Reaction mass of N1,N1'-(methanediyldibenzene-4,1 -diyl)diaryldiamine and 2-{4-[(4-aminophenyl)amino]benzyl}-N4-(4-{4-[(4-aminophenyl)amino]benzyl}phenyl)aryldiamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 September 2014 to 04 November 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

transient deviations from target values for relative humidity with no impact on results or integrity of the study (see below)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

transient deviations from target values for relative humidity with no impact on results or integrity of the study (see below)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid

Details on test material:

- Appearance/physical state: Black solid
- Storage conditions: Room temperature, protected from light under nitrogen

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Wistar Han:RccHan:WIST

Details on species / strain selection:

- The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

ANIMALS AND ANIMAL HUSBANDRY
- A sufficient number of male and female Wistar Han:RccHan:WIST strain rats were obtained from Harlan Laboratories U.K. Ltd., Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatised for seven days during which time their health status was assessed.
- A total of sixty animals (thirty males and thirty females) were accepted into the study.
- At the start of treatment the males weighed 211 to 241 g, the females weighed 151 to 187g, and were approximately six to eight weeks old.
- The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
- Animals were allowed free access to food and water. A pelleted diet (Rodent 2014C Teklad Global Certified Diet, Harlan Laboratories U.K. Ltd., Oxon, UK.) was used.
- Mains drinking water was supplied from polycarbonate bottles attached to the cage.
- Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK).
- The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
- The animals were housed in a single air-conditioned room within the Harlan Laboratories Ltd., Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerised system, and print-outs of hourly temperatures and humidities were included in the study records.
- The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20 % respectively. There were no deviations from these ranges for temperature and two transient deviations for relative humidity during the acclimatisation period were considered not to have affected the purpose or integrity of the study.
- Animals were randomly allocated to treatment groups using a stratified body weight randomisation procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomised. The animals were uniquely identified within the study by an ear punching system routinely used in the laboratories.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

arachis oil

Details on oral exposure:

PROCEDURE
- Animals were allocated to treatment groups as shown in the study group assignment table below.
- Initial dose levels were chosen, in collaboration with the sponsor, based on available toxicity data including a rat seven day range finder toxicity study In this preliminary study, a dosage of 250 mg/kg bw/day was shown to be too high for further long term investigation of toxicity and a dosage of 125 mg/kg bw/day was associated with effects on bodyweight and food consumption for both sexes. It was concluded that the high dosage for further investigation should lie slightly below 125 mg/kg bw/day and dosages of 0 (control), 10, 30 and 100 mg/kg bw/day were therefore selected for use on this twenty eight day toxicity study. Subsequently dose levels for high dosage animals were based on the findings apparent as the study was proceeding.
- As arachis oil had been successfully employed during the preliminary study, this vehicle was also used for this main study, however as stomach findings had been apparent for rats at 250 mg/kg bw/day in the preliminary study, the dose volume was increased to 5 ml/kg for this main study (thereby reducing the required concentrations of the dosing formulations).
- The test item was administered daily by gavage using a stainless steel cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 mL/kg of Arachis oil BP. Control, low (10 mg/kg bw/day) and intermediate (30 mg/kg bw/day) group animals were dosed for twenty-eight consecutive days. High dosage group animals were dosed initially at 100 mg/kg bw/day, treatment was reduced to 60 mg/kg bw/day on Day 9 of the study with dosing suspended for males and females on Day 12 and Day 11 respectively. Dosing recommenced for both sexes on Day 13 at 50 mg/kg bw/day and continued to Day 28. Recovery group animals were maintained for a further fourteen days treatment-free period following termination of treatment.
- The volume of test and control item administered to each animal was based on the most recent scheduled body weight and was adjusted at weekly intervals.

Analytical verification of doses or concentrations:

yes

Remarks:

see Appendix 19 (attached)

Details on analytical verification of doses or concentrations:

TEST ITEM PREPARATION
- The test item was ground and prepared at the appropriate concentrations as a suspension in Arachis oil BP.
- Stability and homogeneity of the test item formulations were determined by Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. Results showed that formulations were stable for at least thirteen days when stored refrigerated (approximately 4 °C in the dark). Formulations were initially prepared daily and then in batches covering up to nine consecutive days of dosing. Daily formulation preparation was resumed for the low dosages from Day 14 of the study.
- Samples of each test item formulations were taken and analysed for concentration of test item at Harlan Laboratories Ltd., Shardlow, UK, Analytical Services. The method used for analysis of formulations and the results obtained are given in Appendix 19 (attached). The results indicate that the prepared formulations for the intermediate and high dosages groups were within 98-109 % of the nominal concentration. For the low dosage group, results were more variable (88-118 % of the nominal concentration); this was considered to reflect the low dose concentration being used and the nature of the test item. Overall the indicated accuracy of the formulation procedure was considered to be acceptable for the purpose of this study.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

Remarks:

reduced to 60 mg/kg bw/day on Day 9 and suspended on Day 11 (females) or Day 12 (males).

