Reaction products of Dihydro-3-(tetrapropenyl)furan-2,5 dione with propane-1,2,diol

Endpoint summary

Description of key information

Repeated dose toxicity: Oral route

Under the conditions of the range-finding study, administration of the test material to Han Wistar (RccHanTM;WIST) rats through  the oral route (gavage) was well tolerated at doses up to 300 mg/kg/day. Treatment at 500 and 1 000 mg/kg/day resulted in a range of signs which necessitated the premature termination of the animals on Days 5 and 2, respectively.

Under the conditions of a sub-acute toxicity study via the oral route, the NOAEL for local effects was 100 mg/kg/day for males and 300 mg/kg/day for females for effects on the stomach. The NOAEL for systemic toxicity under the conditions of this study was considered to be 300 mg/kg/day for both sexes.

 

Repeated dose toxicity: Inhalation route

In accordance with Section 8.6.1. of Column 2 of Annex VIII of the REACH Regulation, Short-term repeated dose toxicity study (28 days) should be conducted on one species, male and female, through the most appropriate route of administration, having regard to the likely route of human exposure which in this case is the oral route.

 

Repeated dose toxicity: Dermal route

In accordance with Section 8.6.1. of Column 2 of Annex VIII of the REACH Regulation, Short-term repeated dose toxicity study (28 days) should be conducted on one species, male and female, through the most appropriate route of administration, having regard to the likely route of human exposure which in this case is the oral route.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

repeated dose toxicity: oral, other

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30 March 2016 to 15 April 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The study was not designed to meet any particular regulatory requirements and is a range-finder for further study.
The purpose of the study was to assess the systemic toxic potential of the test material by oral administration to Han Wistar rats for 14 days at initial test concentrations of 100, 300 and 1 000 mg/kg/day. During the study, clinical condition, body weight, food consumption, visual water consumption, organ weight and macropathology investigations were undertaken.

GLP compliance:

no

Remarks:

No claim for compliance with Good Laboratory Practice was made, although the work performed generally followed Good Laboratory Practice principles.

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

RccHan™:WIST rat.

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 Han Wistar (RccHan™;WIST) 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
- Age at study initiation: Animal ages ranged from 74 to 96 days at start of treatment, specifically:
Group 1 and 2: 74 to 84 days
Group 3: 81 to 91 days
Group 4: 86 to 96 days
- Weight at study initiation: Weight ranged from 296 to 369 g for male animals and 197 to 241 g for female animals; specifically:
Group 1 and 2: Males: 296 to 356g; females: 197 to 232 g.
Group 3: Males: 335 to 369 g; females: 213 to 229 g.
Group 4: Males: 351 to 365g; females: 209 to 241 g.
- Housing: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals. Males and females were blocked by sex and the cages constituting each group were dispersed in a single battery so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The position of the cage batteries in the room were changed weekly, following a rotation plan, to further minimise possible effects of spatial variations. Animals were caged in groups of three (groups 1 to 3) or two (group 4) of the same sex.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 14 to 26 days before commencement of treatment; specifically:
Group 1 and 2: 14 days before commencement of treatment.
Group 3: 21 days before commencement of treatment.
Group 4: 26 days before commencement of treatment.
On day 1 (before dosing) variations in body weight of the animals were checked to ensure that they did not exceed ± 20 % of the mean for the appropriate sex. No replacements were required in Groups 1 to 3. No check was performed for Group 4, since no spare animals were remaining.
During the acclimatization period observations of animals and their cages were recorded at least once per day.

DETAILS OF FOOD AND WATER QUALITY
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are routinely provided by the water supplier. No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 ºC
- Humidity (%): 40-70 %
- Air changes (per hr): Filtered fresh air which was passed to atmosphere and not recirculated. At least 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): Artificial lighting, 12 hours light : 12 hours dark.

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. The test item was administered by gavage.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS
- Method of preparation: Starting with the lowest concentration, the required amount of test item was weighed into a suitable container. Approximately 50 % of vehicle was added; it was placed in a water bath and heated up to 40 °C if necessary. It was then stirred by hand using a spatula until the formulation was at the correct consistency and then magnetically stirred until uniformly mixed. The remaining vehicle was then added to make up to the required volume and then magnetically stirred until homogenous. The formulation was refrigerated (nominally 2 - 8 °C).
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
- Frequency of preparation: Weekly

VEHICLE
- Concentration in vehicle: Formulated concentrations of 20, 60, 200, and 100 mg/mL.
- Amount of vehicle: Dose volume of 5 mL/kg/day. Individual dose volumes were calculated from the most recently recorded scheduled body weight.

Analytical verification of doses or concentrations:

no

Remarks:

No formulation analysis was performed on this study.

Details on analytical verification of doses or concentrations:

No formulation analysis was performed on this study.
Before the commencement of treatment the suitability of the proposed mixing procedures was determined as part of another study. The homogeneity of the test item in the vehicle (at concentrations of 2 and 200 mg/mL) was achieved following refrigerated (2 – 8 °C) storage for up to 15 days and at ambient temperature (nominally 21 °C) storage for up to seven days.

Duration of treatment / exposure:

14 days

Frequency of treatment:

Once daily at approximately the same time each day.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Dose / conc.:

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

No. of animals per sex per dose:

3 animals per sex per dose for the animals dosed at 100, 300 and 1 000 mg/kg/day and 2 animals per sex per dose for the animals dosed at 500 mg/kg/day.

Control animals:

yes, historical

Details on study design:

DOSE SELECTION RATIONALE
Doses were selected based on the results from an acute oral toxicity study in the rat. In this acute toxicity study, a single oral dose of the test item at 2 000 mg/kg (given at a dose volume of 10 mL/kg using arachis oil as the vehicle) was associated with marked clinical signs comprising hunched posture, tip-toe gait, pilo-erection, lethargy, diarrhoea, dehydration and hypothermia and body weight loss necessitating the euthanasia of the animal. Macroscopic findings at necropsy comprised gaseous stomach, haemorrhagic non-glandular and gastric mucosa of the stomach and dark liver and kidneys. At 300 mg/kg, hunched posture was observed for up to two hours after dosing. There were no further signs and no macroscopic findings were apparent at necropsy on completion of the observation period. Consequently, it was considered that that the LD50 was > 300 < 2 000 mg/kg.
Considering the limited information available, it was considered appropriate to use a cautious approach to dosing and treatment commenced initially at 100 and 300 mg/kg/day, with treatment for Group 3 commencing one week later. Following 6-days of dosing at 100 and 300 mg/kg/day, there was no overt evidence of toxicity (food consumption was lower than that recorded pre-treatment, approximately 80 % of pre-dose, but without dose-relationship and there was no clear effect on body weight) and, therefore, a high dose level of 1 000 mg/kg/day for Group 3 was considered appropriate.
Following two days of dosing at 1 000 mg/kg/day, this treatment group was terminated because of excessive toxicity. Since no overt evidence of toxicity was apparent at 300 mg/kg/day, the spare animals ordered for the study (two males and two females) were used to investigate a dose level of 500 mg/kg/day.
The range of dose levels was selected to provide sufficient information for dose level selection for the associated 4-week toxicity study.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes.
Signs are considered in two parts: Detailed observations recorded at times in relation to dose administration, classified as ‘signs associated with dosing’ and extended changes in condition, classified as ‘clinical signs’.
Signs associated with dosing are presented for each animal that showed signs, providing detail of type of sign, day of occurrence and information on the duration of the sign applicable.
Clinical signs are presented for each animal, providing detail of type of sign, day of occurrence and information on the duration of the sign applicable.
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. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

Signs associated with dosing:
Detailed observations were recorded daily at the following approximate times in relation to dose administration:
- Pre-dose.
- After completion of dosing each group.
- One to two hours after dosing.
- As late as possible in the working day.
Additional observations were performed on Day 2 on Group 3 to monitor the condition of the animals.

Clinical signs:
A detailed weekly physical examination was performed on each animal to monitor general health.

MORTALITY
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary. A complete necropsy was performed in all cases.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded before treatment commenced on Day -3 (Groups 1 to 3 only) and Day -1, on the day that treatment commenced (Day 1), twice weekly thereafter throughout the study and before necropsy.
Group mean weight changes were calculated from the weight changes of individual animals surviving the specified period.

