2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs.

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 January 2014 - 28 November 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Remarks:

Two animals were incorrectly identified resulting in the animals being dosed for two days based on the incorrect body weights.

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Monitoring authority, Dept. of Health UK, 2014

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs.

EC Number:

272-221-2

EC Name:

2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs.

Cas Number:

68784-12-3

Molecular formula:

Due to a mixture of numerous isomers a specific molecular formular cannot be provided ( UVCB-substance) .

IUPAC Name:

2,5-Furandione, dihydro-, mono-C15-20-alkenyl derivs.

Test material form:

other: Liquid

Details on test material:

- Name of test material (as cited in study report): AS1100
- Description: Amber coloured liquid
- Purity: 98%
- Batch Number: 5500020562
- Date Received: 15 January 2014
- Storage Conditions: Ambient temperature 10 °C to 30 °C in the dark
- No correction for purity was made.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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 acclimatized for five 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 161 to 192g, the females weighed 141 to 174g, 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 Appendix 23. 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 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 computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study; see deviations from Study Plan.

The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

Test item preparation
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 Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results from the previous study show the formulations to be stable for at least twenty one days. Formulations were therefore prepared weekly and stored at approximately 4 °C in the dark.

Samples of each test item formulation were taken and analyzed for concentration of AS1100 at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The results indicate that the prepared formulations were within ± 7% of the nominal concentration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The test item concentration in the test samples was determined by gas chromatography using an external standard technique. The test item gave a chromatographic profile consisting of a profile of multiple peaks.
The formulations investigated were found to comprise test item in the range of 97 to 107 % and thus the required content limit with reference to the nominal content was met.
The test item was found to be stable in the formulations when kept for 21 days in the refrigerator (4 °C) due to results which met the variation limit of 10 % from the time-zero mean.
The results indicate the accurate use of the test item and vehicle during the study. The formulations were found to be homogeneously prepared and sufficient formulation stability under storage conditions was approved.

Duration of treatment / exposure:

Ninety consecutive days

Frequency of treatment:

Daily

Doses / concentrations

Remarks:

Doses / Concentrations:
10, 100 and 300 mg/kg bw/day
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
Dose levels were selected in collaboration with the Sponsor based on previous toxicity information from a study of a significantly shorter duration which indicated adverse stomach and liver findings at 300 and 1000 mg/kg bw/day, where the incidence and severity of findings were dose-related and the No Observed Adverse Effect Level (NOAEL) assigned in this OECD 422 study was 300 mg/kg bw/day. Therefore, 300 mg/kg bw/day was selected as a high dose level due to the potential for the prolonged exposure period to exacerbate any similar effects.
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 scheduled body weight and was adjusted at weekly intervals.

Examinations

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 throughout the treatment period. 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/behavioral toxicity. During Week 12 functional performances tests were also performed on all animals together with an assessment of sensory reactivity to different stimuli.

Behavioral Assessment
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait, Hyper/Hypothermia, Tremors, Skin color, Twitches, Respiration, Convulsions, Palpebral closure, Bizarre/Abnormal/Stereotypic behavior, Urination, Salivation, Defecation, Pilo-erection, Transfer arousal, Exophthalmia, Tail elevation, Lachrymation.

This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.


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

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


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

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


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

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


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


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
Hematological 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.

Hematology
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
- Methylene blue stained slides were prepared but reticulocytes were not assessed

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

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

Urea, Inorganic phosphorus (P), Glucose, Aspartate aminotransferase (ASAT), Total protein (Tot.Prot.), Alanine aminotransferase (ALAT), Albumin, Alkaline phosphatase (AP), Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat), Sodium (Na+) ,Total cholesterol (Chol), Potassium (K+), Total bilirubin (Bili), Chloride (Cl-), Bile acids, Calcium (Ca++)

Sacrifice and pathology:

Pathology
Necropsy
On completion of the dosing period all animals were killed by intravenous overdose of a suitable barbiturate agent followed by exsanguination.