Doses / concentrations 5

Dose / conc.:

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

Remarks:

administered to males and females in the high dose group on Days 13 to 28

No. of animals per sex per dose:

Five males and five females

Control animals:

yes, concurrent vehicle

Positive control:

Not applicable

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS
- All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing, up to thirty minutes post dosing and one hour after dosing during the working week, weekends and public holidays.
- In addition, as far as possible, individual clinical observations were also performed approximately four hours after dosing during the normal working day (i.e. excluding weekends).
- During the treatment-free period, animals were observed daily.
- All observations were recorded.

FUNCTIONAL OBSERVATIONS
- Prior to the start of treatment and on Days 7, 14, 21 and 26, all animals were observed for signs of functional/behavioural toxicity.
- Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.
- Observations were carried out from approximately two hours after dosing on each occasion.

BEHAVIOURAL ASSESSMENT
- Detailed individual clinical observations were performed for each non-recovery animal using a purpose built arena.
- The parameters listed in the table below were observed.
- The test was developed from the methods used by Irwin (1968) and Moser et al (1988).
- The scoring system used is outlined in the key to scoring system and explanation for behavioural assessments and sensory reactivity tests (attached).

FUNCTIONAL PERFORMANCE TESTS
- Motor Activity: Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Non-recovery animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).
- Forelimb/Hindlimb Grip Strength: An automated grip strength meter was used. Each non-recovery animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

SENSORY REACTIVITY
- Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli.
- The parameters listed in the table below were observed.
- This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988).
- The scoring system used is outlined in the key to scoring system and explanation for behavioural assessments and sensory reactivity tests (attached)

BODY WEIGHT
- Individual body weights were recorded prior to dosing on Day 1 and at weekly intervals thereafter.
- Body weights were also performed prior to terminal kill and, in the case of recovery group animals, on Days 36 and 43 prior to terminal kill.

FOOD CONSUMPTION
- Food consumption was recorded for each cage group at weekly intervals throughout the study.
- Food conversion efficiency was calculated retrospectively.

WATER CONSUMPTION
- Water intake was measured and recorded daily for each cage group.

LABORATORY INVESTIGATIONS
- Hematological and blood chemical investigations were performed on all non-recovery animals from each test and control group at the end of the treatment period (Day 28) and on all recovery group animals at the end of the treatment-free period (Day 42).
- Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Days 29 and 43. Animals were not fasted prior to sampling.
- Urinalytical investigations were performed on all non-recovery test and control group animals during Week 12 and on all recovery group animals during Week 6. Urine samples were collected overnight by housing the rats in metabolism cages. Animals were maintained under conditions of normal hydration during collection but without access to food.

HEMATOLOGY
- Parameters assessed are listed in the table below.
- Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

BLOOD CHEMISTRY
- Parameters listed in the table below were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant.

URINALYSIS
- Parameters listed in the table below were measured on collected urine.

Sacrifice and pathology:

NECROPSY
- On completion of the dosing period or in the case of recovery group animals, at the end of the treatment-free period all animals were killed by intravenous overdose of a suitable barbiturate followed by exsanguination.
- All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.
- At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored frozen at approximately -20 °C for thyroid hormone assessment. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.

Other examinations:

ORGAN WEIGHTS
- Organs listed in the table below were removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation.