FOOD CONSUMPTION: Yes
The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for three days before treatment started (Groups 1 to 3 only) and twice weekly for throughout the study.
As there was only one cage per group and sex, only individual cage consumption values are presented.

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

Sacrifice and pathology:

NECROSCOPY
All animals were subject to a necropsy. Only the thoracic and abdominal cavities were opened. The cranial cavity was not opened as there were no observations during the study to indicate a possible neurotoxic action. 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 fixative. The retained tissues were checked before disposal of the carcass.
The organs weighed and tissue samples fixed. The spleen, liver and kidneys were weighed and fixed (left and right kidney weighed individually). Any tissues with abnormalities were also fixed. Tissues were preserved in 10 % Neutral Buffered Formalin.

METHOD OF SACRIFICE
Carbon dioxide asphyxiation with subsequent exsanguination.

ORGAN WEIGHTS
Organ weights were presented both as absolute and relative for terminal body weight, using the weight recorded on the day of necropsy.

Statistics:

No statistical analysis of the data was performed on this study.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Treatment at 100 or 300 mg/kg/day was well-tolerated. Salivation was observed from Day 5 for males and females receiving 300 mg/kg/day and there was an isolated occurrence on Day 5 for two males receiving 100 mg/kg/day. Chin rubbing was also occasionally observed for animals receiving 300 mg/kg/day. These signs became apparent on completion of dosing and were usually absent by 1 to 2 hours after dosing.
Salivation is often a common occurrence in studies where the test material is administered by oral gavage and is not usually considered of toxicological importance.
Vocalization was also recorded on Days 6, 13 and 15 for the majority of females receiving 100 or 300 mg/kg/day. This was considered not to preclude the use of these doses in future studies.
Treatment at 500 or 1 000 mg/kg/day was not tolerated. Following the second dose administration at 1 000 mg/kg/day, animals showed signs comprising elevated, swaying and/or unsteady gait, decreased activity, irregular and slow breathing, piloerection, partially closed eyes and one female became prostrate. This group was terminated on Day 2 because of the severity of the signs observed.
Treatment at 500 mg/kg/day was tolerated for the first 4-days of treatment, but animals showed deterioration in condition following the fifth dose. Signs observed comprised piloerection, abnormally cold to the touch, decreased activity and hunched posture. It was considered that this dose level would not be tolerated for further doses and, therefore, this group was terminated on Day 5 for welfare reasons.

Mortality:

no mortality observed

Description (incidence):

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Bodyweight gains (Days 1 to 14) were variable with no clear effect of treatment for animals receiving 100 or 300 mg/kg/day.
Bodyweight stasis or small bodyweight losses were observed for animals which received 500 mg/kg/day (Days 1 to 4) and for animals which received 1 000 mg/kg/day (Day 1 compared to terminal bodyweight on Day 2).

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption for animals receiving 100 or 300 mg/kg/day was lower than that recorded pre-treatment (approximately 74-83 % pre-dose) but there was no clear dose-relationship and this may have been influenced by the calorific value of the corn oil vehicle.
Food consumption measured over the first 4 days of treatment for animals receiving 500 mg/kg/day was lower than expected compared to pretreatment values of the other groups. Pretreatment food consumption was not recorded for this group as they were not initially assigned to the study.
Food consumption could not be assessed for animals receiving 1 000 mg/kg/day which were killed prematurely on Day 2.

Food efficiency:

not examined

Description (incidence and severity):

not examined

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

no effects observed

Description (incidence and severity):

A visual assessment of water consumption did not reveal any treatment-related effect

Ophthalmological findings:

not examined

Description (incidence and severity):

not examined

Haematological findings:

not examined

Description (incidence and severity):

not examined

Clinical biochemistry findings:

not examined

Description (incidence and severity):

not examined

Urinalysis findings:

not examined

Description (incidence and severity):

not examined

Behaviour (functional findings):

not examined

Description (incidence and severity):

not examined

Immunological findings:

not examined

Description (incidence and severity):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

There were no clear effects of treatment on organ weight for animals receiving 100, 300, 500 or 1 000 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination at necropsy for animals given 1 000 mg/kg/day, killed prematurely on Day 2 of treatment, revealed findings in the stomach (distended appearance, abnormal contents and depressions, thickening and irregular surface of the non-glandular mucosa) and intestinal tract (distended ileum, jejunum and/or caecum and abnormal contents (gas or thin yellow fluid) in the ileum and jejunum). In addition, a dark pancreas was recorded for one male and all three females and a dark liver for one female.
Macroscopic examination at necropsy for animals given 500 mg/kg/day, killed prematurely on Day 5 of treatment, also revealed findings in the stomach (abnormal contents, distended appearance, irregular surface and thickening of the non-glandular mucosa) and intestinal tract (abnormal contents in the duodenum, ileum and jejunum, distended jejunum and abnormal color of the colon, duodenum, ileum, and jejunum. A dark pancreas was recorded in one male and two females.
For animals killed on completion of the 14-day treatment period, depressions on the non-glandular mucosa was recorded for one male which received 300 mg/kg/day and thickened non-glandular mucosa was recorded for one male and two females which received 300 mg/kg/day.
There were no treatment-related macroscopic findings for animals which received 100 mg/kg/day.

Neuropathological findings:

not examined

Description (incidence and severity):

not examined

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

not examined

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

not examined

Other effects:

not examined

Description (incidence and severity):

not examined

Details on results:

It is concluded that the oral (gavage) administration the test material to Han Wistar (RccHanTM;WIST) rats was well tolerated at doses up to 300 mg/kg/day.
Treatment at 500 and 1 000 mg/kg/day resulted in a range of signs which necessitated the premature termination of the animals on Days 5 and 2, respectively. The main target organs were the stomach and small intestine.
Whilst changes in the stomach were apparent at 300 mg/kg/day, they were not associated with any adverse clinical signs or any effect on bodyweight gain or food consumption. Based on the findings in this study, 300 mg/kg/day was considered to be a suitable high dose level in the forthcoming 28-day study.

Dose descriptor:

NOAEL

Remarks on result:

not measured/tested

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

not specified

Treatment related:

not specified

Discussion

The test material was administered orally by gavage to Han Wistar (RccHanTM;WIST) rats for up to 14 days at dose levels of 100, 300, 500 and 1 000 mg/kg/day.

The doses of 500 and 1 000 mg/kg/day resulted in a range of signs which necessitated in the premature termination of the animals on Day 5 and 2, respectively. Necropsy findings largely comprised abnormalities of the stomach (distended appearance, abnormal colour, irregular surface, depressions or thickening of the non-glandular mucosa) and intestinal tract (abnormal content, abnormal colour and/or distended appearance predominantly in the ileum, jejunum and duodenum). In addition, dark appearance of the pancreas was reported for animals receiving 500 or 1 000 mg/kg/day and a dark liver for one female receiving 1 000 mg/kg/day.

The dose of 300 mg/kg/day was well tolerated with no deaths, minor transient signs and no effects on bodyweight gain or food consumption. Necropsy examination revealed changes in the stomach comprising thickening and depressions on the non-glandular mucosa for one male and thickening of the non-glandular mucosa for two females.

There were no findings of significance at 100 mg/kg/day.

Conclusions:

Under the conditions of the study, administration of the test material to Han Wistar (RccHanTM;WIST) rats through the oral route (gavage) was well tolerated at doses up to 300 mg/kg/day. Treatment at 500 and 1 000 mg/kg/day resulted in a range of signs which necessitated the premature termination of the animals on Days 5 and 2, respectively.

Executive summary:

The purpose of this study was to assess the systemic toxic potential of the test material by oral gavage administration to Han Wistar rats for 14 days.

The initial doses selected for this study were 100, 300 and 1 000 mg/kg/day. Males and females at 1 000 mg/kg/day were killed for reasons of animal welfare on Day 2 of dosing due to the severity of signs observed. An additional two males and two females were assigned to the study and dosed at 500 mg/kg/day to gain further information on the dose response.

During the study, clinical condition, body weight, food consumption, visual water consumption assessment, organ weight and macropathology investigations were undertaken.