All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

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

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

Adrenals, Ovaries Aorta (thoracic), Pancreas, Bone & bone marrow (femur including stifle joint)•, Pituitary, Bone & bone marrow (sternum), Prostate, Brain (including cerebrum, cerebellum and pons), Rectum, Caecum, Salivary glands (submaxillary), Colon, Sciatic nerve, Duodenum, Seminal vesicles, Epididymides ♦, Skin (hind limb), Esophagus, Spinal cord (cervical, mid-thoracic and lumbar), Eyes*, Gross lesions, Spleen, Heart, Stomach, Ileum (including Peyer’s patches), Testes ♦, Jejunum, Thymus, Kidneys, Thyroid/Parathyroid, Liver, Tongue• Lungs (with bronchi) # Trachea, Lymph nodes (mandibular and mesenteric), Urinary bladder, Mammary glands, Uterus (with cervix), Muscle (skeletal)•, Vagina.
* Eyes fixed in Davidson’s fluid
♦ Preserved in Modified Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative
• Retained only and not processed

All tissues were dispatched to the Test Site (Propath UK Ltd, Willow Court, Netherwood Road, Rotherwas, Hereford, HR2 6JU) for processing (Principal Investigator: N Fower). All tissues from control and 300 mg/kg bw/day dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed.

Microscopic examination was conducted by the Study Pathologist (Peter Millar at Peter Millar Associates Ltd., 3 Queen Charlotte Lane, Edinburgh, EH6 6AY).

Statistics:

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 variance were assessed using suitable ANOVA, or if required, ANCOVA with appropriate covarities. Any transformed data were analyzed to find the lowest treatment level that showed a significant effect using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found but the data shows non-homogeneity of means, the data were analyzed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests was performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).

Probability values (p) are presented as follows:

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

Results and discussion

Results of examinations

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):

no effects observed

Food efficiency:

not examined

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:

no effects observed

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):

See results

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

Mortality
There were no unscheduled deaths.


Clinical Observations
Neither the type, incidence or distribution of clinical signs apparent indicated an adverse effect of treatment at 10, 100 or 300 mg/kg bw/day.

Animals of either sex treated with 300 mg/kg bw/day had increased salivation post-dose from Day 22 (Females) and Day 23 (Males). Eight males and six females treated with 100 mg/kg bw/day also showed incidences of increased salivation from Day 44 (Females) and Day 45 (Males). Observations of this nature are commonly experienced following the oral administration of an unpalatable or slightly irritant test item formulation and in isolation are considered not to be of toxicological importance.

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

Functional Observations
Behavioral Assessments
There were no treatment-related changes in the behavioural parameters measured.

All inter and intra group differences in urination and defecation were considered to be a result of normal variation for rats of the strain and age used, and the differences were of no toxicological importance.

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

Males treated with 10 mg/kg bw/day had a statsitically significant reduction in overall activity. No dose relationship was apparent and in the absence of any associated supporting clinical observations to suggest a neurotoxic effect of treatment, this intergroup difference was considered not to be toxicologically significant.

Females treated with 100 mg/kg bw/day had a statistically significant reduction in overall mobility. No dose relationship was evident, therefore the intergroup differences were considered to represent normal biological variation rather than an effect of treatment.

Sensory Reactivity Assessments
There were no treatment-related changes in sensory reactivity.

All inter and intra group differences in sensory reactivity scores were considered to be a result of normal variation for rats of the strain and age used and were of no toxicological importance.


Body Weight
Overall body weight gain was lower than control for males at 300 mg/kg bw/day. Group mean body weight gains were generally lower than control throughout the treatment period with differences from control attaining statistical significance during Weeks 7, 10 and 11. Whilst an effect of treatment on body weight development was apparent in males at this dose level, the magnitude of the effect observed was considered insufficient in severity to consider the effect adverse.

No effects of treatment on body weight development were apparent for females treated with 300 mg/kg bw/day or animals of either sex treated with 10 or 100 mg/kg bw/day.


Food Consumption
There was no obvious effect of treatment on food consumption at 10, 100 or 300 mg/kg bw/day.


Water Consumption
Daily visual inspection of water bottles did not reveal any overt differences in water consumption at 10, 100 or 300 mg/kg bw/day.

Ophthalmscopic Examination
There were no treatment-related ocular effects.

Laboratory Investigations
Hematology
Assessment of hematology parameters did not indicate any obvious effect of treatment at 10, 100 or 300 mg/kg bw/day.

