1-(5-propyl-1,3-benzodioxol-2-yl)-1-ethanone

Endpoint summary

Description of key information

Repeated dose toxicity oral: NOAEL = 300 mg/kg bw/d (OECD 407, GLP, K, rel. 1)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 December 2013 to 16 June 2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted in compliance with OECD Guideline No. 407 without any deviation.

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Compliance Programme (inspected on 01-03 July 2014/ signed on 15 September 2014)

Limit test:

no

Specific details on test material used for the study:

- Purity test date: 30 October 2013
- Storage Conditions: Refrigerated (4 ºC), in the dark under nitrogen

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was chosen as the test species because it is accepted as a predictor of toxic change in man and the requirement for a rodent species by regulatory agencies. The CD strain was used because of the historical control data available at this laboratory.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan (UK) Ltd.
- Age at study initiation: 40-46 days
- Weight at study initiation: Males: 141-186 g; Females: 102-135 g
- Housing: Five animals/sex/cage (main study and recovery) in polycarbonate cages with a stainless steel mesh lid
- Diet: Rat and Mouse No. 1 Maintenance Diet (Special Diet Services), ad libitum; Non-restricted (removed overnight before blood sampling for haematology or blood chemistry and during the period of urine collection).
- Water: Potable water from the public supply, ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-23 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 17 December 2013 to 16 June 2014

Route of administration:

oral: gavage

Details on route of administration:

The oral gavage route of administration was chosen to simulate a potential route of accidental exposure.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Method of preparation: A series of homogenous suspensions at the required concentrations were prepared by dilution of individual weighings of the test substance.
Frequency of preparation: Weekly
Storage of preparation: Refrigerated (2-8 °C)
Stability: 15 days refrigerated (2-8 °C) and 1 day ambient (approximately 21 °C)

VEHICLE
- Concentration in vehicle: 6, 60, 150 and 100 mg/L
- Amount of vehicle: 5 mL/kg bw/day

DOSE VOLUME: 5 mL/kg bw/day

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability and homogeneity: Before commencement of the programme of work, the suitability of the proposed mixing procedures was determined and specimen formulations at 1 mg/mL and 200 mg/mL were analysed to assess the stability and homogeneity of the test substance in the liquid matrix.
Achieved concentration: Samples of each formulation prepared for administration in Weeks 1, Week 1, Day 3 (for Group 4 only) and Week 4 of treatment were analysed for achieved concentration of the test substance.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily at approximately the same time each day

Dose / conc.:

30 mg/kg bw/day (actual dose received)

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Remarks:

Day 1-2: 750 mg/kg bw/day
Day 3-28: 500 mg/kg bw/day

No. of animals per sex per dose:

Main study: 5/sex/dose (0, 30, 300 and 750/500 mg/kg bw/day)
Recovery phase: 5/sex/dose (0 and 750/500 mg/kg bw/day)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected after consultation with the Sponsor and were based on data from a 7-day preliminary toxicity study (Huntingdon Life Sciences Study Number HIK0032).
In the preliminary study, test item ST 14 C 12 was administered by gavage to 3 male and 3 female CD rats at doses of 250, 500 or 1000 mg/kg bw/day for 7 days. Clinical signs, body weight and food consumption were examined at regular intervals throughout the study. Terminal investigations included macroscopic pathology and organ weight measurements.
Signs of toxicity were seen in in all animals receiving 1000 mg/kg bw/day, clinical signs consisted of decreased activity, unsteady gait, unresponsive behaviour, irregular breathing, rapid breathing, piloerection, flattened posture and reduced body tone. Macroscopic irritation (thickening and/or depressions) in the stomach was noted for all females and one male receiving 1000 mg/kg bw/day. Due to the clinical signs and macroscopic stomach findings seen following administration of 1000 mg/kg, this level was considered not to be suitable for the subsequent 28 Day toxicity study.
Clinical signs at 500 mg/kg bw/day consisted of flattened posture, unsteady gait, decreased activity, reduced body tone, piloerection and irregular breathing which were mostly seen on Day 1 only. The low body weight gains for females receiving 500 mg/kg bw/day were considered to be a result of low food consumption noted for this group. There were no toxicologically significant treatment related clinical signs seen in animals receiving 250mg/kg bw/day . There were no macroscopic finings for animals receiving 500 or 250 mg/kg bw/day .
Due to the clinical signs and macroscopic stomach findings seen in the preliminary study following administration of 1000 mg/kg bw/day, this level was considered unsuitable for 4 weeks of treatment. The dose level of 500 mg/kg bw/day was well tolerated therefore the high dose level for the this 4 week study was selected as 750 mg/kg bw/day , this being the mid point between 500 and 1000 mg/kg bw/day to ensure the findings would be tolerated for the longer treatment period. The dose levels of 30 and 300 mg/kg bw/day were selected based on GHS labelling points.