HISTOPATHOLOGY
- Samples of the tissues listed in the table below were removed from all animals and preserved in buffered 10% formalin, except where stated.
- All tissues were dispatched to the histology processing Test Site (Propath UK Ltd, Hereford, UK) for processing (Principal Investigator: N Fower).
- Any macroscopically observed lesions were also processed. In addition, sections of testes from all Control and 100/60/50 mg/kg bw/day males were stained with Periodic Acid-Schiff (PAS) stain and examined.
- Following the results of the initial histopathological examinations of control and high dosage non-recovery animals, histopathology examinations were extended to cover the eyes, liver, spleen and mesenteric lymph nodes for both sexes and the seminal vesicles and prostate for males of the low and intermediate dosage groups and also of the control and high dosage recovery groups.
- Microscopic examination was conducted by the Study Pathologist, Wendy Henderson. A peer review of the findings observed was conducted by Peter Millar (Peter Millar Associates Ltd. Edinburgh) at the histopathology peer review test site. A complete histopathology phase report was provided and represents the consensus view of both pathologists.

Statistics:

- Refer to description of statistical methods below.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- A summary incidence of daily clinical observations is given in Table 1 (attached).
- Initial treatment at 100 mg/kg bw/day was not associated with any adverse clinical signs even though body weight losses observed on Day 8 necessitated the lowering of this dosage to 60 mg/kg bw/day on Day 9. On Day 11, seven of the ten males and all females showed hunched posture, four females also showed piloerection. Dosing was suspended for females on Day 11 and for males on Day 12, with dosing re-commencing for both sexes on Day 13 at 50 mg/kg bw/day.
- No adverse clinical signs were apparent for males from Day 13; however hunched posture was apparent for one female on Day 13 and another female on Days 13 and 17.
- Two females at 100/60/50 mg/kg bw/day showed generalised fur loss from Day 13 and Day 15 respectively; for both females this fur loss persisted throughout the remaining treatment period and most or all of the treatment free recovery period. Such fur loss is not unremarkable but could indicate that the animals were possibly under a degree of physiological stress (see Justification of No Observed Adverse Effect Level).
- At 100/60/50 mg/kg bw/day all animals showed increased post-dosing salivation, this clinical sign first being observed from Day 6 for both sexes. One control male showed similar increased salivation on Day 26 and one female receiving 30 mg/kg bw/day also showed increased postdosing salivation on Day 18 (see Justification of No Observed Adverse Effect Level).
- There were no other clinical signs apparent during the study.

Mortality:

no mortality observed

Description (incidence):

- There were no unscheduled animal deaths during the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- Group mean weekly body weights and standard deviations are given in Table 6 (attached) and statistically
significant differences are indicated.
- Group means are presented graphically in Figure 1 and Figure 2 (attached).
- Group mean weekly body weight gains and standard deviations are given in Table 7 (attached) and statistically significant differences are indicated.
- Treatment at 100 mg/kg bw/day was associated with body weight loss for both sexes during the first week of treatment. Following the afore mentioned lowering of dosages and cessation of treatment during Week 2, body weight gain of females was similar to control but gain for males was lower than control, with differences attaining statistical significance. Body weight gain for both sexes during Weeks 3 and 4 and for selected animals during the two week recovery period was similar or slightly superior to control.
- For females at 30 mg/kg bw/day, body weight gain was lower than control during the first week of treatment with differences from control attaining statistical significance. Subsequent body weight gain during the remainder of the study was similar to control and there was no statistically significant difference in overall body weight gain at the end of the treatment period compared to non-recovery control.
- For both sexes at 10 mg/kg bw/day and males at 30 mg/kg bw/day, body weight gains were similar to control and differences from control did not attain any statistical significance.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Group mean weekly food consumptions are given in Table 8 (attached) and are presented graphically in Figure 3 and Figure 4 (attached).
- Weekly food efficiencies are given in Table 9 (attached).
- Treatment at 100 mg/kg bw/day was associated with lower food consumption compared to control for both sexes during the first week of treatment. Following the lowering of dosages and cessation of treatment during Week 2, food intake of both sexes was still lower than control but to a lesser magnitude to that observed previously. Food consumption for both sexes during Weeks 3 and 4 and for selected animals during the two week recovery period was similar to control.
- For females at 30 mg/kg bw/day, food consumption was lower than control during the first week of treatment but subsequent food intake was similar to control throughout the remainder of the study.
- For both sexes at 10 mg/kg bw/day and males at 30 mg/kg bw/day, food consumption was similar to control.

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

- The value of evaluating food conversion efficiency where a mean body weight loss has been observed is equivocal.
- Treatment at 100 mg/kg bw/day was associated with inferior food conversion efficiency compared to control for both sexes during the first week of treatment; a period when notable body weight loss was apparent at this dosage.
- Following the lowering of dosages and cessation of treatment during Week 2, food conversion efficiency for both sexes was not adversely affected by treatment for the remainder of the study (see Justification of No Observed Adverse effect Level).
- At 10 and 30 mg/kg bw/day, food conversion efficiency for both sexes was generally similar to control and did not indicate any effect of treatment.