Males treated with 300 mg/kg bw/day showed a statistically significant reduction in mean corpuscular hemoglobin concentration and a statistically significant increase in hematocrit, neutrophil count and platelet count. Males treated with 100 mg/kg bw/day also showed a statistically significant increase in platelet count. All treated females showed a statistically significant reduction in mean corpuscular hemoglobin and mean corpuscular hemoglobin concentration. Females treated with 300 and 100 mg/kg bw/day showed a statistically significant reduction in mean corpuscular volume. Females treated with 300 mg/kg bw/day also showed a statistically significant increase in erythrocyte count. Females treated with 100 mg/kg bw/day also showed a statistically significant reduction in hemoglobin. The majority of individual values were within the historical control range and in the absence of any supporting microscopic findings to suggest a treatment-related effect in the spleen, the intergroup differences were considered to be of no toxicological importance.

Blood Chemistry
Animals of either sex treated with 300 mg/kg bw/day had increased alanine aminotransferase and aspartate animotransferase levels at the end of the treatment period. Males treated with 100 mg/kg bw/day also had increased alanine aminotransferase. With the exception of aspartate aminotransferase in the females, all intergroup differences achieved statistical significance and the majority of individual parameters exceeded the normal background ranges for rats of the strain and age used.

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

Males treated with 300 mg/kg bw/day also had a statistically significant reduction in bilirubin, however, all of the individual parameters were within the historical control range; therefore the intergroup differences were considered not to be toxicologically significant. All treated males had a statistically significant increase in chloride. Males treated with 300 mg/kg bw/day also had a statistically significant reduction in urea and males treated with 10 mg/kg bw/day had a statistically significant reduction in creatinine. All individual parameters were within normal background ranges and in the absence of a true dose related response in chloride and creatine or any histopathological correlates, the intergroup differences were considered not to be of toxicological significance.

Pathology
Necropsy
Neither the type, incidence nor distribution of macroscopic findings observed at necropsy indicated any effect of treatment at dosages up to 300 mg/kg bw/day.

One male treated with 300 mg/kg bw/day, three males and two females treated with 100 mg/kg bw/day, one control male and two control females had reddened lungs at necropsy. In the absence of a true dose related response and due to the presence of the finding in control animals, the intergroup differences were considered unrelated to treatment. Two females treated with 10 mg/kg bw/day had unilateral increased pelvic space of the kidney. In the absence of a similar effect at 300 mg/kg bw/day or any microscopic changes to suggest an effect of treatment in the kidney, these findings were considered not to be of toxicological importance.

Organ Weights
Animals of either sex treated with 300 mg/kg bw/day and males treated with 100 mg/kg bw/day showed an increase in liver weights both absolute and relative to terminal body weight with differences in males at 300 mg/kg bw/day achieving statistical significance. Although a true dose related response was not evident, the majority of individual body weight relative values at 300 mg/kg bw/day exceeded the background control ranges. In males at 100 mg/kg bw/day, individual weights exceeded background control ranges for absolute liver weights in three animals and in five animals for body weight relative liver weights.

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

Females treated with 100 mg/kg bw/day showed a statistically significant reduction in liver weights, the majority of individual values were within normal background ranges and therefore were considered not to be of toxicological importance.


Histopathology
There were no treatment-related microscopic findings.

All of the histopathological findings encountered were considered to have arisen spontaneously or at post mortem.

Discussion
Administration of AS1100 by oral gavage at dose levels of 10, 100 and 300 mg/kg bw/day resulted in treatment related effects in animals of either sex at 300 mg/kg bw/day and in males at 100 mg/kg bw/day.

Males treated with 300 mg/kg bw/day had statistically significantly lower body weight gains on three occasions and overall body weight gains were reduced when compared to control animals. The magnitude of the difference from control, however, was not substantial enough to consider the effect adverse.

Animals of either sex treated with 300 mg/kg bw/day and males treated with 100 mg/kg bw/day had an increase in liver weights, both absolute and relative to terminal body weight, and an associated increase in alanine aminotransferase and aspartate aminotransferase. Individual values for both liver weights and hepatic enzymes exceeded background control ranges for the majority of animals, however, there were no microscopic abnormalities detected in the liver. The intergroup differences in liver weight and hepatic enzyme leves were, therefore, considered to represent an adaptive response to treatment and consequently were considered not to represent an adverse effect of treatment.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose tested

2

Dose descriptor:

NOEL

Effect level:

10 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: body weight; organ weights; clinical chemistry

3

Dose descriptor:

NOEL

Effect level:

100 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: body weight; organ weights; clinical chemistry

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The oral administration of AS1100 to rats by gavage for a period of 90 consecutive days at dose levels of 10, 100 and 300 mg/kg bw/day resulted in treatment related effects in animals of either sex at 300 mg/kg bw/day and in males at 100 mg/kg bw/day. The No Observed Effect Level (NOEL) was considered to be 10 mg/kg bw/day for males and 100 mg/kg bw/day for females.