Following the second dose administration at 1 000 mg/kg/day, signs observed included elevated, swaying and/or unsteady gait, decreased activity, irregular and slow breathing, piloerection, partially closed eyes and one female became prostrate. All animals were killed prematurely on Day 2 because of the severity of the signs observed.

Necropsy examination revealed findings in the stomach (distended appearance, abnormal contents and depressions, thickening and irregular surface of the non-glandular mucosa) and intestinal tract (distended ileum, jejunum and/or caecum and abnormal contents in the ileum and jejunum). A dark pancreas was reported for one male and all three females and a dark liver for one female.

Treatment at 500 mg/kg/day was tolerated for the first 4-days of treatment, but animals deteriorated following the fifth dose. Signs comprised piloerection, abnormally cold to the touch, decreased activity and hunched posture. Bodyweight gain and food consumption were low. It was considered that this dose level would not be tolerated for further doses and, therefore, this group was terminated on Day 5 for welfare reasons.

Necropsy examination revealed findings in the stomach (abnormal contents, distended appearance and irregular surface and thickening of the non-glandular mucosa) and intestinal tract (abnormal contents in the duodenum, ileum and jejunum, distended jejunum and abnormal color of the colon, duodenum, ileum, and jejunum). A dark pancreas was recorded in one male and two females.

Treatment of animals with 300 mg/kg/day was well-tolerated and there were no deaths. The only signs comprised transient salivation and chin rubbing. There were no effects on bodyweight gain or food consumption. There was no clear effect of treatment on organ weights. Necropsy examination revealed changes in the stomach comprising thickening and depressions on the non-glandular mucosa for one male and thickening of the non-glandular mucosa for two females.

With the exception of an occasional incidence of transient salivation, the appearance and behavior of the animals were unaffected by treatment dose of 100 mg/kg/day treatment and there were no deaths. There were no effects on bodyweight gain, food consumption or organ weights and no findings at necropsy.

It is concluded that the oral (gavage) administration of the test material to Han Wistar (RccHanTM;WIST) rats was well tolerated at doses up to 300 mg/kg/day.

Treatment at 500 and 1 000 mg/kg/day resulted in a range of signs which necessitated the premature termination of the animals on Days 5 and 2, respectively. The main target organs were the stomach and small intestine.

Whilst changes in the stomach were apparent at 300 mg/kg/day, they were not associated with any adverse clinical signs or any effect on bodyweight gain or food consumption. Based on the findings in this study, 300 mg/kg/day was considered to be a suitable high dose level in the 28-day study.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

02 March 2013 to 25 August 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050,

Version / remarks:

July, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

RccHan™:WIST rat

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 Han Wistar 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
- Age at study initiation: 43 to 49 days.
- Weight at study initiation: Males: 137 to 183 g and Females: 123 to 160 g.
- Housing: Polycarbonate body with a stainless steel mesh lid, changed at appropriate intervals. Five animals of the same sex (main study and recovery) were housed per cage. Wood based bedding which was changed at appropriate intervals each week was added to the cages. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimise possible effects of spatial variations. Chew blocks and plastic shelters were provided as enrichment.
Males and females were blocked by sex and the cages constituting each group were dispersed in batteries so that possible environmental influences arising from their spatial distribution were equilibrated, as far as was practicable. The positions of the cage batteries in the room were changed weekly, following a rotation plan, to further minimize possible effects of spatial variations.
- Diet: Non-restricted. Animals did not have access to food overnight during the period of urine collection which preceded blood sampling procedures for haematology and blood chemistry investigations on the following day.
- Water: Non-restricted except during urine collection.
- Acclimation period: 14 days before commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24 °C
- Humidity: 40 to 70 %.
- Air: Filtered fresh air which was passed to atmosphere and not recirculated, with a minimum of 15 air changes per hour.
- Photoperiod: Artificial lighting, 12 hours light: 12 hours dark.

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration is generally recognised as the primary means of developing general toxicity data.

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
- Starting with the lowest concentration, the required amount of test material was weighed into a suitable container. Approximately 50 % of vehicle was added; it was placed in a water bath and heated up to 40 °C if necessary. It was then stirred by hand using a spatula until the formulation was at the correct consistency and then magnetically stirred until uniformly mixed. The remaining vehicle was then added to make up to the required volume and then magnetically stirred until homogenous.
- The formulation was prepared weekly.
- The formulated test material was stored in a refrigertor, nominally at 2 to 8 °C.
- Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

STABILITY AND HOMOGENEITY
- Before commencement of treatment, the suitability of the proposed mixing procedures was determined and specimen formulations at 2 and 200 mg/mL were analysed to assess the stability and homogeneity of the test material in the liquid matrix. Homogeneity and stability was confirmed following refrigerated (2 to 8 °C) storage for up to 15 days and at ambient temperature (nominally 21 °C) for up to 7 days.

ACHIEVED CONCENTRATION
- Samples of each formulation prepared for administration in Weeks 1 and 4 of treatment were analysed for achieved concentration of the test material.

PREPARATION OF LINEARITY STANDARDS
- For the weighing procedure, during preparation of standards and recovery samples, the test material was heated to 80 °C to enhance the mobility of the test material. Volumetric flasks were pre-weighed, test material added and the volumetric flasks re-weighed.
- A primary standard solution (1 000 μg/mL) was prepared by dissolving an accurately weighed quantity (ca. 50 mg) of test material in ethanol (50 mL). A secondary standard was prepared by diluting 0.2 mL of primary standard to 10 mL using ethanol. Solutions for instrument calibration were prepared by appropriate dilution of the secondary standard using ethanol and contained the test material at nominal concentrations of 400, 800, 1 000, 1 200, 1 600 and 2 000 ng/mL. Calibration standards were matrix-matched by including 250 µL of the initial control vehicle extract.
- Standard solutions were injected onto the LC-MS/MS at the beginning of each sample analysis sequence as a minimum, to assess linearity using the conditions detailed in the chromatographic section. A calibration standard (1 000 ng/mL) was injected throughout the run bracketing a maximum of three samples.

PREPARATION OF TEST SAMPLES
- A representative sample of test material formulation (1 mL, accurately weighed) was dispersed using ultrasonic vibration in a suitable volume of ethanol. The extract was diluted using ethanol, to provide a solution containing the test material at an expected concentration of approximately 1 000 ng/mL. Samples were matrix-matched by including an appropriate volume of the initial control vehicle extract in the final sample dilution where necessary. The concentration of test material in the final solution was quantified by LC-MS/MS.

PREPARATION OF RECOVERY SAMPLES
- Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (corn oil) with known amounts of test material. The prepared procedural recoveries were analysed in accordance with the analytical procedure.

INSTRUMENTAL PARAMETERS
Liquid chromatograph with mass spectrometry detection (LC-MS/MS): Agilent 1100 Binary pump, Perkin Elmer PE200 Autosampler and Sciex API 3000 mass spectrometer or Ionics EP10+ mass spectrometer
Column: Agilent Poroshell SB C18, 2.7 μm, 100 × 4.6 mm
Column temperature: 60 °C
Sample temperature: Ambient
Mobile Phase A: Propan-2-ol/ Water/ Formic Acid 25/75/0.1 v/v/v
Mobile Phase B: 0.1% Formic Acid in Propan-2-ol
Gradient:
- Time (min) 0.0, 4.0, 7.0, 7.1, 10.0
- %A: 50, 5, 5, 50, 50
- %B: 50, 95, 95, 50, 50
Flow rate: 0.35 mL/minute
Rinse solvent/Needle wash: Propan-2-ol
Injection volume: 10 μL
Run time: 10 minutes
Approximate retention time: 6.23 minutes
MS Conditions:
Ionisation Mode: Turbo IonSpray Positive Ion Detection
Monitored Ions: m/z 617.7 to 617.7

CALCULATIONS
- The peak area response for the test material in each calibration standard chromatogram was measured. Calibration curves were constructed by linear regression, weighing factor 1/x, of calibration standard response versus calibration standard concentration. The area response of the peak observed at the characteristic retention time for the test material in the sample and procedural recovery chromatograms was measured. The response factor for the single bracketing standard was determined using the following equation: Response factor (RF) = Calibration standard peak response (Ac) / Concentration of calibration standard (ng/mL)
- The concentration of the test material was determined using the following equation:

Analysed concentration (mg/mL) = (As/RF) × (V/W) × (D/1 000 000)

- Procedural recovery values were determined using the following equation:

Procedural recovery = (Analysed concentration (mg/mL)/ Fortified concentration (mg/mL)) x 100

- Sample concentrations were corrected for procedural recoveries using the following equation:

Corrected concentration, mg/mL = Analysed concentration, mg/mL x (100/ R)

Where:
Ac = Peak response for test material standard.
As = Peak response for test material in sample chromatogram.
RF = Mean response factor of appropriate set of bracketing standard
V = Dilution volume (mL)
W = Sample weight (g)
R = Mean procedural recovery value at analysis
D = Density of sample (g/mL)

VALIDATION OF THE ANALYTICAL PREOCEDURE
- The analytical procedure was validated by determining the following parameters:
- The specificity of the chromatographic analysis in control sample chromatograms.
- The linearity of detector response over the calibration standard concentration range.
- The repeatability of the lowest and highest concentration calibration standards.
- The method accuracy and precision, by determining five procedural recoveries at nominal concentrations of 2 mg/mL and 200 mg/mL during the method validation.

