Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity study: via oral route: NOAEL = 150 mg/kg bw/day (rat, gavage, OECD 407, GLP, K. rel.1). However, this NOAEL was not used for the risk assessment because it is considered too low, resulting from the wide dose range used in this study (15, 150, 1000 mg/lkg bw/day). In an OECD 421 reproductive toxicity study a NOAEL of 250 mg/kg bw/day was identified and used for the risk assessment.

A marked difference in clinical signs and toxicity was observed in the OECD 407 repeat dose study and the OECD 421 reproductive toxicity screening study. In the OECD 407 the test substance was tolerated quite well at 1000mg/kg bw/day, whereas in the OECD 421 after 3 days 3 animals died or were terminated prematurely and surviving animals showed severe clinical signs; the dose level of the substance was reduced to 500 mg/kg bw/day so that the study could proceed to completion. Differences between the studies included the strain of rat used (Sprague Dawl;ey in the OECD 407 and Wistar in the OECD 421), and the vehicle used (carboxy methyl cellulose in the OECD 407 and arachis oil in the OECD 421). A 5 -day investigative study using both strains of rat and two batches of the test substance formulated in arachis oil was performed to establish the reason for the difference in toxicity between the two studies. The results were not conclusive, showing perhaps a modest increase in sensitivity of Wistar rats and females versus males. Other factors such as the age and weight of the animals may have also had an influence on the results.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From June 15 to December 08, 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted according to OECD 407 Guideline without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
other: OPPTS 870.3050, Repeated Dose 28-Day Oral Toxicity Study in Rodents, July 2000.
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on March 07, 2005/ signed on June 09, 2005)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The rat was chosen as the test species because of its acceptance as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The Crl:CD® (SD)IGS BR strain was used because of the historical control data available in this laboratory
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd, Margate, Kent, England
- Age at study initiation: 41 – 45 days
- Weight at study initiation: 220-253 g (males), 162-191 g (females)
- Fasting period before study: Not applicable
- Housing: The animals were housed five of one sex per cage. The cages were made of a stainless steel body with a stainless steel mesh lid and floor, and were suspended above absorbent paper which was changed at appropriate intervals.
- Diet: Standard rodent diet (Rat and Mouse No. 1 Maintenance Diet from Special Diets Services Ltd., Witham, Essex, England), ad libitum
- Water: Potable water taken from the public supply, ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Each cage was kept at positive pressure with respect to the outside by its own supply of filtered fresh air, which was passed to atmosphere and not re-circulated.
- Photoperiod: 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Details on route of administration:
The oral route of administration was chosen to simulate the conditions of potential human exposure
Vehicle:
other: 1.0% w/v methylcellulose in water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The required amount of test substance, as supplied, was ground into a fine powder in a mortar and small amounts of vehicle were then mixed with it to form a smooth paste. Further amounts of the vehicle were gradually added to the paste and mixed with it. The final suspension was mixed using a high-shear homogenizer before being transferred to final containers. All formulations were prepared freshly each week and stored at 4°C in the dark.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The tested substance is stable and forms homogeneous mixtures with vehicle.
- Concentration in vehicle: 0, 1.5, 15 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of each formulation prepared for administration in Week 1 of treatment were analysed for achieved concentration of the test substance. The concentrations of the tested substance in test formulations were analyzed during the study with High performance liquid chromatograph (HPLC) fitted with a UV-Vis detector.

Typical chromatographic conditions:
Analytical column: Symmetry C18, 3.5 μm, 150 x 4.6mm.
Column temperature: Ambient, nominally +21 ºC.
Mobile phase: Acetonitrile / water (80/20 v/v).
Flow rate: 1.0 mL/minute.
Detector wavelength: UV, 279 nm.
Injection volume: 10 µL
Approximate retention time: 2.5 min.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Animals were dosed once each day at approximately the same time each day, seven days per week.
Dose / conc.:
15 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosages used were selected with reference to previous work where the tested substance was tolerated over a period of seven consecutive days up to 1000 mg/kg bw/day. Based on this result, the dosages of 0, 15, 150 and 1000 mg/kg bw/day were selected.
- Rationale for animal assignment (if not random): sequential allocation (1 per sex dose group) until all cages filled as designated
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): no data available
Positive control:
Not applicable
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice a day

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded one week before treatment commenced (Week -1), on the day that treatment commenced (Week 0), weekly throughout the treatment and before necropsy.