- Rationale for animal assignment: Randomly allocated on arrival.
- Post-exposure recovery period in satellite groups: 14 days

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. During the acclimatisation and recovery periods, observations of the animals and their cages were recorded at least once per day. A viability check was performed near the start and end of each working day. Animals were isolated or killed for reasons of animal welfare where necessary.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during Week 1 of treatment, detailed observations were recorded at the following times in relation to dose administration: pre-dose observation; at the end of each dosing group; one to two hours after completion of dosing all groups; as late as possible in the working day.
Daily during Week 2 of treatment, detailed observations were recorded at the following times in relation to dose administration: pre-dose observation; one to two hours after completion of dosing of all groups

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced (Day P1), on the day that treatment commenced (Day 1), weekly throughout the study and before necropsy.

FOOD CONSUMPTION: Yes; 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 for each week throughout the study.

WATER CONSUMPTION: Yes
- Time schedule for examinations: Fluid intake was assessed by daily visual observation. No effect was observed and consequently quantitative measurements were not performed.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 29 (all main study animals); Recovery Day 15 (all recovery phase animals)
- Anaesthetic used for blood collection: Yes; light general anaesthesia induced by isoflurane
- Animals fasted: Yes; Blood samples were collected after overnight withdrawal of food and prior to dosing
- Parameters checked:
Haematology: Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics using a Bayer Advia 120 analyser: Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), Mean cell volume (MCV), Red cell distribution width (RDW), Total leucocyte count (WBC), Differential leucocyte count: Neutrophils (N), Lymphocytes (L), Eosinophils (E), Basophils (B), Monocytes (M) and Large unstained cells (LUC), Platelet count (Plt), Morphology: Anisocytosis, Microcytosis, Macrocytosis, Hypochromasia and Hyperchromasia
Additional blood samples (nominally 0.5 mL) were taken into tubes containing citrate anticoagulant and examined using an ACL series analyser and appropriate reagent in respect of: Prothrombin time (PT) and Activated partial thromboplastin time (APTT)
Clinical chemistry: Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as anticoagulant. After separation, the plasma was examined using a Roche P Modular Analyser in respect of: Alkaline phosphatase (ALP), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transferase (gGT), Total bilirubin (Bili), Total bile acids (Bi Ac), Urea, Urea Nitrogen (BUN), Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Calcium (Ca), Inorganic phosphorus (Phos), Total protein (Total Prot), Albumin (Alb).
Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analysed albumin concentration.

URINALYSIS: Yes
- Time schedule for collection of urine: Week 4 (all main study animals); Recovery Week 2 (all recovery phase animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes; Animals were placed in an individual metabolism cage overnight, without food or water (ca 16 h).
- Parameters checked:
Using manual methods: Clarity and Colour - by visual assessment, Volume - using a measuring cylinder, pH - using a pH meter, Specific gravity - by direct refractometry using a SG meter
Using Multistix reagent strips, interpreted using a Clinitek®500 instrument: Ketones, Bilirubin/bile pigments, Urobilinogen, Blood pigments
Using a Roche P Modular analyser: Protein, Creatinine, Glucose, Sodium, Potassium, Chloride

