difference from control observed for total leucocyte count.

     

Blood Chemistry

For males at 150 mg/kg bw/day, lower levels of total protein, accompanied by lower blood albumin levels and an increase for albumin/globulin ratio, all attained statistical significance compared with control. The majority of individual values for total protein were outside the historical control range, but only a few values for albumin and albumin/globulin ratio were outside this historical range.     

Higher levels of bile acids for males at 150 mg/kg bw/day also attained statistical significance compared with control. All individual values for treated animals exceeded the historical control range but so did the majority of individual control values.

Additionally males at 150 mg/kg bw/day showed a decrease in creatinine levels, although only individual values for three animals were outside the historical control range. Lower levels of blood urea and total cholesterol also attained statistical significance compared to control but all individual values were within the historical control range.   

For males at 5 and 30 mg/kg bw/day statistically significant differences in blood chemistry parameters from control were restricted to lower creatinine levels and all individual values for treated animals were within the historical control range.

There were no statistically significant differences in blood chemistry parameters from control for females at 5, 30 or 150 mg/kg bw/day.

Pathology

Necropsy

There were no findings observed at macroscopic necropsy examination that indicated any adverse effect of treatment.

Two control females and one female at 150 mg/kg bw/day showed reddened lungs, while another female at 150 mg/kg bw/day showed pale foci on the lungs at necropsy. In the absence of any treatment related effects being detected for the lungs during microscopic examination, these findings are considered incidental and unrelated to treatment.

Organ Weights

At 150 mg/kg bw/day, absolute liver weights were lower than control for males but higher than control for females, with differences from control for both sexes attaining statistical significance. Body weight-relative values probably represent a more accurate indicator of treatment effect for this organ and for both sexes mean values were increased compared with control; differences attained statistical significance and the majority of individual body weight relative values exceeding the historical control range. 

For females at 150 mg/kg bw/day, higher absolute and body weight-relative kidney weights attained statistical significance when compared with control, with the majority of individual body weight relative values for treated animal exceeding the historical control range. 

Additionally for females at 150 mg/kg bw/day higher absolute and body weight-relative uterus weights attained statistical significance when compared to control. However, these mean values at 150 mg/kg bw/day were influenced by one female with a particularly high uterus weight; no statistically significant difference from control was apparent when this animal was excluded. In the absence of any supporting histopathological change at this dosage this finding was considered incidental and of no toxicological significance.       

For males at 30 mg/kg bw/day, higher absolute and body weight-relative kidney weights attained statistical significance when compared with control. However, there were no statistically significant differences in kidney weights for males at 150 mg/kg bw/day and the majority of individual values at 30 mg/kg bw/day were within the historical control range. In the absence of any evidence of histopathological kidney change at 150 mg/kg bw/day, the increases in kidney weights were considered incidental and unrelated to treatment.

There were no statistically significant differences in organ weights for females at 30 mg/kg bw/day or either sex at 5 mg/kg bw/day.

Histopathology

Treatment at 150 mg/kg bw/day produced minimal or mild diffuse epithelial hyperplasia and hyperkeratosis of the forestomach in most animals; minimal or mild focal erosion of the forestomach, minimal or mild submucosal inflammatory cell infiltration, submucosal oedema and minimal focal dyskeratosis were also present in the forestomach of a few animals. In addition, minimal to marked foamy macrophages were present in the lamina propria of the small intestine (principally the jejunum but also in the duodenum and ileum) and also in the sinuses of the mesenteric lymph node for both sexes at this dosage.

At 30 mg/kg bw/day treatment-related changes were confined to the forestomach in which there were incidences of minimal focal epithelial hyperplasia, minimal focal erosion, submucosal oedema and minimal or mild submucosal inflammatory cell infiltration.

No treatment-related changes were observed in the small intestine or mesenteric lymph node.

No treatment-related changes were observed for either sex at 5 mg/kg bw/day.

All of the other histopathological findings encountered were considered to have arisen spontaneously or atpost mortem.

Applicant's summary and conclusion

Conclusions:
At 150 mg/kg bw/day, the extent of microscopic changes to the fore stomach and gastro-intestinal tract apparent for both sexes was considered to exclude this dosage from being a No Observed Adverse Effect Level (NOAEL). At 30 mg/kg bw/day, microscopic changes were restricted to the fore stomach and was considered to represent a local irritancy effect of the test item rather than systemic toxicity. As such, a dosage of 30 mg/kg bw/day may be considered the NOAEL for systemic toxicity.