FOOD CONSUMPTION:
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started (Week -1), and each week throughout the treatment. From these records the mean weekly consumption per animal (g/rat/week) was calculated for each cage.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29
- Anaesthetic used for blood collection: Isoflurane
- Animals fasted: Yes; overnight fasting
- How many animals: All animals
- Parameters checked (haematology): Haemoglobin (Hb), Erythrocyte count (RBC), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Total white cell count (WBC), Differential WBC count - Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M), Large unstained cells (LUC), Platelet count (Plt), Prothrombin time (PT) and Activated partial thromboplastin time (APTT)
- Parameters checked (clinical chemistry): Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Sodium (Na), Potassium (K), Total protein (Total Prot), Albumin (Alb) and Albumin/globulin ratio (A/G Ratio)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: daily
- Dose groups that were examined: 5 males and 5 females from each group.
- Battery of functions tested: sensory reactivity, grip strength, motor activity

Sacrifice and pathology:
NECROPSY: All animals were killed by carbon dioxide asphyxiation at the end of the treatment period.
GROSS PATHOLOGY: Yes; all animals were subject to a detailed necropsy.
HISTOPATHOLOGY: Yes (All tissues: Control and High-dose groups)
- Testes and epididymides were fixed in Bouin’s solution prior to transfer to 70% industrial methylated spirit. Samples (or the whole) of the other tissues listed below from all animals were preserved in 10% neutral buffered formalin:
Adrenals, brain, caecum, colon, duodenum, epididymides, femurs+, head#, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes – mandibular and mesenteric, oesophagus#, ovaries, pancreas#, prostate, rectum, sciatic nerves+, seminal vesicles, spinal cord, spleen, sternum#, stomach, testes, thymus, thyroid with parathyroids, trachea, urinary bladder, uterus and cervix

+ Only one processed for examination
# Not processed for examination

Tissue samples were dehydrated, embedded in paraffin wax, sectioned at approximately four to five micron thickness and stained with haematoxylin and eosin, except the testes which were stained using a standard periodic acid/Schiff (PAS) method.
Other examinations:
ORGAN WEIGHTS: adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus
Statistics:
All statistical analyses were carried out separately for males and females.

All analyses were carried out using the individual animal as the basic experimental unit.

The following data types were analyzed at each time point separately:
Grip strength and motor activity
Bodyweight, using gains over appropriate study periods
Blood chemistry and haematology
Organ weights, both absolute and adjusted for terminal bodyweight
Pathological findings, for the number of animals with and without each finding

For categorical data, including pathological findings, the proportion of animals was analysed using Fisher’s Exact test (Fisher 1973) for each treated group versus the control.
For continuous data, Bartlett’s test (Bartlett 1937) was first applied to test the homogeneity of variance between the groups. Using tests dependent on the outcome of Bartlett’s test, treated groups were then compared with the Control group, incorporating adjustment for multiple comparisons where necessary.