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before treatment commenced and during each week of treatment and recovery, detailed physical examination and arena observations were performed on each animal. On each occasion, the examinations were performed at approximately the same time of day (before dosing during the treatment period), by an observer unaware of the experimental group identities.
- Battery of functions tested: sensory activity / grip strength / motor activity
Sensory reactivity and grip strength: Sensory reactivity and grip strength assessments were performed (before dosing) on all Main study animals in Groups 2 and 3 and all Recovery phase animals during Week 4 of treatment. In addition, all recovery phase animals were tested during Week 2 of recovery.
Motor activity: During Week 4 of treatment (before dosing), the motor activity of all Main study animals in Groups 2 and 3 and all Recovery phase animals was measured using a Rodent Activity Monitoring System (Version 2.0.5), with hardware supplied by Pearson Technical Services and software developed and maintained by Huntingdon Life Sciences. In addition, all recovery phase animals were tested during Week 2 of recovery.

IMMUNOLOGY: No

Sacrifice and pathology:

SACRIFICE: Animals were killed by carbon dioxide asphyxiation with subsequent exsanguination. Main study animals were killed following 28 days of treatment, on Day 29 of the study. Recovery animals were killed following 28 days of treatment and 14 days of recovery on Day 43 of the study (Day 15 of recovery ). All Main phase and Recovery phase animals were subject to a detailed necropsy.

GROSS PATHOLOGY: Yes; after a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

ORGAN WEIGHTS: For bilateral organs, left and right organs were weighed together. The requisite organs were weighed for Main phase and Recovery phase animals killed at scheduled termination.

HISTOPATHOLOGY: Yes
Fixation: Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of those detailed below:
Testes: In modified Davidson’s fluid; Eyes: In Davidson’s fluid.
Histology:
Processing: Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Full List: All animals killed or dying prematurely. Main study animals of Groups 1 and 4 killed at a scheduled interval.
Abnormalities and stomach: All animals
Routine staining: Sections were stained with haematoxylin and eosin; in addition samples of the testes were stained using a standard periodic acid/Schiff (PAS) method.
Light microscopy: Tissues preserved for examination were examined as follows:
Premature deaths: All animals from all groups
Scheduled kill:
Main study: All animals of Groups 1 and 4 (all specified in Table 7.5.1/1)
All animals of Groups 2 and 3 (abnormalities only)
Recovery: All animals of Groups 1 and 4 (abnormalities only)
The following tissues, which were considered to exhibit a reaction to treatment at the high dose, were examined for all main study and recovery animals: Stomach

Other examinations:

None

Statistics:

See "Any other information on materials and methods incl. tables"

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Unsteady gait and decreased activity was noted from Day 1 to 7 for most males and females receiving 750/500 mg/kg bw/day. These signs were generally noted a few minutes following dosing; recovering by the end of the working day.
- Transient piloerection, partially closed eyelids, unresponsive, swaying and flattened or prostrate posture were seen in a few males or females receiving 750/500 mg/kg bw/day between Days 1 and 6. These signs were noted from the end of dosing of group observation; recovery was seen by the end of the working day on most occasions.
- Transient chin rubbing (Day 5) and salivation (Days 12 and 13) were noted in females at 750/500 mg/kg bw/day. These signs are not uncommon on oral gavage studies and are believed to be a response to the taste of the test material and are considered not to be of toxicological importance.
- No clinical signs were seen in any animals receiving 30 or 300 mg/kg bw/day.
CONCLUSION :

Mortality:

mortality observed, treatment-related

Description (incidence):

- There were two deaths on the study at 750/500 mg/kg bw/day, one female (750 mg/kg bw/day on Day 1) and one male (at 750 mg/kg bw/day on Days 1 and 2 and 500 mg/kg bw/day on Days 3-5) were euthanized for welfare reasons on Day 1 and 5, respectively, due to persistent unresponsive behaviour. Clinical signs prior to death consisted of unresponsive behaviour, decreased activity, prostrate posture and cold to touch (both animals), unsteady gait (female), irregular breathing and partially closed eyelids (male).
CONCLUSION: Adverse effects