HOMOGENEITY AND STABILITY IN CORN OIL FORMULATIONS
- The homogeneity and stability of the test material in corn oil formulations was assessed at nominal concentrations of 2 mg/mL and 200 mg/mL, during ambient and refrigerated storage. Freshly prepared specimen formulations (300 mL) were equally sub divided (3 × 100 mL) into 3 amber glass screw top bottles by Pharmacy personnel and submitted for analysis.

AMBIENT TEMPERATURE STORAGE (NOMINALLY 21 °C)
- On receipt, the contents of one bottle of each formulation were mixed by 20-fold inversion followed by magnetic stirring. After stirring for 20 minutes (representing 0 hour) and 4 hours, single samples (nominally 1 mL) were removed for analysis from approximately one quarter, one half and three quarters the depth (representing the top, middle and bottom) of the continuously stirred formulation.
- The remainder of the bottle was stored at ambient temperature and after 2 days and 7 days storage the contents were remixed and sampled as detailed above.

REFRIGERATED STORAGE (NOMINALLY +4 °C)
- The remaining bottles were refrigerated on receipt and on Day 2, Day 7 and Day 15; the appropriate bottle was removed from storage and equilibrated to ambient temperature. The contents of the bottle were mixed by 20-fold inversion followed by magnetic stirring for a minimum of 20 minutes and single samples (nominally 1 mL) were removed for analysis from the top, middle and bottom of the stirred formulation.

CONCENTRATION OF DOSE FORMULATIONS
- For Week 1 and Week 4, freshly prepared test formulations were sampled (4 × 1 mL, accurately weighed) by Pharmacy personnel and submitted for analysis. Duplicate samples were analysed in accordance with the analytical procedure, and the remaining samples were retained for contingency. Samples were disposed of once satisfactory results were achieved. In Week 1, the results for Groups 3 and 4 did not meet acceptance criteria, Group 3 had a low RME from nominal concentration and Group 4 had high percent difference from mean between duplicate samples, contingency samples were analysed for both groups. Both groups are reported from the mean of four results. In Week 4, contingency analysis was performed for Groups 2, 3 and 4, as the original results did not meet acceptance criteria. As for the Week 1 formulations, these groups are reported from the mean of four results.


ANALYTICAL RESULTS
METHOD VALIDATION
The analytical procedure was successfully validated for the test material in corn oil with respect to the specificity of chromatographic analysis, the linearity of detector response, repeatability, method accuracy and precision.
-The specificity of the LC-MS/MS assay was demonstrated by the absence of a peak with a response >20 % of the response of the lowest calibration standard at the characteristic retention time for the test material in the control sample chromatogram.
-Linearity was confirmed over the nominal concentration range 400 ng/mL to 2 000 ng/mL with a correlation coefficient >0.99.
-The repeatability was <6 % for six replicate injections of standard solutions containing the test material at a nominal concentration of 400 ng/mL. The repeatability was <5 % for six replicate injections of standard solutions containing the test material at a nominal concentration of 2 000 ng/mL.
-Method accuracy and precision were confirmed, a mean procedural recovery value of 100.4 % (CV = 0.79 %, n = 5) was obtained for 2 mg/mL and 106.2 % (CV = 1.55 %, n = 5) was obtained for 200 ng/mL.
-Procedural recovery results throughout the entirety of the study were variable on a run by run basis. This is considered to be a result of the inherent variability of the LC-MS/MS method used for analysis of the test material. After a review of the completed study data, it is considered that an acceptance limit of 90 – 110 % of the nominal fortification is acceptable for recoveries. This has been applied to all analytical occasions and was reviewed for each run.
 
HOMOGENEITY AND STABILITY OF DOSE FORMULATIONS
-The homogeneity and stability of the test material in corn oil formulations was assessed with respect to the level of concentration at nominal concentrations of 2 mg/mL and 200 mg/mL.
-Homogeneity was confirmed during distribution between the bottles, during magnetic stirring for 4 hours, and on re-suspension following storage at ambient temperature for 7 days and refrigeration for up to 15 days. At each time-point, the mean analysed concentration for the three samples remained within 10 % of the initial time zero value and the coefficient of variation was less than 5 %.
-Recovery results during the trial remained within applied limits of 90 to 110 % recovery, showing the continued accuracy of the method with the exception of a 2 mg/mL recovery prepared on Day 2 which showed a recovery of 87.8 %. This was excluded from calculation, and the Day 2 results are corrected for the mean of the remaining three acceptable recoveries prepared on this occasion as per SOP.
 
CONCENTRATION OF DOSE FORMULATIONS
-The mean concentrations were within applied limits +10 / -15 %, confirming the accuracy of formulation. The coefficient of variation for Group 3 in Week 1, and all treated groups in Week 4 is >5 %, this is considered to be due to the variability of the LC-MS/MS method and formulations are deemed to have been prepared correctly.

UNFORESEEN EVENTS
Study Plan Amendment 1 states that formulation stability will be assessed after 7 days storage as the results of Day 2 were deemed to be unreportable due to two of four procedural recoveries being outside validated limits. After a review of the completed study the procedural recovery acceptance limits were amended to 90 to 110 % of the nominal concentration. After this review, three of four procedural recoveries were within limits, meaning the Day 2 results could be reported in line with SOP. Both the Day 2 and Day 7 results are reported for the stability trial, Day 2 results meet all acceptance criteria.

CONCLUSION
-The analytical procedure was successfully validated with respect to specificity of chromatographic analysis, linearity of detector response, repeatability, method accuracy and precision.
-The homogeneity and stability was confirmed for the test material in corn oil formulations at nominal concentrations of 2 mg/mL and 200 mg/mL during distribution between the bottles, during magnetic stirring for 4 hours, ambient temperature storage for up to 7 days and refrigerated storage for up to 15 days.
-The mean concentrations of test material in test formulations analysed for the study were within +10 / -15 % of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

Minimum treatment period was four weeks followed by a two week recovery period.

Frequency of treatment:

Once daily, at approximately the same time each day.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

5 animals per sex per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels selected for this study (30, 100 and 300 mg/kg/day) were selected based on the results of a 14-day dose-range finding study.
In that study, doses of 500 and 1 000 mg/kg/day were not tolerated. Following the second dose administration at 1 000 mg/kg/day, animals showed signs comprising elevated, swaying and/or unsteady gait, decreased activity, irregular and slow breathing, piloerection, partially closed eyes and one female became prostrate. This group was terminated on Day 2 because of the severity of the signs observed.
Treatment at 500 mg/kg/day was tolerated for the first 4-days of treatment, but animals showed deterioration in condition following the fifth dose. Signs observed comprised piloerection, abnormally cold to the touch, decreased activity and hunched posture. It was considered that this dose level would not be tolerated for further doses and, therefore, this group was terminated on Day 5 for welfare reasons.
Predominant findings at necropsy at 500 and/or 1 000 mg/kg/day included abnormalities of the gastro-intestinal tract, comprising a range of findings in the stomach including, but not limited to, distended appearance, depressions, irregular surface and thickening of the non-glandular region, distended ileum, jejunum and caecum with abnormal contents, and dark liver (1 000 mg/kg/day only) and dark pancreas.
At 100 and 300 mg/kg/day, the only sign seen was transient salivation. Macroscopic findings at 300 mg/kg/day comprised thickened stomach wall for one male and two females and depressions in the stomach wall for one male. There were no treatment-related macroscopic findings for animals which received 100 mg/kg/day.
Based on these findings, it was considered appropriate to investigate a high dose level of 300 mg/kg/day in this study. The intermediate and low dose levels were selected to assess any dose-responsiveness of any test substance-related finding.
- Rationale for selecting satellite groups: 5 animals per sex in the control and 5 animals per sex at the highest dose level were used for the recovery phase.
- Post-exposure recovery period in satellite groups: 2 weeks

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- Cages were inspected daily for evidence of animal ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupants. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization and recovery periods, observations of animals and their cages were recorded at least once per day.
During the treatment period, detailed observations were recorded daily at the following times in relation to dose administration:
Week 1 of treatment: Pre-dose observation, at the end of dosing each group, one to two hours after completion of dosing of all groups, as late as possible in the working day.
Week 2 of treatment to termination: Pre-dose observation and one to two hours after completion of dosing of all groups.