Significant differences between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01) level.
a - p<0.05; b - p<0.01 - using categorical or parametric tests
A - p<0.05; B - p<0.01 - using non-parametric tests
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment related clinical sign of underactivity was observed occasionally in males and females receiving 1000 mg/kg/day during the treatment period. This sign was generally observed on isolated occasions from approximately 1 hour to 4 hours after dosing. This time onset might indicate that underactivity resulted from a systemic effect of ST 06 C 05, leaving time for some absorption to occur first.
Mortality:
no mortality observed
Description (incidence):
There was no unscheduled deaths during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Low bodyweight gains with associated low efficiency of food utilisation observed in males treated at 1000 mg/kg/day were considered to be due to treatment. However, due to the small magnitude of difference from controls, no actual bodyweight losses being apparent and some males having overall gains that were within the concurrent control range, these low bodyweight gains were not considered to be detrimental to the overall health of the animals.
Therefore, they were considered not to represent overt toxicity within the context of this study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
The food consumption over the study period for treated groups was generally similar to that of control thus indicating no effect of treatment.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Low bodyweight gains with associated low efficiency of food utilisation observed in males treated at 1000 mg/kg/day were considered to be due to treatment. However, due to the small magnitude of difference from controls, no actual bodyweight losses being apparent and some males having overall gains that were within the concurrent control range, these low bodyweight gains were not considered to be detrimental to the overall health of the animals.
Therefore, they were considered not to represent overt toxicity within the context of this study.
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Analysis for animals receiving treatment at 1000 mg/kg bw/day revealed lower mean white blood cell counts for males (reflected in lower mean eosinophil counts and lymphocyte counts) and higher mean prothrombin time for animals of both sexes, when compared with their respective control mean values. No relevant effects were recorded for other dose levels.
No histopathological findings could be linked either to the lower than control white blood cell (WBC) count observed for males or to the higher than control mean prothrombin time noted for animals of both sexes receiving treatment at 1000 mg/kg/day. Additionally, the values measured for these parameters were within expected background range (7.99 to 18.29 in males for WBC and 12.3 to 16.5 in females for prothrombin time) thus, although the changes may be attributable to treatment, they are considered to be of no toxicological importance in the context of this study.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Analysis showed that animals receiving treatment at 1000 mg/kg bw/day had lower urea concentrations and higher albumin concentrations with concomitant higher A/G ratio for animals of both sexes, as well as lower creatinine concentration for females when compared with their respective control mean values. No relevant effects were recorded for other dose levels.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
All animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena and no deviation from normal was recorded.
The motor activity assessment revealed a reduced activity (both cage floor and rearing activity) in males receiving 1000 mg/kg/day. Despite these recordings occurring approximately 24 hours after dosing, there was some correlation with the occasional post-dose underactivity seen at this level (Animal number 19 was noted to be underactive after dosing on the day the motor activity was performed). The differences from controls in motor activity were minor as indicated by only being seen on a few occasions in individual animals after dosing and only being clearly evident during the first part of the 1-hour motor activity sessions, when the animals, including controls, were showing the highest activity. Although females receiving 1000 mg/kg/day were also noted to show a low level of post-dose underactivity, they did not show any clear treatment-related effects on motor activity. There was no indication that the slightly lower motor activity in males was a neurotoxic effect. It is possible that the slightly lower activity observed in males receiving 1000 mg/kg/day was due to a combination of general toxicity (as indicated by the low bodyweight gains and inefficient food utilisation) and possibly some slight abdominal discomfort due to the higher liver weights and hepatic enlargement seen in these animals. The aetiology of the low rearing activity noted in males treated at 150 mg/kg/day is not clear from this study. In the absence of any other corroborative findings at this level in males, the low activity is considered not to be of toxicological importance.
Sensory reactivity and grip strength were considered to be unaffected by treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
In the liver, centrilobular hepatocyte hypertrophy was found in the majority of animals, of both sexes, receiving treatment at 1000 mg/kg/day. This treatment-related finding correlates with the reported macroscopic finding at necropsy of liver enlargement, and with the high liver weights recorded for these animals. The liver is one of the major organs involved in xenobiotic metabolism and enlargement/increased weight due to hepatocyte hypertrophy is a typical adaptive response. Therefore, the changes reported in the liver are considered not to be adverse but do indicate the liver as a target organ.
The increased weights of the kidney, recorded for females treated at 1000 mg/kg/day, might have reflected an increased efficiency of the kidneys in animals treated at 1000 mg/kg/day. A consequence of such an increased efficiency would have been to lower waste concentrations in plasma of affected animals. There was evidence of a reduction in urea and creatinine plasma concentrations, which are waste products, in the females treated at 1000 mg/kg/day and of a reduction in plasma urea concentration in the males, although no increase in the males’ kidney weights was noted. No correlated histopathology findings were observed, thus the higher weights recorded for females at 1000 mg/kg/day were not indicative of toxicity. Alternatively, the lower plasma urea concentrations might have been linked with the higher plasma albumin concentrations observed in males and females receiving 1000 mg/kg/day. As urea is a breakdown product of protein, if a reduction in the breakdown of protein (e.g. albumin) in the plasma occurred, lower plasma urea concentrations concomitant with higher plasma albumin concentration would not be unexpected.
Statistically significantly lower than control bodyweight-adjusted mean ovaries weights were recorded for all the treated female groups. There was, however, no evidence of a dose relationship and no histopathology findings were observed in the ovaries of the treated females. It was therefore concluded that these organ-weight differences were the reflection of the stage of oestrus each cage of animals was in and that these changes were not related to treatment.
Although lower than control mean adrenals and spleen weights (adjusted for bodyweight) were noted for males treated at 1000 mg/kg/day, in the absence of any histopathology findings in the adrenals or in the spleen, these changes were considered to be of no toxicological importance in the context of this study.
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Key result
Dose descriptor:
NOAEL
Effect level:
150 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
behaviour (functional findings)
Key result
Critical effects observed:
no

The homogeneity and stability was confirmed for test item in 1% methylcellulose formulations at nominal concentrations of 1 mg/mL and 100 mg/mL during distribution between the bottles, during magnetic stirring for 2 hours, at ambient temperature storage for 2 days and at refrigerated storage for 8 days. The storage times represented the maximum time from preparation to completion of administration.

 

Formulation analysis showed that the mean concentrations of the test substance in dose formulations were within ±6% of nominal concentrations, confirming accurate formulation.

Conclusions:
Under the test conditions, the NOAEL for test item is 150 mg/kg bw/day in rats. The test item is not classified according to the annex I of the Regulation (EC) No. 1272/2008 (CLP) and of the GHS.
Executive summary:

In a repeated dose oral toxicity study conducted according to the OECD Guideline 407 and in compliance with GLP, test item was administered daily by oral gavage to groups of Sprague-Dawley rats (5/sex/dose) at the dose-levels of 0 (vehicle), 15, 150 and 1000 mg/kg bw/day in the vehicle (1.0% w/v methylcellulose in water) with the dose volume of 10 mL/kg bw/day for 28 days. Examinations during the study included: clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, haematology, blood chemistry, organ weight, gross pathology and histopathology

 