Body weight and weight changes:

no effects observed

Description (incidence and severity):

- There was considered to be no effect of treatment on body weight gain during the treatment or recovery period.
- The body weight gain was lower than control for treated females during the recovery period (0.6X control), however, analysis of the individual bodyweight gains (Day R1 to R14) revealed that the majority of values for the treated females were within the control range indicating this reflected biological variation.
CONCLUSION : Non-adverse effects

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect of treatment on food consumption.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

Fluid intake was assessed by daily visual observation during the course of this study. As no treatment related effects were noted during the treatment phase no precise measurements were taken.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- The haematological examination on Day 29 indicated, a higher group mean lymphocyte count for treated animals, with the group mean value for males receiving 750/500 mg/kg bw/day attaining statistical significance (p < 0.05), consequently the total white blood cell counts were also higher than concurrent controls. The majority of lymphocyte values were similar to control following the recovery period.
- After 15 days of recovery period, a higher group mean basophil and monocyte count for treated animals when compared to controls with males attaining statistical significance at 750/500 mg/kg bw/day at the end of the recovery period, consequently the total WBC remained higher than control.
- All other differences from control values, including those attaining statistical significance, were minor or lacked dose-relationship and were therefore attributed to normal biological variation. Such changes included higher than control mean cell haemoglobin and mean cell volume for females treated at 750/500 mg/kg bw/day following 28 days of treatment and lower than control reticulocyte count and activated partial thromboplastin time for males receiving 750/500 mg/kg bw/day following 28 days of treatment and two weeks recovery.
CONCLUSION: Non-adverse effects / partially reversible within the recovery period / no biological or toxicological significance

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Blood chemistry investigations on Day 29 revealed a high group mean alanine amino-transferase activity when compared to control animals for both sexes receiving 300 or 750/500 mg/kg bw/day. The group mean alkaline phosphatase concentration, aspartate aminotransferase activity and plasma cholesterol levels were statistically significantly higher than control for treated males receiving 750/500 mg/kg bw/day. Recovery was evident following 2 weeks off dose.
- The group mean plasma potassium ion concentration was higher than control on Day 29 for both sexes given 750/500 mg/kg bw/day; attaining statistical significance (p < 0.05) in females only. Recovery was evident following 2 weeks off dose.
- All other differences including those attaining statistical significance were confined to one sex, lacked dose relationship or were not apparent during the treatment phase. Therefore, these were attributed to normal biological variation. Such changes included a statistically lower than control sodium concentration for males treated at 750/500 mg/kg bw/day at the end of the treatment period, a higher than control potassium level for females treated at 750/500 mg/kg bw/day following 28 days of treatment and a higher than control blood glucose level for females receiving 750/500 mg/kg bw/day following 28 days of treatment 2 weeks recovery.
CONCLUSION : Non-adverse effects / partially reversible within the recovery period / no biological or toxicological significance

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

- Urinalysis investigations in Week 4 revealed the following changes: a higher group mean glucose concentration for all treated groups in both sexes when compared to controls with males and females receiving 300 or 750/500 mg/kg bw/day attaining statistical significance. After 2 weeks of recovery the glucose levels for animals receiving 750/500 mg/kg bw/day was similar to that of control animals.
- Following 4 weeks of treatment, a higher than control group mean chloride concentration was seen for all treated animals, with both sexes receiving 750/500 mg/kg bw/day attaining statistical significance. Due to an oversight this parameter was not assessed at the end of the recovery period. If this finding had been present at recovery, in isolation it would have not been considered as adverse, therefore the loss of this data does not affect the overall scientific validity of the study.
- All other differences from control values, including those attaining statistical significance, were minor or lacked dose-relationship and were therefore attributed to normal biological variation. Such changes included higher than control sample volume, protein concentration and urinary creatinine concentration for females receiving 750/500 mg/kg bw/day following 28 days of treatment.
CONCLUSION : Non-adverse effects / partially reversible within the recovery period / no biological or toxicological significance

