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:
The in-life phase of the study was conducted between 08 September 2011 (first day of treatment) and 08 December (final day of necropsy).
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do not affect the quality of relevant results.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Sponsor's identification : AS 100 (Di-isopropyl xanthogen polysulphide)
Description : yellow liquid
Batch number : 246041110023
Date received : 05 August 2011
Storage conditions : room temperature in the dark
Expiry date : 18 July 2012

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
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 six 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 179 to 224g, the females weighed 140 to 171g, and were approximately five 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 . 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. 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.


ENVIRONMENTAL CONDITIONS
The animals were housed in a single air-conditioned room. 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 mean temperatures and humidities are included in the study records. The temperature and relative humidity controls were set to achieve target values of 21 ± 2ºC and 55 ± 15% respectively. Short term deviations from these targets were considered not to have affected the purpose or integrity of the study.








Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
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 previously determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results show ed the formulations to be stable for at least twenty one days. Formulations were initially prepared weekly and thereafter fortnightly and stored at 4ºC in the dark.

Samples of each test item formulation were taken and analysed for concentration of AS 100 (Di-isopropyl xanthogen polysulphide).

The results indicate that the prepared formulations were within ±7 % of the nominal concentration.

The test item was administered daily, for up to ninety consecutive days, by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 4 ml/kg bw/day 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.


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 AS 100 (Di-isopropyl xanthogen polysulphide).

The concentration of AS 100 Di-isopropyl xanthogen polysulphide in the test item formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique.

The results indicate that the prepared formulations were within ±7 % of the nominal concentration.

The full method used for analysis of formulations and the results obtained are given in Appendix 16: Chemical Analysis of Test Item Formulations, Methods and Results (see attached background material).
Duration of treatment / exposure:
Up to 90 consecutive days.
Frequency of treatment:
The test item was administerd daily.
Doses / concentrations
Remarks:
Doses / Concentrations:
control (0 mg/kg bw/day), 10 mg/kg bw/day, 50 mg/kg bw/day, 250 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 males and 10 females per dose.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were selected following review of available toxicity information provided by the study sponsor.

- Rationale for animal assignment (if not random): 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.
Positive control:
No positive control.

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. 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

FUNCTIONAL PERFORMANCE TEST:
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 day, 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. 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. 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 following parameters were observed:
Grasp response
Touch escape
Vocalisation
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach

BODYWEIGHT:
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:
Daily visual inspection of water bottles during the first week of dosing revealed possible intergroup differences. Water consumption was, therefore, measured and recorded for each cage group from Day 8 onwards.

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

HAEMATOLOGY:
Haemoglobin (Hb)
Erythrocyte count (RBC)
Haematocrit (Hct)
Erythrocyte indices - mean corpuscular haemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular haemoglobin concentration (MCHC)
Total leucocyte count (WBC)
Differential leucocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic) - Methylene blue stained slides were prepared and reticulocytes 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 (Bile)
Calcium (Ca++)










Sacrifice and pathology:
PATHOLOGY:
On completion of the dosing period all surviving 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 (cervical and mesenteric), Trachea, Mammary glands, Urinary bladder, Muscle (skeletal), Uterus, Oesophagus

* Eyes fixed in Davidson’s fluid
♦ Preserved in Bouin’s fluid transferred to Industrial Methylated Spirits (IMS) approximately 48 hours later
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before immersion in fixative

All tissues were despatched for processing. All tissues from control and 250 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. Any macroscopically observed lesions were also processed.

Since there were indications of treatment-related changes in animals treated at 250 mg/kg bw/day, examination was subsequently extended to include similarly prepared sections from animals in the low and intermediate groups.

Microscopic examination was conducted by the Study Pathologist. A peer review of the histopathology examination was performed by the Principal
Investigator.























Statistics:
Data were processed to give group mean values and standard deviations where appropriate.