There were no unscheduled deaths during the study and treatment related effects observed were confined to animals receiving 1000 mg/kg bw/day. Sensory reactivity and grip strength were considered to be unaffected by treatment for all animals. Animals of both sexes receiving treatment at 1000 mg/kg bw/day were observed with low transient incidences of post-dose salivation, underactivity, abnormal gait and flat posture. Motor activity assessment revealed a slightly reduced activity (both cage floor and rearing activity) for the first half hour of the 1-hour recording period in males receiving 1000 mg/kg bw/day, whereas males at 150 mg/kg bw/day exhibited a reduced rearing activity. Low bodyweight gain and low efficiency of food utilisation, recorded in male animals receiving 1000 mg/kg bw/day, were considered to be related to treatment, whereas food consumption was considered not to be affected by treatment. At the end of the treatment period, analysis of the haematological parameters for animals receiving treatment at 1000 mg/kg bw/day revealed lower than control white blood cell count for males and higher than control mean prothrombin time for animals of both sexes. Analysis of the blood chemistry parameters, on this occasion, revealed for animals treated at 1000 mg/kg bw/day, lower than control urea concentrations and higher than control albumin concentrations with concomitant higher albumin to globulin (A/G) ratios for animals of both sexes, as well as lower creatinine concentrations for females. In males and females receiving treatment at 1000 mg/kg bw/day, higher than control mean liver weights were measured, enlargement of the liver was noted at the gross pathology examination and liver centrilobular hepatocyte hypertrophy was found in the majority of animals at the histopathology examination. Additionally, higher than control mean kidney weights were recorded for the females and lower than control mean adrenals and spleen weights were recorded for the males. Liver was identified as a target organ on this study with adaptive but not adverse treatment-related changes.

 

In summary, the only findings at 1000 mg/kg bw/day which were considered to be indicative of toxicity were slightly lower bodyweight gains and motor activity in males and occasional underactivity and abnormal gait in both sexes. Due to these findings of minor toxicity, the dose level of 1000 mg/kg bw/day could not be classed as a clear No Observed Adverse Effect Level (NOAEL). No adverse effects of treatment were observed at the preceding dose level of 150 mg/kg bw/day. A difference in male rearing behaviour (slightly low rearing activity in test versus control animals) were observed at 150 mg/kg bw/day, but in the absence of statistical significance or any other findings, this activity was considered not to be of toxicological importance. Thus 150 mg/kg bw/day can be classed as the study NOAEL.

 

Under the test conditions, the NOAEL for test item is 150 mg/kg bw/day in rats. The test item is not classified according to the annex I of the Regulation (EC) No. 1272/2008 (CLP) and of the GHS.

This study is considered as acceptable and satisfies the requirement for subacute repeated dose toxicity endpoint.

Endpoint:
repeated dose toxicity: oral, other
Remarks:
Five-day repeated dose oral (gavage) comparative assessment toxicity study in the rat
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
From Mars 7 to 12, 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline required
Principles of method if other than guideline:
The purpose of this study was to evaluate possible differences in response to treatment using two batches of the same test item and two strains of rats: Wistar Han™ RccHan™:WIST and Sprague-Dawley Crl:CD® (SD) IGS BR. The results of the study will support the discussion on the differences in response to treatment that were identified between the following two studies: Reproduction/Developmental Toxicity Screening Test in the Wistar Han™ RccHan™:WIST strain rat and Twenty-Eight Day Repeated Dose Toxicity Study in the Sprague-Dawley Crl:CD® (SD) IGS BR strain rat.
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on July 19-21, 2011/ signed on August 31, 2011)
Limit test:
no
Species:
rat
Strain:
other: Wistar Han™ RccHan™:WIST and Sprague-Dawley Crl:CD® (SD) IGS BR
Details on species / strain selection:
The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Wistar from Harlan Laboratories U.K. Ltd., Oxon, UK and SD from Charles River (UK) Limited, Margate,
Kent, UK respectively.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately six to eight weeks old.
- Weight at study initiation: At the start of treatment the Wistar males weighed 173 to 189g, the Wistar females weighed 142 to 166g, the Sprague-Dawley males weighed 199 to 222g, and the Sprague-Dawley females weighed 145 to 168g.
- Fasting period before study: no
- Housing: in groups of up to three by sex and by strain in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum): ad libitum. Pelleted diet (Rodent 2018C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK)
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: at least five days