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

- Week 4 motor activity investigations revealed higher than control group mean high beam scores (rearing activity) for males receiving 750/500 mg/kg bw/day attained statistical significance (p<0.05) at three of the 6-minute intervals (24, 30 and 54 minutes) and the total score, however statistical significance (p<0.01) was limited to the 54-minute interval for low beam scores. Individual high and low beam scores for all treated groups showed high variability especially for males receiving 750/500 mg/kg bw/day, and as the high group mean scores seen in this group were largely due to high levels of activity in two males (numbers 21 and 25) this was considered unlikely to be an effect of treatment. Scores for males receiving 30 or 300 mg/kg bw/day were similar to that of controls.
- Female individual high and low beam scores for all groups, including controls, showed high variability during the Week 4 investigations when compared to historical background data, however there was no clear effect of treatment. Group mean high beam scores for all treated female groups at the 18-minute interval were low compared with controls and attained statistical significance (p<0.05), however, these isolated differences did not reflect a change in the overall pattern of activity.
- Following the recovery period, group mean high and low beam scores and total scores were similar to those seen in control animals indicating recovery for the majority of males previously treated at 750/500 mg/kg bw/day. Male number 25 continued to show high levels of activity with total scores for both high and low beams being higher than all other individuals within the group.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- After 4 weeks of treatment the body weight adjusted group mean liver weight was marginally but statistically significantly higher than control for both sexes receiving 300 or 750/500 mg/kg bw/day. The body weight adjusted kidney weights were also higher than control for both sexes receiving 750/500 mg/kg bw/day, attaining statistical significance (p < 0.05) in females only.
- After 2 weeks of recovery the body weight adjusted liver weight for females remained higher than control, whereas there was evidence of recovery in the males. The body weight adjusted kidney weights were similar to control for both sexes.
CONCLUSION : Non-adverse effects

Gross pathological findings:

no effects observed

Description (incidence and severity):

- The macroscopic examination performed after 28 days of treatment and a two week recovery period revealed no test article-related lesions.
- The incidence and distribution of all findings were consistent with common background changes previously seen in control CD rats at Huntingdon Life Sciences’ laboratories.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Changes related to treatment were seen in the stomach: Minimal or slight hyperplasia of the non-glandular epithelium of the stomach was seen in individual males from each treated group and an individual female receiving 750/500 mg/kg bw/day. Minimal hyperplasia of the non-glandular epithelium was seen in a single male receiving 300 or 750/500 mg/kg bw/day. In one male receiving 30 mg/kg bw/day, slight hyperplasia was associated with slight ulceration and inflammation and minimal oedema, and in one female receiving 750/500 mg/kg bw/day slight hyperplasia was associated with slight hyperkeratosis and minimal parakeratosis.
- Incidental findings: The nature and incidence of all other findings were consistent with the commonly seen background of microscopic changes in CD rats at Huntingdon Life Sciences’ laboratories.
Findings following 4 weeks of treatment and 2 weeks of recovery:
- Examination of the stomach from Control animals and animals previously receiving 750/500 mg/kg bw/day did not reveal any changes which were considered to be related to previous treatment.
- A limited number of macroscopic abnormalities were examined from animals killed at the end of the 2 week recovery period. The findings seen in these tissues were considered to be incidental and unrelated to previous treatment.
CONCLUSION : adverse effects

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

water consumption and compound intake

Key result

Critical effects observed:

no

Administration of 750 mg/kg/day resulted in unforeseen clinical signs (unsteady gait, decreased activity, piloerection, partially closed eyelids, unresponsive and abnormal posture (swaying/flattened/prostrate)), one female was killed for humane reasons on Day 1 and further male on Day 5 due to persistent unresponsive behaviour. As a precautionary measure the high dose level was reduced to 500 mg/kg/day from Day 3 onwards.

There was evidence of adaptive changes in the liver at 750/500 mg/kg/day and to a lesser extent at 300 mg/kg/day. The increased cholesterol, alanine amino-transferase, alkaline phosphatase and aspartate amino-transferase levels, and increased adjusted liver weight are indicative of increased activity in the liver. Orally administered substances are metabolised by the liver, therefore this increase in activity is a normal adaptive response following administration of a test substance. There was general evidence of recovery and no histopathological changes in the liver.