Where appropriate, quantitative data were analysed by the Provantis™ Tables and Statistics Module. For each variable, the most suitable transformation of the data was found, the use of possible covariates checked and the homogeneity of means assessed using ANOVA or ANCOVA and Bartlett’s test. The 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 nonparametric data. If no dose response was found, but the data showed non-homogeneity of means, the data were analysed by a stepwise Dunnett (parametric) or Steel (nonparametric) test to determine significant differences from the control group. Finally, if required, pair-wise tests were 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:
effects observed, treatment-related
Description (incidence and severity):
see details on results for discussion of results
Mortality:
mortality observed, treatment-related
Description (incidence):
see details on results for discussion of results
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see details on results for discussion of results
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
see details on results for discussion of results
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Description (incidence and severity):
see details on results for discussion of results
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results for discussion of results
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results for discussion of results
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see details on results for discussion of results
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
see details on results for discussion of results
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY:
One 250 mg/kg bw/day female (No. 71) was found dead on the morning of Day 69. Pulmonary congestion was identified at necropsy and when the lungs were examined microscopically. For this reason the premature death of this animal was considered a result of a mal-dose and not test item toxicity. The test item was known to be irritant in nature and any material deposited in the upper oesophagus during dosing or by reflux from the stomach could conceivably induce asphyxiation.
No further unscheduled deaths occurred during the study.

CLINICAL OBSERVATIONS:
Clinical findings were confined to transient episodes of increased salivation observed shortly after dosing in test animals from the first (250 mg/kg bw/day) or second (50 mg/kg bw/day) week of treatment persisting through to study termination. Sporadic instances of soiled fur associated with excessive salivation were also occasionally observed. Transient episodes of increased salivation were occasionally also observed in a small number of 10 mg/kg bw/day animals.

FUNCTIONAL OBSERVATIONS:-
BEHAVIOURAL ASSESSMENTS:
There were no treatement-related changes in the behavioural assessments observed.

FUNCTIONAL PERFORMANCE TESTS:
In comparison with the concurrent control, males from all test groups showed a statistically significant increase (p<0.05) in overall activity while females from all test groups showed a reduction (p<0.01) in this parameter. The isolated nature of this change in the absence of a dose dependent trend was considered not to be of neurotoxicological importance but more likely, an indirect effect of abdominal discomfort associated with the irritancy of the test item formulation.

SENSORY REACTIVITY ASSESSMENTS:
All inter and intra group differences in sensory scores were considered to be a result of normal variation for rats of the species and strain used and, therefore, were considered to be of no toxicological significance.

BODY WEIGHT:
Body weight development throughout the study in 250 mg/kg bw/day males was consistently below that of the concurrent control and frequently achieved statistical significance (p<0.05 or p<0.01). Females at this dosage also showed slightly lower weight gains in comparison with female controls achieving statistical significance (p<0.01) at Week 7. Males and females receiving 50 mg/kg bw/day also on occasion displayed slightly low weight gains attaining statistical significance (p<0.05) in males at Weeks 5, 6 and 12 in comparison with controls

Body weight development in males and females receiving 10 mg/kg bw/day generally remained similar to that of the controls throughout the study.

FOOD CONSUMPTION:
There was no overt evidence of adverse changes in food consumption in test animals throughout the study. However, food efficiency appeared effected in 250 mg/kg bw/day males and to a lesser degree in females at this dosage. Food efficiency in animals from the 10 and 50 mg/kg bw/day treatment groups remained similar to controls throughout the treatment period.

WATER CONSUMPTION:
Gravimetric measurement of water intake undertaken from the second week of treatment revealed an overall increase of ca. 40% for 250 mg/kg bw/day males and females in comparison with controls. Water consumption in animals dosed at 10 and 50 mg/kg bw/day remained similar to that of the controls throughout the treatment period.

OPHTHALMOSCOPIC EXAMINATION:
No ocular changes were detected in any control or high dose animal prior to start of treatment or prior to termination.