DETAILS OF FOOD AND WATER QUALITY:
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 CONDITIONS
- Temperature (°C): 21 ± 2ºC
- Humidity (%): 55 ± 15%
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 07 March 2012 To: 12 March 2012
Route of administration:
oral: gavage
Details on route of administration:
The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item.
Vehicle:
arachis oil
Details on oral exposure:
- PREPARATION OF DOSING SOLUTIONS:
For the purpose of this study, the test item batches were prepared at the appropriate concentrations as solutions in Arachis oil BP. The test item batches were administered within two hours of being formulated. It is assumed that the formulations were stable for this duration.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): Suitable vehicle.
- Concentration in vehicle: 250 mg/mL
- Amount of vehicle (if gavage): 4 mL/kg
- Lot/batch no. (if required): not reported
- Purity: not reported
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Up to 5 consecutive days
Frequency of treatment:
Daily
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Following the observation of adverse clinical signs, the 1000 mg/kg bw/day dose level was reduced to 750 mg/kg bw/day in female Wistar rats administered Batch #1 on Day 3, and further reduced to 500 mg/kg bw/day from Day 4 onwards. Dose reduction from 1000 mg/kg bw/day to 500 mg/kg bw/day also occurred from Day 4 onwards for female Wistar rats administered Batch #2.
No. of animals per sex per dose:
3 per sex, per strain, per Batch
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on available toxicity information (Harlan Laboratories Ltd., Project Number: 41103156)
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable
- Section schedule rationale (if not random): not applicable
Positive control:
Not required
Observations and examinations performed and frequency:
CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to thirty minutes after dosing and one hour after dosing. Additional observations were also made five hours following dosing whenever possible (not at the weekend).

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION: Yes, daily

FOOD EFFICIENCY: Yes

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY:No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, internal and external macroscopic examination with special attention paid to the stomach

HISTOPATHOLOGY: Yes (stomach)
Other examinations:
None
Statistics:
None
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Of the surviving animals, one female Wistar rat (No.6) treated with Batch #1 at 1000 mg/kg bw/day reduced to 750 mg/kg bw/day on Day 3 of the study showed a decreased respiratory rate, prostration and lethargy on Days 2 and 3 of treatment. Two female Wistar rats (Nos.13 and 14) treated with Batch #2 at 1000 mg/kg bw/day showed a decreased respiratory rate, ataxia and lethargy on Day 3 only. No such observations were recorded after a reduction of the dose level to 500 mg/kg bw/day on Day 4 of the study.
All male Wistar rats and one female Wistar rat (No.8) treated with Batch #1 at 1000 mg/kg bw/day did not show any clinically observable signs of toxicity.
Two male Sprague-Dawley rats (Nos.17 and 19) treated with Batch #1 at 1000 mg/kg bw/day showed increased salivation immediately after dosing on Day 5 and two male Sprague-Dawley rats (Nos.24 and 25) treated with Batch #2 at 1000 mg/kg bw/day showed increased salivation immediately after dosing on Day 2. One female Sprague-Dawley rat (No.28) treated with Batch #2 at 1000 mg/kg bw/day showed ataxia 1 hour after dosing on Day 4 whilst a further female (No.26) from this treatment group showed increased salivation immediately after dosing on Day 5 only.
There were no clinically observable signs of toxicity detected in one male Sprague- Dawley rat (No.18) treated with Batch #1 at 1000 mg/kg bw/day, one male Sprague- Dawley rat (No.23) treated with Batch #2 at 1000 mg/kg bw/day, all female Sprague- Dawley rats treated with Batch #1 at 1000 mg/kg bw/day and in one female Sprague- Dawley rat (No.27) treated with Batch #2 at 1000 mg/kg bw/day.
Mortality:
mortality observed, treatment-related
Description (incidence):
One female Wistar rat (No.7) treated with Batch #1 at 750 mg/kg bw/day (reduced from 1000 mg/kg bw/day on Day 3 of the study) and one female Wistar rat (No. 12) treated with Batch #2 at 1000 mg/kg bw/day were killed at Study Director request on Day 3 due to clinical signs that exceeded the severity limits for this type of study as follows:
- One female Wistar rat (No.7) treated with 1000 mg/kg bw/day with Batch #1 showed decreased respiratory rate, prostration and lethargy on Day 2 of the study. This dose level was reduced to 750 mg/kg bw/day on Day 3 of the study, however, clinical observations for this female showed all the above findings and also ataxia. Due to the excessive severity of these findings this female was killed in extremis on Day 3 of the study.
- One female Wistar rat (No.12) treated with 1000 mg/kg bw/day with Batch #2 showed ataxia on Day 2 of the study. Day 3 clinical observations showed decreased respiratory rate, prostration, lethargy and ataxia. Due to the excessive severity of the above findings this female was also killed in extremis on Day 3 of the study.
Following the further reduction of the dose level to 500 mg/kg bw/day in female Wistar rats treated with Batch #1 or #2 on Day 4 there were no further unscheduled deaths during the study period.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Neither of the test item batches had an obvious effect on body weight in either sex or strain of rat. Compared with the single control animal/sex/strain, slight changes in body weight gain were observed among the treatment groups, as follows.
Slight reductions in overall body weight gains were observed in all male Wistar rats, in all male Sprague-Dawley rats, and in female Sprague-Dawley rats treated with Batch #2.
Slight increases in overall body weight gains were observed in female Wistar rats treated with Batch #2 and female Sprague-Dawley rats treated with Batch #1.
The toxicological significance of the changes in body weight gain are unclear given their small magnitude and comparison of values for treated groups were made with those from a single control animal/sex/strain.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Female Wistar rats treated with Batch #1 or #2 at 1000 mg/kg bw/day showed a reduction in food consumption on Day 2 of the study when compared to the control female Wistar rat. No such effects were evident on any other study day.
No obvious effects on food consumption were detected in remaining treated animals when compared to controls throughout the treatment period
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The two female Wistar rats that were killed in extremis on Day 3 (one each from the groups administered Batch #1 and Batch #2) were observed to have localized epithelial sloughing of the glandular region of the stomach at necropsy.
At terminal kill necropsy two female Sprague-Dawley rats treated with Batch #1 at 1000 mg/kg bw/day each had a raised non-glandular region of the stomach, inflammatory processes were the histological correlate.
Macroscopic examinations did not reveal any abnormalities in any treated male rats or in female Sprague-Dawley rats treated with Batch #2 at 1000 mg/kg bw/day.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Description (incidence and severity):
Stomach: Findings in the glandular stomach were considered to be of minor severity and were limited to mononuclear cell foci (observed in one Wistar and one Sprague-Dawley male administered Batch #1) and inflammatory cell infiltrates (observed in a control Sprague-Dawley male and one Sprague-Dawley female at 1000 mg/kg bw/day). Thus, there were no clearly test item-related findings in the glandular stomach. While sloughing of the glandular region of the stomach was observed at necropsy in the two female Wistar rats killed in extremis, there were no histological correlates. Inflammatory findings were detected in the forestomach of some Wistar and Sprague-Dawley rats treated with Batch #1 and #2. The inflammatory findings consisted mainly of squamous epithelial hyperplasia, hyperkeratosis, inflammation in the submucosa and a few cases of minor degrees of ulceration. The total number of affected animals was higher for Wistar rats (two females treated with Batch #1 and three females treated with Batch #2 both at 1000 mg/kg bw/day reduced to 500 mg/kg bw/day by Day 4, and also one male treated with Batch #2 at 1000 mg/kg bw/day) when compared to total number of affected animals for Sprague-Dawley rats (two females treated with Batch #1 at 1000 mg/kg bw/day and one male treated with Batch #2 at 1000 mg/kg bw/day); however, there was no clear difference between the strains with respect to severity of the findings.
All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strains employed, and there were no differences in incidence or severity between control and treatment groups that were considered to be of toxicological significance.
Conclusions:
The data generated for this study indicate that female Wistar rats appear to be more sensitive to treatment with ST 20 C 11. Adverse clinical signs were noted starting on Day 2 following the oral administration of either batch of the test item in female Wistar rats; however, similar responses were not observed in male Wistar rats or in Sprague- Dawley rats of either sex. Male Wistar rats and Sprague-Dawley rats of either sex occasionally showed treatment-related responses such as decreased body weight gain; however, these findings were of slight or minimal severity.
Executive summary:

Introduction. The purpose of this study was to evaluate possible differences in response to treatment using two batches of the same test item (Batch #1 = 95.9% pure and Batch #2 = 99.4% pure) and two strains of rats: Wistar Han™ RccHan™:WIST and Sprague-Dawley Crl:CD® (SD) IGS BR. The results of the study will support the discussion on the differences in response to treatment that were identified between the following two studies: Reproduction/Developmental Toxicity Screening Test in the Wistar Han™ RccHan™:WIST strain rat (Harlan Laboratories Ltd., Project Number 41103156) and Twenty-Eight Day Repeated Dose Toxicity Study in the Sprague-Dawley Crl:CD® (SD) IGS BR strain rat (Huntington Life Sciences Ltd., Study Number FIR/062).

Methods. Two batches of the test item were administered by gavage to two groups, each of three male and three female Wistar Han™:RccHan™:WIST (Wistar) or Sprague- Dawley Crl:CD® (SD) IGS BR (Sprague-Dawley) strain rats, for up to five consecutive days, at an initial dose level of 1000 mg/kg bw/day. Following the observation of adverse clinical signs, the 1000 mg/kg bw/day dose level was reduced to 750 mg/kg bw/day in female Wistar rats administered Batch #1 on Day 3, and further reduced to 500 mg/kg bw/day from Day 4 onwards. Dose reduction from 1000 mg/kg bw/day to 500 mg/kg bw/day also occurred from Day 4 onwards for female Wistar rats administered Batch #2. Two control groups of one male and one female per group were dosed with vehicle alone (Arachis oil BP).

Clinical signs, body weight change and dietary intake were monitored during the study.  All animals were subjected to gross necropsy examination followed by histopathology examinations of the stomach.

Results.

Mortality. One female Wistar rat (No. 7) treated with Batch #1 at 1000 mg/kg bw/day (reduced to 750 mg/kg bw/day on Day 3) and one female Wistar rat (No. 12) treated with Batch #2 at 1000 mg/kg bw/day were killed in extremis approximately five hours postdosing on Day 3.

There were no further unscheduled deaths.