A dosage of 5 mg/kg bw/day was considered to represent the No Observed Effect Level (NOEL).
Executive summary:

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

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

ii)        The EU Annex B Method B26 - “Subchronic Oral Toxicity Test - Repeated Dose 90-Day Oral Toxicity Study in Rodents” - updated 21 August 2001.

iii)       The United States Environmental Protection Agency (EPA), Health Effects Test Guidelines, OPPTS 870.3100 - 90 Day Oral Toxicity in Rodents.

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 continuously by gavage to three groups, each of ten male and ten female Wistar Han™: RccHan™: WIST strain rats, for ninety consecutive days, at dose levels of 5, 30 and 150 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP) over the same treatment period.

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. Ophthalmoscopic examination was also performed on control group and high dose animals.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. These examinations were extended for the stomach, duodenum, jejunum, ileum and mesenteric lymph nodes to include animals from the low and intermediate dosage groups. 


Results.

Mortality.There were no unscheduled deaths on the study.

Clinical Observations.There were no clinical signs observed that were considered to be of any toxicological significance at 5, 30 or 150 mg/kg bw/day during the study.

Behavioural Assessment.Behavioural assessments did not reveal any effect of treatment at 5, 30 or 150 mg/kg bw/day. 

Functional Performance Tests.Functional performance tests did not indicate any effects of treatment at dosages of 5, 30 or 150 mg/kg bw/day.

Sensory Reactivity Assessments.Sensory reactivity assessments did not indicate any effects of treatment at dosages of 5, 30 or 150 mg/kg bw/day.

Body Weight.At 150 mg/kg bw/day body weight gain of males was lower than control throughout the treatment period with overall body weight gain being approximately 71% of control by the end of treatment.

Food Consumption.At 150 mg/kg bw/day food consumption of males was generally lower than control throughout the treatment period.

Food Conversion Efficiency.Food conversion efficiency was considered to have been unaffected by treatment at 5, 30 or 150 mg/kg bw/day.

Water Consumption.Visual assessment of water intake did not indicate any effect of treatment at 5, 30 or 150 mg/kg bw/day.

Ophthalmoscopy.Ophthalmic examination did not indicate any effect of treatment at 150 mg/kg bw/day.

Haematology.For females at 150 mg/kg bw/day, mean cell haemoglobin concentration, mean cell haemoglobin and mean cell volume were statistically significantly lower than control and the number of neutrophils was statistically significantly higher than control.

Blood Chemistry.For males at 150 mg/kg bw/day, lower levels of total protein, accompanied by lower blood albumin levels and an increase for albumin/globulin ratio, all attained statistical significance compared with control. Higher levels of bile acids and lower levels of creatinine also attained statistical significance compared with control.

Necropsy.There were no macroscopic findings observed at necropsy examination that were considered to be associated with treatment.

Organ Weights.At 150 mg/kg bw/day, higher absolute liver weight for males and lower absolute liver weight for females attained statistical significance compared to control. Body weight-relative values were statistically significantly higher than control for both sexes. 

At 150 mg/kg bw/day, higher absolute and body weight-relative female kidney weights attained statistical significance when compared with control.

Histopathology.At 150 mg/kg bw/day minimal or mild diffuse epithelial hyperplasia and hyperkeratosis of the forestomach were observed in most animals with minimal or mild focal erosion of the forestomach, minimal or mild submucosal inflammatory cell infiltration, submucosal oedema and minimal focal dyskeratosis being present in the forestomach for a few animals. In addition, minimal to marked foamy macrophages were present in the lamina propria of the small intestine (principally the jejunum but also in the duodenum and ileum) and also in the sinuses of the mesenteric lymph node for both sexes.

At 30 mg/kg bw/day treatment-related changes were restricted to incidences of minimal focal epithelial hyperplasia, minimal focal erosion, submucosal oedema and minimal or mild submucosal inflammatory cell infiltration of the forestomach. No treatment-related changes were observed in the small intestine or mesenteric lymph node.

Conclusion.At 150 mg/kg bw/day, the extent of microscopic changes to the fore stomach and gastro-intestinal tract apparent for both sexes was considered to exclude this dosage from being a No Observed Adverse Effect Level (NOAEL). At 30 mg/kg bw/day, microscopic changes were restricted to the fore stomach and were considered to represent a local irritancy effect of the test item rather than systemic toxicity. As such, a dosage of 30 mg/kg bw/day may be considered the NOAEL for systemic toxicity.

A dosage of 5 mg/kg bw/day was considered to represent the No Observed Effect Level (NOEL).