LABORATORY INVESTIGATIONS:
HAEMATOLOGY:
Males and females treated with 250 mg/kg bw/day showed a reduction (p<0.01) in circulating erythrocytes together with associated changes in mean corpuscular haemoglobin and mean cell volume (both p<0.01). An increase in reticulocytes in each sex at 250 mg/kg bw/day was further evidence of an anaemia in either sex at this dose level. An increase (p<0.01) in platelet count was also seen in males and females from this dose group with females also showing elevated numbers of leucocytes (p<0.05) namely in the neutrophil fraction (p<0.01) in comparison with controls.

Both sexes receiving 50 mg/kg bw/day showed slightly low numbers of erythrocytes (RBC) in comparison with the concurrent control achieving statistical significance (p<0.01) in 50 mg/kg bw/day females. This finding was further evidenced in both sexes by an increase (p<0.01) in mean cell volume (MCV). However, almost all individual values for both RBC and MCV were within the historical ranges. The reticulocyte counts in males at 50 and 10 mg/kg bw/day achieved statistical significance (p<0.01). However, as this finding was absent in females from these test groups the toxicological relevance of this change was considered to be minimal.

A reduction in mean corpuscular haemoglobin concentration (MCHC, p<0.05 or p<0.01) was identified in all male test groups and in 250 mg/kg bw/day females. However, the variance from control was minimal and the majority of individual values were within the background ranges for rats of the age and strain used this finding was considered of no toxicological importance.

BLOOD CHEMISTRY:
Elevated levels of alanine aminotransferase (p<0.01) were detected in males at 250 mg/kg bw/day and increased plasma bilirubin levels in both sexes at 250 mg/kg bw/day (males: p<0.01, females: p<0.05) and in males at 50 mg/kg bw/day (p<0.01). Further changes included elevated levels of sodium and chloride and a reduction in inorganic phosphate (p<0.05 or p<0.01) in 250 mg/kg bw/day males. Females at 250 mg/kg bw/day also showed an increase (p<0.05) in potassium and a reduction in creatinine levels when compared to controls. Males from all test groups showed an increase in calcium levels in comparison with controls; however, it was the individual values at 50 and 250 mg/kg bw/day that were in most instances above the anticipated historical ranges.

Males at 250 and 50 mg/kg bw/day showed reductions in plasma urea accompanied in males receiving 250 mg/kg bw/day by lowered plasma glucose and cholesterol levels in comparison with controls. Reductions in these parameters are not generally indicative of an adverse effect of treatment therefore these fluctuations were considered not to be of toxicological importance. An increase (p<0.05 or p<0.01) in urea was evident in all female test groups in comparison with controls however as individual values were almost all within the acceptable historical ranges this finding was considered not to be of toxicological importance. A statistically significant reduction (p<0.05) was identified for albumin/globulin ratio in 250 mg/kg bw/day females in comparison with controls. However, with no convincing effect on total protein, this finding was considered associated with reduce diet intake.
PATHOLOGY
ORGAN WEIGHTS:
Males and females treated with 50 and 250 mg/kg bw/day showed statistically significant increases (p<0.05 or p<0.01) in the absolute and/or relative weights of the liver, kidney and spleen weights.

Males receiving 10 mg/kg bw/day also showed increases in the absolute and relative weights of the liver and spleen. However, individual values differed only slightly from the corresponding control and in the absence of any histopathological correlates; these changes were considered to be of minimal significance.

Males from all test groups showed a reduction in absolute brain weight in comparison with control males. However, in view that most of the individual values were within the background ranges for rats of the age and strain used and in the absence of any histopathological correlates, this isolated finding was considered spurious and unrelated to test item toxicity.

NECROPSY:
Examination of animals killed at study termination revealed reddened lungs in one female at 10 mg/kg bw/day, white fibrous adhesions between the lungs and heart in one female at 50 mg/kg bw/day with similar changes together with reddened lungs in three females at 250 mg/kg bw/day.

Examination of the one unscheduled death (No.71) showed fluid filled reddened lungs (indicating a mal-dose as the cause of death). There were no other macroscopic findings identified in this animal.

HISTOPATHOLOGY:
Under the conditions of this study, AS 100 (Di-isopropyl xanthogen polysulphide) caused treatment-related effects in the liver, kidneys, urinary bladder, stomach and duodenum.