DETAILED CLINICAL OBSERVATIONS: Yes
- 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.
- After removal from the home cage, animals were assessed for physical condition and behaviour during handling and after being placed in a standard arena. Any deviation from normal was recorded with respect to the nature and, where appropriate, degree of severity. Particular attention was paid to possible signs of neurotoxicity, such as convulsions, tremor and abnormalities of gait or behaviour. Findings were reported as "present" or assigned a severity grade - slight, moderate or marked.
Signs are considered in two parts: Detailed observations recorded at times in relation to dose administration, classified as ‘signs associated with dosing’ and extended changes in condition, classified as ‘detailed physical examination and arena observations’.
Clinical observations are presented for each animal that showed signs, providing detail of type of sign, week of occurrence and information on the duration of the sign applicable.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced, on the day that treatment commenced (Week 0), weekly throughout the study and before necropsy. The last scheduled body weight was recorded on Day 28 for the main study animals and Day 14 of recovery for the recovery phase animals prior to overnight deprivation of food and water for clinical pathology investigations.
Group mean weight changes were calculated from the weight changes of individual animals.

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 and for each week throughout the study. Food remaining for the last week of the treatment and recovery periods was recorded on Day 28 or 14, respectively, (i.e. prior to overnight deprivation of food for clinical pathology investigations).
Weekly group mean food consumptions and standard deviations were derived from unrounded cage values. Overall mean food consumption values were calculated from the weekly group mean values.

WATER CONSUMPTION: Yes
- Fluid intake was initially assessed by daily visual observation. This indicated a possible treatment-related effect and, therefore, quantitative measurements were performed during Weeks 3 and 4 of treatment and Weeks 1 and 2 of recovery. Water consumption was recorded by weight (over a 2 day period during Week 3 of treatment and a 3 day period for Week 4 of treatment and for each week of the recovery period) for each cage of animals, using water bottles fitted with sipper tubes.
Water consumption was calculated from measurements of initial and final weights (g) of the water bottle and its contents for each cage (it was assumed that 1 mL of water weighed 1 g).

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
Day 29 of treatment (prior to termination): All main study animals;
Day 15 of recovery (prior to termination): All recovery males;
Day 16 of recovery (prior to termination): All recovery females.
- Anaesthetic used for blood collection: Yes, animals were held under light general anesthesia induced by isoflurane.
- Animals fasted: Yes, blood samples were collected after overnight withdrawal of food and prior to dosing (where appropriate). Sampling was performed on the morning after overnight collection of urine. Animals were, therefore, also deprived of water overnight but had access to water for a minimum period of one hour prior to the commencement of procedures.
- Parameters checked: Blood samples (nominally 0.5 mL) were withdrawn from the sublingual vein, collected into tubes containing EDTA anticoagulant and examined for the following characteristics. Haematocrit (Hct), Haemoglobin concentration (Hb), Erythrocyte count (RBC), Absolute reticulocyte count (Retic), Mean cell hemoglobin (MCH), Mean cell hemoglobin, 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), Large unstained cells (LUC), Platelet count (Plt).
Blood film (prepared for all samples) - Romanowsky stain, examined for abnormalities by light microscope, in the case of flags from the Advia 120 analyser. Confirmation or a written description from the blood film was made where appropriate.
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) - using IL PT Fibrinogen reagent, and
Activated partial thromboplastin time (APTT) - using IL APTT reagent.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Samples were cllected at the following occassions:
Day 29 of treatment (prior to termination): All main study animals,
Day 15 of recovery (prior to termination): All recovery males,
Day 16 of recovery (prior to termination): All recovery females.
- Animals fasted: Yes. Blood samples were collected after overnight withdrawal of food and prior to dosing (where appropriate). Sampling was performed on the morning after overnight collection of urine. Animals were therefore also deprived of water overnight, but had acces to water for a minimum period of one hour prior to the commencement of procedures.
- Anaesthetic used for blood collection: Animals were held under light general anaesthesia induced by isoflurane. Blood samples (nominally 0.7 mL) were withdrawn from the sublingual vein and collected into tubes containing lithium heparin as an anticoagulant.
- Parameters checked: After separation, the plasma was examined using a Roche P Modular Analyser in respect of: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Gamma-glutamyl transferase (gGT), Total bilirubin (Bili), Total bile acids (BiAc), Urea, Creatinine (Creat), Glucose (Gluc), Total cholesterol (Chol), Triglycerides (Trig), Sodium (Na), Potassium (K), Chloride (Cl), Inorganic phosphorus (Phos), Total protein (Total Prot) and Albumin (Alb). Albumin/globulin ratio (A/G Ratio) was calculated from total protein concentration and analysed albumin concentration.
Albumin to globulin ratio (A/G Ratio) was calculated as:

A/G Ratio = Albumin concentration / (total protein – albumin concentration)

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected overnight at the following occasions:
Day 29 of treatment (prior to termination): All main study animals,
Day 15 of recovery (prior to termination): All recovery males,
Day 16 of recovery (prior to termination): All recovery females.
- Metabolism cages used for collection of urine: Yes, animals were placed in individual metabolism cages.
- Animals fasted: Yes animals were housed without food and water.
- Parameters checked: The individual samples were examined for the following characteristics:
Clarity and Colour (App) - by visual assessment,
Volume (Vol) - using a measuring cylinder,
pH - using a pH meter, and
Specific gravity (SG) - by direct refractometry using a SG meter.
Using Multistix reagent strips interpreted using Clinitek(R) 500 instrument: Ketones (Keto), Bile pigments (Bili) and Blood pigments (UBld).
Using a Roche P Modeular Analyser: Protein (T-Prot), Creatinine (T-Creat), Glucose (T-Gluc), Sodium (T-Na), Potassium (T-K) and Chloride (T-Cl).
A microscpoic exmination of the urine sediment was performed. An aliquot of the urine sample was centrifuged, stained with Kova stain and the resulting deposit spread on a microscope slide. The number of elements seen in nine high or low power fields (HPF or LPF) were recorded in the raw data and entered onto the database and the number seen /HPF or /LPF was derived from these data as follows: Epithelial cells (Epi), Leucocytes (WBC), Erythrocytes (RBC), Casts and Other abnormal components (A). The slide was also examined for abnormalities in spermatozoa and crystals.
Group means and standard deviations are presented for volume, pH, specific gravity, protein, creatinine, glucose and electrolytes only.