Clinical Observations. The two female Wistar rats (Nos. 7 and 12) treated with either Batch #1 or #2 that were killed in extremis on Day 3 of the study showed decreased respiratory rate, prostration, lethargy and/or ataxia after dosing on Days 2 and/or 3 of treatment.

One female Wistar rat (No. 6) treated with Batch #1 at 1000 mg/kg bw/day (reduced to 750 mg/kg bw/day on Day 3) showed a decreased respiratory rate, prostration and lethargy on Days 2 and 3 of treatment. Two female Wistar rats (No. 13 and 14) treated with Batch #2 at 1000 mg/kg bw/day showed a decreased respiratory rate, ataxia and lethargy on Day 3 only. There were no adverse clinical signs observed in female Wistar rats following a reduction in the dose level from 1000 or 750 mg/kg bw/day to 500 mg/kg bw/day.

Male Wistar rats treated with Batch #1 and #2 at 1000 mg/kg bw/day did not show any clinically observable signs of toxicity.

Two male Sprague-Dawley rats treated with Batch #1 at 1000 mg/kg bw/day showed increased salivation on Day 5 and two male Sprague-Dawley rats treated with Batch #2 at 1000 mg/kg bw/day showed increased salivation on Day 2. One female Sprague- Dawley rat treated with Batch #2 at 1000 mg/kg bw/day showed ataxia one hour postdosing on Day 4. Given the transient nature of ataxia observed in a single female Sprague-Dawley rat only on Day 4 of the study, this finding also was considered to be of minimal toxicological significance. One female rat from this same treatment group showed increased salivation on Day 5 only; however, as salivation is often due to an unpalatable test item formulation this finding was not considered to be of toxicological importance.

There were no clinically observable signs of toxicity detected in female Sprague-Dawley rats treated with Batch #1 at 1000 mg/kg bw/day.

Body Weight. Slight reductions in overall body weight gains were evident in male Wistar rats and male Sprague-Dawley rats treated with either Batch #1 or #2 at 1000 mg/kg bw/day and also in female Sprague-Dawley rats treated with Batch #2 at 1000 mg/kg bw/day.

There were no obvious effects on overall body weight gains in female Wistar rats treated with Batch #1 or #2 (1000 mg/kg bw/day reduced to 500 mg/kg bw/day), or in female Sprague-Dawley rats administered Batch #1 at 1000 mg/kg bw/day.

Overall, there were no notable differences in body weights observed among groups at

the end of the study.

Food Consumption. Female Wistar rats treated with Batch #1 or #2 at 1000 mg/kg bw/day had a lower food consumption than the control animal on Day 2. No obvious effects on food consumption were detected in these animals on other study days or in any other animals on study.

Necropsy. The two female Wistar rats killed on Day 3 of the study that had received either Batch #1 or Batch #2 were observed to have localized epithelial sloughing of the glandular region of the stomach at necropsy. There was no histopathological correlate to this finding.

Two female Sprague-Dawley rats treated with Batch #1 at 1000 mg/kg bw/day each had a raised non-glandular region of the stomach at necropsy.

No such effects were detected in any treated male rats or in female Sprague-Dawley rats treated with Batch #2 at 1000 mg/kg bw/day.

Histopathology. The following treatment related changes were identified:

Stomach: No test item-related histopathological findings were observed in the glandular stomach. Inflammatory findings were detected in the forestomach of some Wistar rats treated with Batch #1 or #2 at either 500 (females) or 1000 (males) mg/kg bw/day and some Sprague-Dawley rats treated with Batch #1 or #2 at 1000 mg/kg bw/day. The inflammatory findings predominantly affected Wistar rats and consisted mainly of squamous epithelial hyperplasia, hyperkeratosis, inflammation in the submucosa and a few cases of minor degrees of ulceration.

Conclusion. Oral administration of the test item ST 20 C 11 Batch #1 or Batch #2 to male and female Wistar and Sprague-Dawley rats by gavage, at dose levels of up to 1000 mg/kg bw/day (reduced to 750 mg/kg bw/day on Day 3 of treatment in female Wistar rats administered Batch #1, and further reduced to 500 mg/kg bw/day on Day 4 of treatment in female Wistar rats administered Batch #1 or #2), resulted in evidence of a difference in response to the test item between strains and between sexes.

The data generated for this study indicate that female Wistar rats appeared to be more sensitive to treatment with ST 20 C 11. Adverse clinical signs were noted starting on Day 2 following the oral administration of both batches of the test item in female Wistar rats; however, similar responses were not observed in male Wistar rats or in Sprague- Dawley rats of either sex. A rapid adverse response was recorded in female Wistar rats treated with Batch #1 and a slower but equally severe response was evident in female Wistar rats treated with Batch #2.