One Group 4 (250 mg/kg bw/day) female was found dead during the study and considered a result of a maldose. The following findings were identified in animals killed as scheduled at the end of the treatment period.

Liver: Hepatocellular hypertrophy affecting the centrilobular and midzonal regions of the liver was seen in animals of Group 4 (250 mg/kg bw/day). In animals of this Group, glycogen content was increased in the cytoplasm of the hepatocytes, and minimal single cell necrosis of the hepatocytes was seen in males of Group 4. Some males of Group 3 (50 mg/kg bw/day) also presented a minimal centrilobular hepatocelluar hypertrophy and glycogen increase in the cytoplasm of the hepatocytes.

Kidneys: Tubular hypertrophy was seen in males and in a few females of Group 4. Minimal diffuse hyperplasia of the transitional epithelium of the urinary bladder was observed in a few males and females of Group 4.

Stomach: Acanthosis of the forestomach was seen in males and a few females of Group 4. Forestomach ulceration was present in one female of Group 4. Diffuse mucosal hyperplasia was observed in the duodenum of males and females of Group 4.

Spleen: Increased hemopoiesis and hemosiderin deposits in the spleen, and increased cellularity of the femur bone marrow were seen in animals of Group 3 and 4 were secondary to the anaemia observed at the hematological investigation in animals of these Groups.

Duodenum: Diffuse mucosal hyperplasia was observed in animals of either sex at 250 mg/kg bw/day.

Urinary Bladder: Minimal diffuse hyperplasia of the transitional epithelium was observed among animals of either sex at 250 mg/kg bw/day.

Effect levels

Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: The treatment-related findings detected at 50 mg/kg bw/day in the absence of degenerative histopathological changes was considered to be a No Observed Adverse Effect Level (NOAEL). See discussion on results for full details.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

DISCUSSION

Oral administration of the test item, AS 100 (Di-isopropyl xanthogen polysulphide), to rats for a period of up to ninety consecutive days, at dose levels of 10, 50 and 250 mg/kg bw/day, resulted in treatment-related changes in animals from the high (250 mg/kg

bw/day) and intermediate (50 mg/kg bw/day) dose groups.

There were no clinical signs to indicate toxicity, however, oral administration of the formulated test item to animals at 250 and 50 mg/kg bw/day and on occasion in 10 mg/kg bw/day animals, induced episodes of transient increased salivation around the time of dosing. Excessive salivation is frequently reported following oral administration of slightly irritant/unpalatable test item formulations and is therefore seldom associated directly with systemic toxicity. A relationship with the irritative properties of the test item may also have contributed to the one interim death (250 mg/kg bw/day female on Day 69) in this study.

Further demonstration of the irritative properties of the test item in high dose group animals involved histopathological evidence of gastric acanthosis and ulceration and duodenal mucosal hyperplasia. It is reasonable to assume a relationship between the intestinal congestion (seen microscopically) and the impaired bodyweight, food efficiency and conspicuous increase in water consumption identified in these animals. Animals tested at 50 mg/kg bw/day also showed slightly low weight gains in comparison with controls. However, in the absence of other overt toxicological findings, the significance of lower bodyweight, in these animals, was thought to be minimal.

Examination of high dose group haematological data revealed a reduction in erythrocyte count, together with associated changes in mean corpuscular haemoglobin and mean cell volume, strongly indicating an induced anaemia; a condition further indicated by a

meaningful increase in reticulocyte numbers. Additionally, high dose group animals showed elevated levels of neutrophils (females only) and an increased platelet count (both sexes). It is conceivable, given the function of these latter parameters, that the increased production may have been stimulated by the intestinal lesions that were identified microscopically. Animals at 50 mg/kg bw/day also exhibited a slight disruption in erythrocytes and mean cell volume; however, reticulocyte numbers appear not to have been greatly affected; indicating the anaemia in these animals to be of lesser relevance. Nevertheless, histopathological examination identified an affect on haemopoiesis, indicated by increased levels of haemosiderin in the spleen in animals treated at 250 mg/kg bw/day and to a lesser extent in those treated at 50 mg/kg bw/day. Haemosiderin results from deposition of iron containing pigment during the destruction of erythrocytes and the presence of this finding was also thought to be the origin for the elevated spleen weights in both sexes at 250 and 50 mg/kg bw/day. Biochemical examinations in high dose males revealed elevated levels of alanine aminotransferase