NEUROBEHAVIOURAL EXAMINATION: Yes
- 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. Animals were tested by an observer who was unaware of the treatment group to which each animal belonged. Before the start of observations, cage labels showing the treatment group were replaced by labels stating only the study, animal and cage numbers. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.
The following measurements, reflexes and responses were recorded.
- Approach response: A blunt probe was brought towards the animal’s head until it was close to the animal’s nose (but not touching the whiskers). The animal’s reaction was recorded as:
- 1: No reaction or ignores probe/walks past probe
- 2: Normal awareness and reaction e.g. approaches and/or sniffs probe
- 3: Active avoidance, abnormally fearful or aggressive reaction

- Pinna reflex: The inside of one ear was touched lightly with a nylon filament and the reaction recorded as:
- 1: No response
- 2: Normal response e.g. ear twitches/flattens or animal shakes its head
- 3: Abnormally fearful or aggressive response

- Auditory startle reflex: The animal’s response to a sudden sharp noise was assessed and scored as:
- 1: No response
- 2: Weak response e.g. ear twitch only
- 3: Normal response e.g. obvious flinch or startle
- 4: Exaggerated response e.g. all feet off floor

- Tail pinch response: The animal’s tail was pinched sharply with forceps approximately one third from the tip and the response graded as:
- 1: No response
- 2: Weak response e.g. turns around slowly or weak vocalisation without moving away
- 3: Normal response e.g. jumps forward or turns around sharply, usually with vocalisation
- 4: Exaggerated response e.g. excessive vocalization, body movement or aggression

- Grip strength: Forelimb and hind-limb grip strength was measured using Mecmesin Basic Force Gauges. Three trials were performed.

At any point during the observations, additional comments were made as free text where considered appropriate.

- 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 2.0.6), with hardware supplied by Pearson Technical Services and software developed and maintained by Envigo. In addition, all Recovery phase animals were tested during Week 2 of recovery.
Animals were tested individually in clear polycarbonate cages and motor activity was measured by counting infra-red beam breaks over ten 6-minute intervals (one hour total). Ten beams were set at two height levels (five low and five high) to detect cage floor and rearing activity respectively. Animals were not necessarily all tested on the same day, but the numbers of animals and the times of testing were balanced across the groups on each day of testing.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY AND HISTOPATHOLOGY: Yes
- All main study and recovery animals were subject to a detailed necropsy. 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 or tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. The retained tissues were checked before disposal of the carcass.
- Main study animals were killed following four weeks of treatment (Day 29). Recovery animals were killed following four weeks of treatment and two weeks of recovery (males on Day 15 of recovery and females on Day 16 of recovery).
- The following organs were fixed and subjected to pathology and histology: Adrenals, Brain (cerebellum, cerebrum, midbrain), Cecum, Colon, Duodenum, Epididymides, Esophagus, Eyes, Femur (femorotibial joint), Head, Heart (including auricular and ventricular regions), Ileum, Jejunum, Kidneys, Liver (section from two lobes), Lungs (section from two major lobes including bronchi), Lymph nodes- mandibular, mesenteric and left axillary, Ovaries, Peyer’s patches, Pituitary, Prostate, Rectum, Sciatic nerves, Seminal vesicles (with coagulating glands), Skeletal muscle, Spinal cord (transverse and longitudinal sections at the cervical, thoracic and lumbar levels), Spleen, Sternum (with bone marrow), Stomach, Submandibular salivary gland, Testes, Thymus, Thyroid with parathyroids, Trachea, Urinary bladder, Uterus with cervix and Vagina.
- Organ weights were recorded for the following: Adrenals, Brain (cerebellum, cerebrum, midbrain), Epididymides, Heart (including auricular and ventricular regions, Kidneys, Liver (section from two lobes), Ovaries, Prostate, Seminal vesicles (with coagulating glands), Spleen, Testes, Thymus, Thyroid with parathyroids and Uterus with cervix. Prostate and seminal vesicles with coagulating gland were weighed together. Thyroid and parathyroid were weighed after partial fixing.
For bilateral organs, left and right organs were weighed together, unless specified. Requisite organs were weighed for main study and recovery animals killed at scheduled intervals.
Organ weights were presented both as absolute/unadjusted and adjusted for terminal body weight, using the weight recorded on the day of necropsy.
- Tissues were routinely preserved in 10 % Neutral Buffered Formalin with the exception of the following:
Testes - In modified Davidson’s fluid, and
Eyes - In Davidson’s fluid.
- Histology: 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.
For main study animals of Groups 1 and 4, the full list was examined. For all main study animals of groups 2 and 3 and all recovery phase animals, only abnormalities and the stomach were examined. Sections were stained with haematoxylin and eosin.
Tissues preserved were examined by light microscopy. Findings were either "present" or assigned a severity grade. In the latter case, one of the following grades was used: Minimal, slight, moderate, marked or severe. a reviewing pathologist undertook a peer review of the microscopic findings.

Statistics:

See below.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The clinical appearance of the animals was not affected by treatment.
Transient salivation and chin rubbing was observed, predominantly during the first week of treatment, for males and females receiving 300 mg/kg/day. Salivation was also observed for a small number of females receiving 100 mg/kg/day. These signs were generally apparent at the end of dosing each group and were mostly absent by 1-2 hours after dosing.
Salivation is a commonly observed sign in studies where the test material is administered by gavage and, as such, is considered of no toxicological importance.

Mortality:

no mortality observed

Description (incidence):

There were no treatment-related deaths.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Bodyweight gains were slightly lower than those of the controls during the treatment period for all treated male groups. There was, however, no dose-relationship.
During the recovery period, the overall body weight gain for male animals that previously received 300 mg/kg/day was similar to that of the control animals, indicating recovery from the previous effect of treatment.
Bodyweight gain for females was considered to be unaffected by treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

During the treatment period, food consumption for all treated groups was slightly lower than that of the controls and this trend continued during the recovery phase. The differences from controls were, however, minor (<10 %) with no dose-relationship and, therefore, were of uncertain relationship to treatment.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

A visual assessment of water consumption did not reveal any treatment-related effect during the first week of treatment. During Week 2 of treatment, treated groups appeared to have consumed less water than the controls. Quantitative measurements were, therefore, performed during Weeks 3 and 4 of treatment.
Quantitative measurements during Weeks 3 and 4 confirmed that males in all treated groups consumed less water than Controls and a dose response was apparent.
During the two week recovery period, water consumption values for males that previously received 300 mg/kg/day continued to be less than controls.
Water consumption for females was considered to be unaffected by treatment.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Haematology investigations after 4 weeks of treatment revealed slightly low mean haematocrit, haemoglobin concentration and erythrocyte count, when compared with the controls, for females receiving 300 mg/kg/day (maximum effect 0.91X of control). Erythrocyte counts were also slightly low compared with controls for females receiving 30 or 100 mg/kg/day but without dose-relationship (0.94X and 0.96X control, respectively). Following two weeks off-dose period, the values were similar to controls indicating full recovery had occurred.
Neutrophil counts for males receiving 300 mg/kg/day were slightly higher than controls following 4 weeks of treatment (1.5X control). Following 2 weeks off-dose, neutrophil counts for previously-treated males were still slightly higher than controls (1.3X control), but this was due to a high value in one animal, whilst values for all other animals were within the range of the controls. The difference did not attain statistical significance and, therefore, recovery was considered to have occurred.
All other differences from controls observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.
Some statistically significant differences were reported at the end of the recovery period for parameters that were unaffected during the treatment period. These were all minor and were considered of no biological or toxicological significance and not to represent an adverse effect of the test material. These included slightly high white blood cell, lymphocyte, monocyte and large unstained cells count for previously treated females and slightly high red cell distribution width and slightly low mean cell haemoglobin and mean cell volume for previously treated males.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

The biochemical analysis of blood plasma following 4 weeks of treatment did not identify any toxicologically significant difference from controls.
A small number differences from controls attained statistical significance, but these were minor, confined to one sex or lacked dose-relationship and were therefore attributed to normal biological variation. Such differences included a small increase of chloride concentration at all doses in males which did not display any dose dependent trend and mean bile acid concentration was high for females receiving 300 mg/kg/day, but this was due to very high values in two animals and attributed to normal variation.
Following two weeks of recovery, females previously receiving 300 mg/kg/day presented lower glucose concentrations, higher triglyceride and sodium concentrations and lower albumin globulin ratios when compared to controls. These differences were not apparent at the end of the treatment period and as such are not attributed to the test material. No significant differences to the control were apparent in males at this sampling occasion.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Urinalysis investigations after 4 weeks of treatment revealed slightly low urinary pH, compared with controls, for males receiving 30 or 300 mg/kg/day (but not 100 mg/kg/day). Specific gravity was also slightly high for males receiving 30 or 300 mg/kg/day (but not 100 mg/kg/day), correlating with reduced urine volume in these animals. These changes were no longer apparent after 2 weeks of recovery.
All other differences from controls observed during the treatment period were minor or lacked dose relationship and were therefore attributed to normal biological variation.
Following two weeks of recovery females which previously received 300 mg/kg/day presented a higher total protein than control. This difference was minor, not apparent at the end of the treatment and, therefore, considered due to normal biological variation.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