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

effects observed, treatment-related

Description (incidence and severity):

- Group mean daily water consumptions are given in Table 10 (attached).
- Treatment of males at 100 mg/kg bw/day was associated with an increase in water consumption compared to control from the start of treatment. Water intake generally remained higher than control despite the lowering of the dosage to 60 mg/kg bw/day on Day 9 and the temporary cessation of dosing on Day 12. Following the re-commencement of dosing at a dosage of 50 mg/kg bw/day on Day 13, higher water consumption persisted until Days 15-16; thereafter water consumption during the treatment period was essentially similar to control.
- Following the cessation of treatment, water consumption during the recovery period was generally lower than control but was considered unlikely to be related to treatment (see Justification of No Observed Adverse Effect Level).
- Treatment of females at 100 mg/kg bw/day was generally associated with an increase in water consumption compared to control from the start of treatment. Following the lowering of the dosage to 60 mg/kg bw/day (Day 9), water consumption was lower than control on Days 9-10 but was essentially similar to control on Days 10-11. On Days 11-12 and 12-13 when dosing was suspended, water intake was notably higher than control. Following the re-commencement of dosing at a dosage of 50 mg/kg bw/day on Day 13, higher intake generally remained for the
remaining treatment period and the treatment-free recovery period.
- For females at 30 mg/kg bw/day, water consumption tended to be higher than control during the first three days of treatment; thereafter there was no obvious effect of treatment on water consumption throughout the treatment period (see Justification of No Observed Adverse Effect Level).
- Water consumption for both sexes at 10 mg/kg bw/day and males at 30 mg/kg bw/day appeared to be unaffected by treatment (see Justification of No Observed Adverse Effect Level).