(an enzyme commonly associated with hepatic function) and increased plasma bilirubin levels (also observed in high dose females and in 50 mg/kg bw/day males). As bilirubin is a breakdown product of haemoglobin it is reasonable to associate the heightened

levels with the accelerated breakdown of erythrocytes (anaemia) that was identified in high and possibly intermediate dose groups animals. Microscopic liver changes in high dose animals involved hepatocellular hypertrophy, increased cytoplasmic glycogen and

minimal single cell necrosis (the latter in males only). Minimal centrilobular hepatocelluar hypertrophy and glycogen increase were also detected in a few 50 mg/kg bw/day males. Hepatocellular hypertrophy is commonly observed in the rodent liver following the

administration of xenobiotics and in the absence of associated inflammatory or degenerative changes a condition generally considered to be an adaptive response to treatment.

The remaining noteworthy treatment-related findings attested to an affect on kidney function that was characterised by blood biochemical changes in high group dose animals (primarily in males) involving statistically significant fluctuations in a number of

electrolyte levels. In addition, an increase in kidney weight was also evident in either sex at 250 and 50 mg/kg bw/day. Histopathologically, tubular hypertrophy was detected in both sexes at 250 mg/kg bw/day. These findings confirm impaired renal function with the cellular changes considered to be adaptive in nature possibly resultant of the presence of high concentrations of the test item. The irritative characteristics of the test item were further indicated by detection of minimal epithelial diffuse hyperplasia in the urinary bladder.

Applicant's summary and conclusion

Conclusions:
Oral administration of AS 100 (Di-isopropyl xanthogen polysulphide) to rats for a period of up to ninety days at dose levels of 10, 50 and 250 mg/kg bw/day resulted in the presence of treatment related changes in both sexes of animals from all dose groups precluding identification of a No Observed Effect Level.

The treatment-related findings detected at 50 mg/kg bw/day in the absence of degenerative histopathological changes was considered to be a No Observed Adverse Effect Level (NOAEL).
Executive summary:

Introduction.

This study was designed to investigate the systemic toxicity of the test item and is compatible with the recommendations of 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 continuously by gavage to three groups, each of ten male and ten female Wistar Han: RccHan: WIST strain rats, for up to ninety consecutive days, at dose levels of 10, 50 and 250 mg/kg bw/day. A control group of ten males and ten females was dosed with vehicle alone (Arachis oil BP).

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. 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 prior to start of treatment and during Week 12 of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from all test and control animals was performed.

Results.

Mortality.

One 250 mg/kg bw/day female (No. 71) was found dead on the morning of Day 69. In the absence of any supporting evidence of toxicity this death was considered a result of a mal-dose and not test item toxicity.

There were no further unscheduled deaths which occurred during the study.

Clinical Observations.

Increased salivation developed in 50 and 250 mg/kg bw/day animals within the first two weeks of treatment and persisted through to study termination. Occasional instances of excessive post dose salivation were also observed in a small number of 10 mg/kg bw/day animals. Associated with the excessive salivation were instances of soiled fur that was seen sporadically in test animals.

Behavioural Assessment.

There were no treatment-related changes in the behavioural assessments observed.

Functional Performance Tests.

Functional performance assessments did not indicate any evidence of neurotoxicological changes.

Sensory Reactivity Assessments.

Sensory reactivity assessments did not indicate any adverse effects of treatment.

Body Weight.

Impaired body weights were evident in either sex at 250 mg/kg bw/day throughout the study. A similar trend albeit of lesser significance was also apparent in either sex at 50 mg/kg bw/day. Body weight development in males and females receiving 10 mg/kg bw/day remained unaffected by treatment.