The increased adjusted kidney weight for females, and to a lesser extent males, receiving 750/500 mg/kg/day is considered to be associated with the increase in plasma potassium, urinary glucose and chloride levels. There was full recovery and no histopathological changes in the kidney.

The histopathological examinations showed minimal or slight hyperplastic and/or degenerative/inflammatory changes in the non-glandular region of the stomach from individual males in each treated group and females at 750/500 mg/kg/day. This is indicative of local irritation and is consistent with the macroscopic stomach findings at higher dose levels in the 7-day preliminary study (Huntingdon Life Sciences study number HIK0032). For some individuals (Group 2 male number 3 and Group 3 male 28 and Group 4 male 16) there is a correlation between this irritation and the increased lymphocyte count at the end of the treatment period, with the increased basophil and monocyte count at the end of the recovery period being a continuation of the response to local insult.

Administration of 750/500 mg/kg/day resulted in the death of 2 rats due to the severity of the clinical signs observed, findings associated with local irritation to the stomach. The combination of these findings indicate that this is an adverse effect level.

At 300 mg/kg/day the findings in the liver, kidney and stomach were observed to a lesser extent than at 750/500 mg/kg/day and in the absence of any deaths or clinical signs, 300 mg/kg/day was the no-observed-adverse effect level (NOAEL).

The findings at 30 mg/kg/day comprised an increase in urinary glucose and lesions in the stomach of animal No. 3. In the absence of any corroborative findings in the kidney at this level the increase in urinary glucose was not adverse and as the stomach findings had no associated findings, they are also considered not to be adverse, thus confirming the higher level of 300 mg/kg/day as NOAEL.

Conclusions:

Under the test conditions, the NOAEL for test substance was considered to be 300 mg/kg bw/day in rats.

Executive summary:

In a repeated dose toxicity study performed in accordance with OECD test guideline No. 407 and in compliance with GLP, test item ST 14 C 12 was administered by gavage to 5 male and 5 female Hsd: Sprague Dawley SD rats at doses of 30, 300 or 750/500 mg/kg bw/day for 28 consecutive days. A similarly constituted control group received the vehicle, corn oil at the volume dose of 5 mL/kg bw/day. A further five male and five female rats were assigned to each of the control and high dose groups. These animals were treated for 28 days, followed by a 14 day period without treatment to assess the potential for any treatment related change to recover. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, haematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

One male and one female, both receiving 750/500 mg/kg bw/day were killed during the first week of the study due to persistent unresponsive behaviour.

 

Clinical signs for surviving animals during the treatment period were only observed for the group receiving 750/500 mg/kg bw/day and consisted of: unsteady gait, decreased activity, piloerection, partially closed eyelids, unresponsive and abnormal posture (swaying/flattened/prostrate) following dosing during the first week of treatment.

 

There were no clear effect of treatment on motor activity, sensory activity and grip strength.

 

There was no effect of treatment on food consumption or body weight gain.

 

The haematological examination on Day 29 indicated slightly higher lymphocyte and consequently total white blood cell counts for males receiving 750/500 mg/kg bw/day. Following the 2 weeks off dose period the lymphocyte values were similar to control, however, the basophil and monocyte counts were higher.

 

Blood chemistry investigations on Day 29 revealed high alanine amino-transferase for both sexes receiving 300 or 750/500 mg/kg bw/day and high alkaline phosphatase, aspartate amino-transferase, plasma cholesterol and potassium for males at 750/500 mg/kg bw/day. Recovery was evident following 2 weeks off dose.

 

Urinary glucose and chloride was high for all treated groups in both sexes when compared to controls, following two weeks off dose glucose was similar to control. Due to an oversight chloride was not assessed at the end of the recovery period. If this finding had been present at recovery, in isolation it would have not been considered as adverse, therefore the loss of this data does not affect the overall scientific validity of the study.