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

- Group mean values and standard deviations for test and control group animals are given in Table 11 (attached) and statistically significant differences are indicated.
MALES
- At 100/60/50 mg/kg bw/day, lower erythrocyte count, hemoglobin and hematocrit at the end of the treatment period attained statistical significance when compared with control; all individual values for these treated animals were outside the historical control range. Additionally, lower mean cell hemoglobin, mean cell volume and mean cell hemoglobin concentration also attained statistical significance when compared with control, but all individual values for these treated animals were within the historical control range. Additionally, at 100/60/50 mg/kg bw/day, higher numbers of neutophils and total leukocytes attained statistical significance compared with control although the majority of individual values were within the historical control range.
- At 30 mg/kg bw/day, lower mean cell hemoglobin, mean cell volume and mean cell haemoglobin concentration at the end of the treatment period attained statistical significance when compared with control, but with the exception of one individual value for mean cell volume, all individual values for these treated animals were within the historical control range.
- At 100/60/50 mg/kg bw/day, lower erythrocyte count, hemoglobin and hematocrit at the end of the treatment period attained statistical significance when compared with control; all individual values for these treated animals were outside the historical control range. Additionally, lower mean cell hemoglobin, mean cell volume and mean cell hemoglobin concentration also attained statistical significance when compared with control, but all individual values for these treated animals were within the historical control range. Additionally, at 100/60/50 mg/kg bw/day, higher numbers of neutophils and total leukocytes attained statistical significance compared with control although the majority of individual values were within the historical control range.
- At 30 mg/kg bw/day, lower mean cell hemoglobin, mean cell volume and mean cell haemoglobin concentration at the end of the treatment period attained statistical significance when compared with control, but with the exception of one individual value for mean cell volume, all individual values for these treated animals were within the historical control range.
FEMALES
- At 100/60/50 mg/kg bw/day, lower erythrocyte count and hemoglobin at the end of the treatment period attained statistical significance when compared with control; all or the majority of individual values for these treated animals were outside the historical control range. Lower mean cell hemoglobin and mean cell hemoglobin concentration also attained statistical significance when compared with control, but all individual values for these treated animals were within the historical control range.
- At 30 mg/kg bw/day, lower mean hemoglobin at the end of the treatment period attained statistical significance when compared with control; all individual values for these treated animals were outside the historical control range. Lower mean cell hemoglobin and mean cell hemoglobin concentration also attained statistical significance when compared with control; the majority of individual values for mean cell hemoglobin were outside the historical control range although all individual values for mean cell hemoglobin concentration were within this historical range.
- At 10 mg/kg bw/day there were no statistically significant differences from control observed for hematology parameters at the end of the treatment period.
- At 100/60/50 mg/kg bw/day, at the end of the two week treatment free recovery period, lower values for mean cell hemoglobin concentration attained statistical significance compared to control but all individual values for these treated animals were within the historical control range.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Group mean values and standard deviations for test and control group animals are given in Table 12 (attached) and statistically significant differences are indicated).
MALES
- At 100/60/50 mg/kg bw/day, higher levels of alanine aminotransferase at the end of the treatment period attained statistical significance when compared with control; the majority of individual values exceeding the historical control range. Higher levels of total billirubin and bile acids also attained statistical significance when compared with control with all or the majority of individual values exceeding the historical control range. Additionally, higher total cholesterol and lower triglyceride levels attained statistical significance when compared with control but all values were within the historical control range.
- At 30 mg/kg bw/day, higher levels of total cholesterol and total billirubin at the end of the treatment period attained statistical significance when compared with control but all individual values were within the historical control range.
- At 10 mg/kg bw/day, higher levels of total cholesterol and total billirubin at the end of the treatment period attained statistical significance when compared with control but all individual values were within the historical control range (see Justification of No Observed Adverse Effect Level).
- At 100/60/50 mg/kg bw/day, higher levels of billirubin and bile acids at the end of the treatment free recovery period attained statistical significance when compared with control; however the majority or all of the individual values for these parameters were within the historical control range.
- At 100/60/50 mg/kg bw/day, higher levels of sodium, potassium and chloride and lower levels of total protein at the end of the treatment free recovery period attained statistical significance when compared to control; however the majority or all of the individual values for these parameters were within the historical control range (see Justification of No Observed Adverse Effect Level).
- Other statistically significant differences from control observed for blood chemistry parameters at the end of the treatment period were considered to be incidental and of no toxicological significance (see Justification of No Observed Adverse Effect Level).
FEMALES
- At 100/60/50 mg/kg bw/day, higher levels of total cholesterol and total billirubin at the end of the treatment period attained statistical significance when compared with control; the majority or all of the individual values exceeded the historical control range. Higher levels of bile acids also attained statistical significance when compared with control but only two individual values exceeded the historical control range. Total protein, albumin and albumin/globulin ratio were statistically significantly lower than control; all individual values for albumin fell below the historical control range but individual values for the other parameters were within their historical
range. Additionally higher levels of alanine aminotransferase and lower triglyceride levels also attained statistical significance when compared with control but all individual values were within the historical control range.
- At 30 mg/kg bw/day, total protein, albumin and albumin/globulin ratio at the end of the treatment period were lower than control with differences attaining statistical significance; all individual values for albumin were below the historical control range but individual values for the other parameters were within this historical range. Higher bile acids levels also attained statistical significance when compared with control with two individual values exceeding the historical control range.
- At 10 mg/kg bw/day, lower albumin and albumin/globulin ratio at the end of the treatment period attained statistical significance when compared with control. There was no accompanying statistically significant difference from control observed for total protein and all individual values for these parameters were within the historical control range (see Justification of No Observed Adverse Effect Level).
- At 100/60/50 mg/kg bw/day, higher levels of alanine aminotransferase and lower albumin/globulin ratio and triglyceride levels at the end of the treatment free recovery period attained statistical significance when compared with control; however all individual values for these parameters were within the historical control range.
- At 100/60/50 mg/kg bw/day, higher levels of alkaline phosphatase and aspartate aminotransferase levels at the end of the treatment free period attained statistical significance but all individual values for these parameters were within the historical control range (see Justification of No Observed Adverse Effect Level).

Urinalysis findings:

no effects observed

Description (incidence and severity):

URINALYSIS
- A summary of urinalytical findings is given in Table 13 (attached).
- Urinalysis assessments did not indicate any obvious effect of treatment for either sex at 10, 30 or 100/60/50 mg/kg bw/day (see Justification of No Observed Adverse Effect Level).