Male Wistar rats and Sprague-Dawley rats of either sex occasionally showed treatment-related responses such as lower body weight gain compared to the respective control animal; however, these findings were of slight or minimal severity.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
The key study is GLP-compliant and of high quality (Klimisch score = 1)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

A subacute study complying with GLP conditions has been conducted to investigate repeated dose oral toxicity in rats (Huntingdon Life Sciences, 2006). The study was conducted according to the OECD Testing Guideline No. 407. The test material was administered by oral gavage at doses of 0, 15, 150 or 1000 mg/kg bw/day daily for 28 consecutive days. A standard list of study endpoints was assessed, including sensory reactivity, grip strength and motor activity. 

There were no unscheduled deaths during the study and treatment-related effects were confined to animals receiving 1000 mg/kg bw/day. Clinical signs were observed post-dosing and included salivation, which was not considered to be of toxicological importance given its transience and that it is often seen in oral gavage studies and is generally associated with the taste of the test substance and/or the dosing procedure. The clinical signs of transient underactivity and, in a few animals on a few occasions, flat posture and abnormal gait, occurring post-dose in high-dose animals together with the reduced motor activity in males were attributed to slight abdominal discomfort. There was no indication that the slightly lower activity observed in males was a neurotoxic effect. Overall, the low activity was considered not to be of toxicological importance

Low body weight gains with accompanying low efficiency of food utilisation was observed in males at the high dose and considered to be due to treatment. However, the magnitude of the changes were small when compared to control values, there were no actual body weight losses, and for some males the overall gains were with the range observed in concurrent controls. Therefore, the slightly lower body weight gains were not considered to represent overt toxicity due to the substance.

Hematological findings, while noted, were within historical range for these values and were not accompanied by histopathology changes; as such, the findings were not considered toxicologically significant.

Centrilobular hepatocyte hypertrophy correlated with liver enlargement and the higher liver weights in animals at the high dose compared to controls. While the liver was identified as a target organ in this study, the findings were considered to be adaptive but not adverse treatment-related changes. There was some limited evidence that the test substance affected the functional capacity of the kidney (based on increased kidney weights in females at 1000 mg/kg bw/day and decreases in urea and creatinine plasma concentrations, indicating a possible increase in efficiency) but there were no indications of adverse effects on this organ when considering the absence of any findings upon microscopic examination. Overall, the increased kidney weights were not indicative of toxicity.

Changes in male adrenal and spleen weights were not accompanied by histopathological findings in these organs and were thus considered to be of no toxicological importance. Changes in ovaries weights in the absence of any histological findings were considered to reflect the stage of oestrus in each cage of animals and not related to treatment.

In summary, the only findings at 1000 mg/kg bw/day which were considered to be indicative of toxicity were slightly lower bodyweight gains and motor activity in males and occasional underactivity and abnormal gait in both sexes. Due to these findings of minor toxicity, the dose level of 1000 mg/kg bw/day could not be classed as a clear No Observed Adverse Effect Level (NOAEL). No adverse effects of treatment were observed at the preceding dose level of 150 mg/kg bw/day. A difference in male rearing behaviour (slightly low rearing activity in test versus control animals) were observed at 150 mg/kg bw/day, but in the absence of statistical significance or any other findings, this activity was considered not to be of toxicological importance. Thus 150 mg/kg bw/day can be classed as the study NOAEL, noting that the NOAEL may be artificially low as no doses were evaluated between 150 and 1000 mg/kg bw/day.

In a 5 -day investigative study, oral administration of two batches of the test item to male and female Wistar and Sprague-Dawley rats by gavage, at dose levels of up to 1000 mg/kg bw/day (reduced to 750 mg/kg bw/day on Day 3 of treatment in female Wistar rats administered Batch #1, and further reduced to 500 mg/kg bw/day on Day 4 of treatment in female Wistar rats administered Batch #1 or #2), resulted in evidence of a difference in response to the test item between strains and between sexes. The data generated for this study indicate that female Wistar rats appeared to be more sensitive to treatment with ST 20 C 11. Adverse clinical signs were noted starting on Day 2 following the oral administration of both batches of the test item in female Wistar rats; however, similar responses were not observed in male Wistar rats or in SpragueDawley rats of either sex. A rapid adverse response was recorded in female Wistar rats treated with Batch #1 and a slower but equally severe response was evident in female Wistar rats treated with Batch #2. Male Wistar rats and Sprague-Dawley rats of either sex occasionally showed treatment related responses such as lower body weight gain compared to the respective control animal; however, these findings were of slight or minimal severity. The results of this study are considered inconclusive.


Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification according to the Regulation (EC) No. 1272/2008.

Self-classification:

Slightly lower bodyweight gains and motor activity in males and occasional underactivity and abnormal gait in both sexes were observed at 1000 mg/kg bw/day. These findings, by themselves, do not indicate "significant" toxicity (CLP Annex I: 3.9.2.8.1). There was no evidence to support the notion that the slightly lower activity observed in males was a neurotoxic effect. There was no histopathological correlates and the responses were transient. As a result, the substance does not meet the criteria for classification according to the Regulation (EC) No. 1272/2008 and to the GHS.