See neuropathological findings below.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Analysis of organ weights for animals killed after 4 weeks of treatment revealed, when compared with the controls, slightly high absolute and body weight-adjusted liver weights at all doses in males and females (maximum effect 1.2 X control). All other inter-group differences from controls were minor or lacked dose-relationship and were therefore attributed to normal biological variation.
Following two weeks of recovery absolute and body weight-adjusted liver weights were similar to control in both male and female animals previously receiving 300 mg/kg/day indicating recovery had occurred.
Female animals killed on completion of the recovery period presented lower than control absolute and body weight-adjusted ovary weights and higher than control absolute and body weight-adjusted spleen weights. These differences were not apparent at the end of the treatment period and were considered fortuitous and not an effect of previous treatment.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

ANIMALS KILLED AFTER 4 WEEKS TREATMENT
Stomach: Dark areas and/or depressions were seen in the non-glandular region of males treated at 300 mg/kg/day with dark areas seen in one male treated at 30 mg/kg/day. Thickening of the stomach was seen in animals treated at 300 mg/kg/day and one male treated at 100 mg/kg/day.
The incidence and distribution of all the other findings were consistent with the common background.

ANIMALS KILLED AFTER 2 WEEKS OF RECOVERY AFTER TREATMENT
The macroscopic examination performed after 2 weeks of recovery revealed no test material related lesions.
All findings were similar to the background of changes commonly seen here.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Sensory activity and grip strength were unaffected by treatment in Week 4 of treatment and no evidence of any delayed affects were apparent in Week 2 of recovery.
There was no effect of the test material on motor activity assessed either during Week 4 of treatment or during Week 2 of recovery. Activity data is highly variable, occasional statistical significances obtained were not part of a larger trend and did not result in any statistically significant effect on the overall scores.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

ANIMALS KILLED AFTER 4 WEEKS TREATMENT
Changes related to treatment with the test material were seen in the stomach.
Stomach: Multifocal hyperplasia and hyperkeratosis of the epithelium of the nonglandular region was seen in males and females treated with 300 mg/kg/day with multifocal degeneration/vacuolation of the epithelium of the nonglandular region seen in males treated with 300 mg/kg/day. Ulceration was seen in one male treated with 300 mg/kg/day. No treatment-related findings were apparent at 30 or 100 mg/kg/day.
Incidental Findings: The incidence and distribution of all other findings were considered to be incidental and unrelated to treatment.

ANIMALS KILLED AFTER 2 WEEKS OF RECOVERY AFTER TREATMENT
Treatment Related Findings: There were no changes related to treatment the test material after 2 weeks recovery, in the stomach.
Incidental Findings: All other histological changes were considered to be unrelated to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Details on results:

It is concluded that oral administration of the test material to Han Wistar rats for 4 weeks at doses up to 300 mg/kg/day was well tolerated and did not result in any treatment-related deaths. Macroscopic and microscopic changes in the stomach were identified, indicative of an irritant effect on the stomach and in males given 300 mg/kg/day the findings (ulceration and degeneration) were considered adverse. Other minor changes were seen in bodyweight gain, food consumption and water consumption, but without any clinical signs. Liver weights were slightly increased at all doses in both sexes, but without any histopathological correlate. Some minor changes were seen in a few hematological and urinalysis parameters, predominantly at 300 mg/kg/day, but none were considered adverse in nature. All changes showed recovery.

Key result

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

Key result

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No adverse effects observed.

Key result

Dose descriptor:

NOAEL

Remarks:

systemic toxicity

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed.

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

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

System:

gastrointestinal tract

Organ:

stomach

Conclusions:

Under the conditions of this study the No-Observed-Adverse-Effect-Level (NOAEL) for local effects was considered to be 100 mg/kg/day for males and 300 mg/kg/day for females, due to the findings seen in the stomach of the high dose males. All other changes were considered non-adverse and, therefore, the No-Observed-Adverse-Effect-Level (NOAEL) for systemic toxicity under the conditions of this study was considered to be 300 mg/kg/day for both sexes.

Executive summary:

The sub-acute repeated dose toxicity of the test material was investigated in a study conducted in accordance with the standardised guidelines OECD 407, EU Method B.7 and OPPTS 870.3050, under GLP conditions.

Male and female Han Wistar rats were treated with the test material daily for 4 weeks by oral gavage. Recovery from any effects was evaluated during a 2-week recovery period. Main study animals received the vehicle (corn oil) or the test material by oral gavage for four weeks. Recovery animals were similarly treated for four weeks followed by a two week off dose period.

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption, haematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

The general appearance, sensory activity, grip strength and motor activity of the animals were not affected by treatment and there were no deaths. Transient salivation and chin rubbing were observed after dosing for animals receiving 300 mg/kg/day and salivation for females receiving 100 mg/kg/day, but were considered of no toxicological significance. 

Bodyweight gain during the treatment period was slightly lower than that of the controls for all treated male groups, with recovery being demonstrated after cessation of treatment. Bodyweight gain for females was unaffected by treatment. 

During the treatment period, food consumption for all treated groups was slightly lower than that of the controls and this trend continued during the recovery phase. The differences from controls were, however, minor (<10 %) with no dose-relationship and, therefore, were of uncertain relationship to treatment.

Water consumption was reduced for all treated male groups and a dose response was apparent. This trend of low water consumption continued during the recovery period. Water consumption for females was unaffected. 

Haematology investigations after 4 weeks of treatment revealed slightly low haematocrit, haemoglobin concentration and erythrocyte count for females receiving 300 mg/kg/day and slightly high neutrophil counts for males receiving 300 mg/kg/day. Recovery was evident following two weeks off-dose.

The biochemical examination of the blood performed at the end of the treatment and recovery periods did not reveal any test item-related findings.

Urinalysis investigations after 4 weeks of treatment revealed slightly low urinary pH for males receiving 30 or 300 mg/kg/day (but not 100 mg/kg/day) and slightly high specific gravity for males receiving 30 or 300 mg/kg/day (but not 100 mg/kg/day), correlating with reduced urine volume in these animals. These changes were no longer apparent after 2 weeks of recovery.

Absolute and body weight-adjusted liver weights were slightly high at all doses in both sexes. There was no associated histopathological correlate and the finding showed complete recovery.

Macroscopic examination after 4 weeks of treatment revealed changes in the stomach comprising dark areas and/or depressions in the non-glandular region of the stomach of males treated at 300 mg/kg/day with dark areas in one male treated at 30 mg/kg/day. Thickening of the stomach was seen in animals treated at 300 mg/kg/day and one male treated at 100 mg/kg/day. The macroscopic examination performed after 2 weeks of recovery revealed no test item related lesions.

Histopathological changes related to treatment were seen in the stomach. Hyperplasia and hyperkeratosis of the epithelium of the non-glandular region was seen in males and females treated with 300 mg/kg/day with degeneration/vacuolation of the epithelium of the non-glandular region seen in males treated with 300 mg/kg/day. Ulceration was seen in one male treated with 300 mg/kg/day. There were no changes related to treatment after 2 weeks recovery, indicating full recovery had occurred.

It is concluded that oral administration of the test material to Han Wistar rats for 4 weeks at doses up to 300 mg/kg/day was well tolerated and did not result in any treatment-related deaths. Macroscopic and microscopic changes in the stomach were identified, indicative of an irritant effect on the stomach and in males given 300 mg/kg/day the findings (ulceration and degeneration) were considered adverse.  Other minor changes were seen in bodyweight gain, food consumption and water consumption, but without any clinical signs. Liver weights were slightly increased at all doses in both sexes, but without any histopathological correlate. Some minor changes were seen in a few haematological and urinalysis parameters, predominantly at 300 mg/kg/day, but none were considered adverse in nature. All changes showed recovery.  

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via inhalation in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with Section 8.6.1. of Column 2 of Annex VIII of the REACH Regulation, Short-term repeated dose toxicity study (28 days) should be conducted on one species, male and female, through the most appropriate route of administration, having regard to the likely route of human exposure which in this case is the oral route.