 

The bodyweight adjusted kidney and liver weights were high for both sexes receiving 750/500 mg/kg bw/day with the liver weight also high at 300 mg/kg bw/day. There was evidence of recovery in the kidney and male liver, however, the female liver weight remained higher than control following the recovery period.

 

The macroscopic examination performed after 28 days of treatment and a two week recovery period revealed no test article-related lesions.

 

The histopathological examinations showed minimal or slight hyperplastic and/or degenerative/inflammatory changes in the non-glandular region of the stomach from individual males in each treated group and females at 750/500 mg/kg bw/day.

Administration of 300 mg/kg bw/day resulted in similar changes liver and stomach findings, but at a lower incidence and severity than at 750/500 mg/kg/day. In the absence of any clinical signs at these levels, 300 mg/kg bw/day was the no-observed-adverse effect level (NOAEL) for this study.

Under the test conditions, the NOAEL for test substance was considered to be 300 mg/kg bw/day in rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

300 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 key study was identified (HLS, 2015, rel. 1). In this subacute toxicity study performed in accordance with OECD test guideline No. 407 and in compliance with GLP, test item ST 14 C 12 was administered by gavage to 5 male and 5 female Crl:CD(SD) strain rats at doses of 30, 300 or 750/500 mg/kg bw/day for 28 consecutive days. A similarly constituted control group received the vehicle, corn oil at the volume dose of 5 mL/kg bw/day.

Administration of 750 mg/kg/day resulted in unforeseen clinical signs (unsteady gait, decreased activity, piloerection, partially closed eyelids, unresponsive and abnormal posture (swaying/flattened/prostrate)), one female was killed for humane reasons on Day 1 and further male on Day 5 due to persistent unresponsive behaviour. As a precautionary measure the high dose level was reduced to 500 mg/kg/day from Day 3 onwards.

There was evidence of adaptive changes in the liver at 750/500 mg/kg/day and to a lesser extent at 300 mg/kg/day. The increased cholesterol, alanine amino-transferase, alkaline phosphatase and aspartate amino-transferase levels, and increased adjusted liver weight are indicative of increased activity in the liver. Orally administered substances are metabolised by the liver, therefore this increase in activity is a normal adaptive response following administration of a test substance. There was general evidence of recovery and no histopathological changes in the liver.

The increased adjusted kidney weight for females, and to a lesser extent males, receiving 750/500 mg/kg/day is considered to be associated with the increase in plasma potassium, urinary glucose and chloride levels. There was full recovery and no histopathological changes in the kidney.

The histopathological examinations showed minimal or slight hyperplastic and/or degenerative/inflammatory changes in the non-glandular region of the stomach from individual males in each treated group and females at 750/500 mg/kg/day. This is indicative of local irritation and is consistent with the macroscopic stomach findings at higher dose levels in the 7-day preliminary study (Huntingdon Life Sciences study number HIK0032). For some individuals (Group 2 male number 3 and Group 3 male 28 and Group 4 male 16) there is a correlation between this irritation and the increased lymphocyte count at the end of the treatment period, with the increased basophil and monocyte count at the end of the recovery period being a continuation of the response to local insult.

Administration of 750/500 mg/kg/day resulted in the death of 2 rats due to the severity of the clinical signs observed, findings associated with local irritation to the stomach. The combination of these findings indicate that this is an adverse effect level.

At 300 mg/kg/day the findings in the liver, kidney and stomach were observed to a lesser extent than at 750/500 mg/kg/day and in the absence of any deaths or clinical signs, 300 mg/kg/day was the no-observed-adverse effect level (NOAEL).

The findings at 30 mg/kg/day comprised an increase in urinary glucose and lesions in the stomach of animal No. 3. In the absence of any corroborative findings in the kidney at this level the increase in urinary glucose was not adverse and as the stomach findings had no associated findings, they are also considered not to be adverse, thus confirming the higher level of 300 mg/kg/day as NOAEL.

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:

Based on the available data, no self-classification is proposed regarding the specific target organ toxicity after oral dose-repeated exposure according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

There were no data regarding the dermal and inhalation route.