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

- A summary incidence of behavioural assessment observations is given in Table 2 (attached) and group mean behavioural assessment scores are given in Table 3 (attached).
- Group mean functional performance test values and standard deviations are given in Table 4 (attached) and statistically significant differences are indicated.
- Group mean sensory reactivity assessments are given in Table 5 (attached).
- Assessment of the animals in a standard arena on Day 7 did not reveal any obvious adverse effects of treatment at dosages of 10, 30 and 100 mg/kg bw/day.
- Subsequent assessments on Days 14, 21 and 26 also did not reveal any obvious adverse effects of treatment at dosages of 10, 30 and 100/60/50 mg/kg bw/day (see Justification of No Observed Adverse Effect Level).
- Assessment of functional performance using grip strength and motor activity did not indicate any obvious effects of treatment for either sex at dosages of 10, 30 and 100/60/50 mg/kg bw/day (see Justification of No Observed Adverse Effect Level).
- Sensory reactivity scores were the same in the control and all treatment groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Group mean absolute and relative organ weights and standard deviations for test and control group animals are presented in Table 15 (attached) and statistically significant differences are indicated.
- At 100/60/50 mg/kg bw/day, mean absolute and body weight relative kidney weights for both sexes at the end of treatment were higher than control, with differences attaining statistical significance. For this organ, body weight relative values are probably the better indicator of toxicological effect and the majority of individual body weight relative values exceeded the historical control range. For males at this dosage following the treatment free recovery period, absolute kidney weights were lower than control although body weight relative values remained
higher than control; differences from control continued to attain statistical significance but the majority of individual values for these treated animals were within the historical control range. No statistically significant differences in kidney weights were apparent for females at the end of the recovery period.
- At 100/60/50 mg/kg bw/day, mean absolute and body weight relative liver weight for females at the end of treatment was higher than control with differences attaining statistical significance. For males at 100/60/50 mg/kg bw/day, mean absolute liver weight was statistically significantly lower than control, but body weight relative liver weight, considered to be a better indicator of toxicological effect for this organ, was higher than control with differences attaining statistical significance. For both sexes, the majority of individual liver values were within the historical control range. For males at 100/60/50 mg/kg bw/day following the treatment free recovery period, absolute and body weight relative liver weights were statistically significantly lower than control; the majority of individual absolute liver values were outside the historical control range but most body weight-relative values were within this historical range. No statistically significant differences in liver weights were apparent for females at the end of the recovery period.
- At 100/60/50 mg/kg bw/day, mean absolute and body weight relative spleen weights for both sexes at the end of treatment were higher than control; differences attained statistical significance and the majority of individual values exceeded the historical control range. Absolute and body weight relative spleen weights were also statistically significantly higher than control for males at 30 mg/kg bw/day although the majority of individual values were within the historical control range. No statistically significant differences in spleen weights were apparent for either sex at 100/60/50 mg/kg bw/day at the end of the recovery period.
- At 100/60/50 mg/kg bw/day, lower absolute and body weight relative prostate and seminal vesicles weights for males at the end of treatment attained statistical significance when compared with control but, the majority of individual values were within the historical control range. Lower absolute and body weight relative prostate and seminal vesicles weights at the end of the recovery period also attained statistical significance when compared with control; all individual values were within the historical control range.
- No statistically significant differences from control were apparent for organ weights from animals of either sex at 10 mg/kg bw/day.
- Other statistically significant differences from control observed for organ weights were considered to be incidental and of no toxicological significance (see Justification of No Observed Adverse Effect Level).