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because exposure of humans via the dermal route in production and/or use is not likely as based on the provided thorough and rigorous exposure assessment

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In accordance with Section 8.6.1. of Column 2 of Annex VIII of the REACH Regulation, Short-term repeated dose toxicity study (28 days) should be conducted on one species, male and female, through the most appropriate route of administration, having regard to the likely route of human exposure which in this case is the oral route.

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: Oral route range finder

The purpose of this study was to assess the systemic toxic potential of the test material by oral gavage administration to Han Wistar rats for 14 days.

The initial doses selected for this study were 100, 300 and 1 000 mg/kg/day. Males and females at 1 000 mg/kg/day were killed for reasons of animal welfare on Day 2 of dosing due to the severity of signs observed. An additional two males and two females were assigned to the study and dosed at 500 mg/kg/day to gain further information on the dose response.

During the study, clinical condition, body weight, food consumption, visual water consumption assessment, organ weight and macropathology investigations were undertaken.

Following the second dose administration at 1 000 mg/kg/day, signs observed included elevated, swaying and/or unsteady gait, decreased activity, irregular and slow breathing, piloerection, partially closed eyes and one female became prostrate. All animals were killed prematurely on Day 2 because of the severity of the signs observed.

Necropsy examination revealed findings in the stomach (distended appearance, abnormal contents and depressions, thickening and irregular surface of the non-glandular mucosa) and intestinal tract (distended ileum, jejunum and/or caecum and abnormal contents in the ileum and jejunum). A dark pancreas was reported for one male and all three females and a dark liver for one female.

Treatment at 500 mg/kg/day was tolerated for the first 4-days of treatment, but animals deteriorated following the fifth dose. Signs comprised piloerection, abnormally cold to the touch, decreased activity and hunched posture. Bodyweight gain and food consumption were low. It was considered that this dose level would not be tolerated for further doses and, therefore, this group was terminated on Day 5 for welfare reasons.

Necropsy examination revealed findings in the stomach (abnormal contents, distended appearance and irregular surface and thickening of the non-glandular mucosa) and intestinal tract (abnormal contents in the duodenum, ileum and jejunum, distended jejunum and abnormal color of the colon, duodenum, ileum, and jejunum). A dark pancreas was recorded in one male and two females.

Treatment of animals with 300 mg/kg/day was well-tolerated and there were no deaths. The only signs comprised transient salivation and chin rubbing. There were no effects on bodyweight gain or food consumption. There was no clear effect of treatment on organ weights. Necropsy examination revealed changes in the stomach comprising thickening and depressions on the non-glandular mucosa for one male and thickening of the non-glandular mucosa for two females.

With the exception of an occasional incidence of transient salvation, the appearance and behavior of the animals were unaffected by treatment dose of 100 mg/kg/day treatment and there were no deaths. There were no effects on bodyweight gain, food consumption or organ weights and no findings at necropsy.

It is concluded that the oral (gavage) administration of the test material to Han Wistar (RccHanTM;WIST) rats was well tolerated at doses up to 300 mg/kg/day.

Treatment at 500 and 1 000 mg/kg/day resulted in a range of signs which necessitated the premature termination of the animals on Days 5 and 2, respectively. The main target organs were the stomach and small intestine.

Whilst changes in the stomach were apparent at 300 mg/kg/day, they were not associated with any adverse clinical signs or any effect on bodyweight gain or food consumption. Based on the findings in this study, 300 mg/kg/day was considered to be a suitable high dose level in the 28-day study.

Repeated dose toxicity: Oral route 28 day study

The subacute repeated dose toxicity of the test material was investigated in a study conducted in accordance with the standardised guidelines OECD 407, EU Method B.7 and OPPTS 870.3050, under GLP conditions. The study was awarded a reliability score of 1 in accordance with the criteria set forth by Klimisch et al. (1997).

Male and female Han Wistar rats were treated with the test material daily for 4 weeks by oral gavage. Recovery from any effects was evaluated during a 2-week recovery period. Main study animals received the vehicle (corn oil) or the test material by oral gavage for four weeks. Recovery animals were similarly treated for four weeks followed by a two week off dose period.

During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, body weight, food consumption, water consumption, haematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

The general appearance, sensory activity, grip strength and motor activity of the animals were not affected by treatment and there were no deaths. Transient salivation and chin rubbing were observed after dosing for animals receiving 300 mg/kg/day and salivation for females receiving 100 mg/kg/day, but were considered of no toxicological significance. 

Bodyweight gain during the treatment period was slightly lower than that of the controls for all treated male groups, with recovery being demonstrated after cessation of treatment. Bodyweight gain for females was unaffected by treatment. 

During the treatment period, food consumption for all treated groups was slightly lower than that of the controls and this trend continued during the recovery phase. The differences from controls were, however, minor (<10 %) with no dose-relationship and, therefore, were of uncertain relationship to treatment.

Water consumption was reduced for all treated male groups and a dose response was apparent. This trend of low water consumption continued during the recovery period. Water consumption for females was unaffected. 

Haematology investigations after 4 weeks of treatment revealed slightly low haematocrit, haemoglobin concentration and erythrocyte count for females receiving 300 mg/kg/day and slightly high neutrophil counts for males receiving 300 mg/kg/day. Recovery was evident following two weeks off-dose.

The biochemical examination of the blood performed at the end of the treatment and recovery periods did not reveal any test item-related findings.

Urinalysis investigations after 4 weeks of treatment revealed slightly low urinary pH for males receiving 30 or 300 mg/kg/day (but not 100 mg/kg/day) and slightly high specific gravity for males receiving 30 or 300 mg/kg/day (but not 100 mg/kg/day), correlating with reduced urine volume in these animals. These changes were no longer apparent after 2 weeks of recovery.

Absolute and body weight-adjusted liver weights were slightly high at all doses in both sexes. There was no associated histopathological correlate and the finding showed complete recovery.

Macroscopic examination after 4 weeks of treatment revealed changes in the stomach comprising dark areas and/or depressions in the non-glandular region of the stomach of males treated at 300 mg/kg/day with dark areas in one male treated at 30 mg/kg/day. Thickening of the stomach was seen in animals treated at 300 mg/kg/day and one male treated at 100 mg/kg/day. The macroscopic examination performed after 2 weeks of recovery revealed no test item related lesions.

Histopathological changes related to treatment were seen in the stomach. Hyperplasia and hyperkeratosis of the epithelium of the non-glandular region was seen in males and females treated with 300 mg/kg/day with degeneration/vacuolation of the epithelium of the non-glandular region seen in males treated with 300 mg/kg/day. Ulceration was seen in one male treated with 300 mg/kg/day. There were no changes related to treatment after 2 weeks recovery, indicating full recovery had occurred.

It is concluded that oral administration of the test material to Han Wistar rats for 4 weeks at doses up to 300 mg/kg/day was well tolerated and did not result in any treatment-related deaths. Macroscopic and microscopic changes in the stomach were identified, indicative of an irritant effect on the stomach and in males given 300 mg/kg/day the findings (ulceration and degeneration) were considered adverse.  Other minor changes were seen in bodyweight gain, food consumption and water consumption, but without any clinical signs. Liver weights were slightly increased at all doses in both sexes, but without any histopathological correlate. Some minor changes were seen in a few haematological and urinalysis parameters, predominantly at 300 mg/kg/day, but none were considered adverse in nature. All changes showed recovery.  

Repeated dose toxicity: Inhalation route

In accordance with Section 8.6.1. of Column 2 of Annex VIII of the REACH Regulation, Short-term repeated dose toxicity study (28 days) should be conducted on one species, male and female, through the most appropriate route of administration, having regard to the likely route of human exposure which in this case is the oral route.

Repeated dose toxicity: Dermal route

In accordance with Section 8.6.1. of Column 2 of Annex VIII of the REACH Regulation, Short-term repeated dose toxicity study (28 days) should be conducted on one species, male and female, through the most appropriate route of administration, having regard to the likely route of human exposure which in this case is the oral route.

Justification for classification or non-classification

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No 1272/2008, the substance does not require classification with respect to repeated dose toxicity via the oral route.