Lower body weight gains in males treated with 300 mg/kg bw/day were considered insufficient in magnitude to represent an adverse effect of treatment. The increased liver weights in animals of either sex treated with 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day, accompanied by associated increases in hepatic enzymes, in the absence of microscopic changes in the liver do not represent an adverse effect of treatment. The effects were considered to represent an adaptive response to treatment and therefore considered not to represent a serious risk to health.

For this reason, 300 mg/kg bw/day may be regarded as a 'No Observed Adverse Effect Level' (NOAEL) for animals of either sex.

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).

 

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

 

Methods…….

The test item was administered by gavage to three groups, each of ten male and ten female Wistar Han™:RccHan™:WIST strain rats, for ninety consecutive days, at dose levels of 10, 100 and 300 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

 

The dose levels were chosen in collaboration with the Sponsor based on previous toxicity work including an OECD 422 study in which the No Observed Adverse Effect Level (NOAEL) was 300 mg/kg bw/day. Therefore, 300 mg/kg bw/day was selected for investigation as a high dose due to the prolonged exposure period.

 

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

 

Results…….

Mortality

There were no unscheduled deaths.

 

Clinical Observations

Neither the type, incidence or distribution of clinical signs apparent indicated an adverse effect of treatment at 10, 100 or 300 mg/kg bw/day.

 

Behavioral Assessment

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

 

Functional Performance Tests

There were no toxicologically significant changes in functional performance.

 

Sensory Reactivity Assessments

There were no treatment-related changes in sensory reactivity.

 

Body Weight

There was no adverse effect of treatment on body weight development at 10, 100 or 300 mg/kg bw/day. 

 

Food Consumption

There was no obvious effect of treatment on food consumption at 10, 100 or 300 mg/kg bw/day.

 

Water Consumption

Daily visual inspection of water bottles did not reveal any overt differences in water consumption at 10, 100 or 300 mg/kg bw/day.

 

Ophthalmoscopy

There were no treatment-related ocular changes.

 

Hematology

There were no toxicologically significant effects detected in the hematological parameters examined.

 

Blood Chemistry

Animals of either sex treated with 300 mg/kg bw/day and males treated with 100 mg/kg bw/day had increased alanine aminotransferase and aspartate animotransferase at the end of the treatment period. No such effects were detected in females treated with 100 mg/kg bw/day or animals of either sex treated with 10 mg/kg bw/day.

 

Necropsy

Neither the type, incidence nor distribution of macroscopic findings observed at necropsy indicated any effect of treatment at dosages up to 300 mg/kg bw/day.

 

Organ Weights

Animals of either sex treated with 300 mg/kg bw/day showed an increase in liver weights both absolute and relative to terminal body weight with differences in males achieving statistical significance. Males treated with 100 mg/kg bw/day also showed an increase in both absolute and body weight relative liver weights. No toxicologically significant effects were detected in females treated with 100 mg/kg bw/day or animals of either sex treated with 10 mg/kg bw/day.

 

Histopathology

There were no treatment related microscopic abnormalities detected.

All of the histopathological findings encountered were considered to have arisen spontaneously or at post mortem.

 

Conclusion

The oral administration of AS1100 to rats by gavage for a period of 90 consecutive days at dose levels of 10, 100 and 300 mg/kg bw/day resulted in treatment related effects in animals of either sex at 300 mg/kg bw/day and in males at 100 mg/kg bw/day. The No Observed Effect Level (NOEL) was considered to be 10 mg/kg bw/day for males and 100 mg/kg bw/day for females.

 

Lower body weight gains in males treated with 300 mg/kg bw/day were considered insufficient in magnitude to represent an adverse effect of treatment. The increased liver weights in animals of either sex treated with 300 mg/kg bw/day and in males treated with 100 mg/kg bw/day, accompanied by associated increases in hepatic enzymes, in the absence of microscopic changes in the liver do not represent an adverse effect of treatment. The effects were considered to represent an adaptive response to treatment and therefore considered not to represent a serious risk to health.

For this reason, 300 mg/kg bw/day may be regarded as a 'No Observed Adverse Effect Level' (NOAEL) for animals of either sex.