Food Consumption and Food Efficiency.

Dietary intake in test animals was comparable to that of the controls throughout the study. However, reduced food efficiency was evident in 250 mg/kg bw/day males and females. Food efficiency in animals from the remaining treatment groups remained similar to that of the controls throughout the treatment period.

Water Consumption.

Gravimetric measurement of water consumption revealed both sexes treated at 250 mg/kg bw/day to have consumed considerably more water than the corresponding controls. Water intake in animals from the remaining treatment groups remained similar to that of the controls throughout the treatment period.

Ophthalmoscopy:

No ocular changes were detected in any of the control or high dose animals prior to start of treatment or prior to termination.

Haematology.

Males and females treated with 250 mg/kg bw/day showed a reduction in circulating erythrocytes together with associated changes in mean corpuscular haemoglobin and mean cell volume together with increases in platelet and reticulocyte counts. In addition, 250 mg/kg bw/day females also showed an increase in leucocyte count in comparison with controls. Both sexes at 50 mg/kg bw/day showed slightly lower numbers of erythrocytes and an increase in mean cell volume in comparison with controls. There were no convincing treatment-related changes in animals treated at 10 mg/kg bw/day.

Blood Chemistry.

Elevated levels of alanine aminotransferase were detected in 250 mg/kg bw/day males accompanied in both sexes by increased plasma bilirubin levels, the latter was also observed in males at 50 mg/kg bw/day. In addition, elevated levels of sodium and chloride and a reduction in inorganic phosphate were detected in 250 mg/kg bw/day males and an increase in potassium and a reduction in creatinine were seen in females at 250 mg/kg bw/day when compared to controls. 250 mg/kg bw/day males and males at 50 mg/kg bw/day showed a noteworthy increase in calcium levels in comparison with controls. There were no other convincing treatment-related changes detected.

Organ Weights.

Males and females treated with 50 and 250 mg/kg bw/day showed treatment-related statistically significant increases in the weights of the liver, kidney and spleen in comparison with controls.

Necropsy.

Macroscopic changes detected in animals at the end of the treatment period were confined to reddened lungs in one female at 10 mg/kg bw/day, white fibrous adhesions between the lungs and heart in one female at 50 mg/kg bw/day with similar changes together with reddened lungs in three females at 250 mg/kg bw/day.

Examination of the one unscheduled death (No.71) revealed fluid filled reddened lungs.

Histopathology.

Examination of animals that survived treatment revealed:

Liver - Hepatocellular hypertrophy in animals at 250 mg/kg bw/day accompanied by increased glycogen content in the cytoplasm of the hepatocytes, and minimal single cell necrosis of the hepatocytes amongst males only. A small number of males treated at 50 mg/kg bw/day also showed minimal centrilobular hepatocelluar hypertrophy and glycogen increase in the cytoplasm of the hepatocytes.

Kidneys - Tubular hypertrophy was seen in 250 mg/kg bw/day males and in a few females from this treatment group. In addition, minimal diffuse hyperplasia of the transitional epithelium of the urinary bladder was observed in a few males and females at 250 mg/kg bw/day.

Stomach - Acanthosis of the forestomach was seen in males and a few females at 250 mg/kg bw/day accompanied in one of the females by ulceration in the forestomach. These changes were accompanied in a number of each sex at 250 mg/kg bw/day by

diffuse mucosal hyperplasia in the duodenum.

Spleen - Increased hemopoiesis and hemosiderin deposits in the spleen, and increased cellularity of the femur bone marrow were seen in animals at 50 and 250 mg/kg bw/day.

Conclusion.

Oral administration of AS 100 (Di-isopropyl xanthogen polysulphide) to rats for a period of up to ninety days at dose levels of 10, 50 and 250 mg/kg bw/day resulted in the presence of treatment related changes in both sexes of animals from all dose groups precluding identification of a No Observed Effect Level.

The treatment-related findings detected at 50 mg/kg bw/day in the absence of degenerative histopathological changes was considered to be a No Observed Adverse Effect Level (NOAEL).