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

- A summary incidence of necropsy findings is given in Table 14 (attached).
- At 100/60/50 mg/kg bw/day, two males showed small prostate and seminal vesicles at the end of the treatment period; no similar findings were apparent for these organs at the end of the two week recovery period.
- Neither the type, incidence or distribution of other macroscopic findings apparent during necropsy at the end of the treatment period or at the end of the treatment-free recovery period indicated any obvious effect of treatment (see Justification of No Observed Adverse Effect Level).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Retinal detachment was present in the eyes of all non-recovery and recovery animals (both sexes) receiving 100/60/50 mg/kg bw/day; this finding was observed at a marked level indicating that the whole retina was affected. At 30 mg/kg bw/day, all females were similarly affected at a moderate or mild level and 4/5 males had minimal retinal degeneration. This finding was not present in animals receiving 10 mg/kg bw/day.
- Centrilobular hypertrophy of the liver was present in 4/5 males and all females receiving 100/60/50 mg/kg bw/day at a minimal or mild level. At 30 mg/kg bw/day, 3/5 males and 3/5 females also showed centrilobular hypertrophy at a minimal level. In the periportal area, minimal or mild increase in cytoplasmic rarefaction, periportal cellular change (minimal appearing as cellular shrinkage, basophilia with occasional lymphocytic infiltration) and minimal periportal dilation (bile duct and vascular) was observed with 4/5 non-recovery male and 4/5 non-recovery females at 100/60/50 mg/kg bw/day having one or more these findings. At 30 mg/kg bw/day, one or more of these findings were present in 2/5 males and 4/5 females. Increased hematopoiesis was present in 3/5 males and 3/5 females receiving 100/60/50 mg/kg bw/day and one male receiving 30 mg/kg bw/day.
- The various liver changes were still present for both sexes at 100/60/50 mg/kg bw/day after the recovery period with 4/5 males and all females being affected to some extent and with 3/5 males and 4/5 females showing one or more of the changes noted for the periportal area. There was also an increase in the number of animals with brown pigment in the periportal area with 3/5 males and 4/5 females being affected. Overall the incidence and severity of the findings indicated some reversibility but not complete recovery in the liver.
- Erythrocytosis, at a minimal or mild level, was present in the mesenteric lymph nodes of all males at end of treatment at 100/60/50 mg/kg bw/day.
- Erythrocytosis, at a minimal or mild level, was also present in the mesenteric lymph nodes of 2/5 females at 100/60/50 mg/kg bw/day and 2/5 males and 1/5 females at 30 mg/kg bw/day at the end of treatment. Additionally this sign was also present at a minimal level in 2/5 males and 1/5 females at the end of the recovery period at 100/60/50 mg/kg bw/day, indicating recovery (see Justification of No Observed Adverse Effect Level).
- Increased amounts of haematopoiesis was present in the spleen of 4/5 males and 4/5 females at 100/60/50 mg/kg bw/day at the end of the treatment period compared with control animals. Haematopoiesis was also observed for 1/5 males and 2/5 females at 30 mg/kg bw/day at the end of treatment and 1/5 males and 1/5 females at 100/60/50 mg/kg bw/day at the end of the recovery period (see Justification of No Observed Adverse Effect Level).
- Reduced secretion in the prostate and seminal vesicles was observed for 3/5 non-recovery males and 2/5 recovery males at 100/60/50 mg/kg bw/day.
- For remaining organs, normal variations in a number of background findings were noted but no consistent variations were apparent that could be attributed to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

1

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

2

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

eye

Organ:

retina

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

JUSTIFICATION OF NO OBSERVED ADVERSE EFFECT LEVEL

- The following differences between treated and control animals were considered not to be indicative of test item toxicity.

CLINICAL OBSERVATIONS

- Two females at 100/60/50 mg/kg bw/day showed generalised fur loss from Day 13 and Day 15 respectively; this fur loss persisted throughout the remaining treatment period and most or all of the treatment free recovery period. While this finding may indicate that the animals were possibly under a degree of physiological stress, such fur loss is not unremarkable and at the level observed in this study was considered not to represent an adverse effect of treatment.

- At 100/60/50 mg/kg bw/day all animals showed increased post-dosing salivation, this clinical sign first being observed from Day 6 for both sexes. One control male showed similar increased salivation on Day 26 and one female receiving 30 mg/kg bw/day also showed increased post-dosing salivation on Day 18. Increased post-dosing salivation is frequently observed when animals are dosed via the oral gavage route and is generally considered to reflect distaste or slight irritancy of the dosing formulations rather than any systemic effect of treatment.

BEHAVIOURAL ASSESSMENTS

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

FUNCTIONAL PERFORMANCE TESTS

- For females at 100/60/50 mg/kg bw/day, higher grip strength measurement on the first of three trials for the hind limb attained statistical significance when compared with control. In the absence of any statistically significant differences from control for the remaining trial for the hind limb or for trials assessing forelimb grip strength this finding was considered to be incidental and unrelated to treatment.

- For females at 100/60/50 mg/kg bw/day, overall mobile activity was lower than control with differences attaining statistical significance when compared with control. In the absence of any supporting findings observed during other assessments of behaviour, this isolated finding was considered to be incidental and of no toxicological significance.

BODY WEIGHT GAIN

- At 100/60/50 mg/kg bw/day, body weight gains of females during Week 6 were statistically significantly higher than control. Such an increase in body weight in the recovery period is considered unlikely to indicate an adverse effect of treatment and therefore was considered to be of no toxicological significance.

FOOD CONVERSION EFFICIENCY

- For females at 100/60/50 mg/kg bw/day, food conversion efficiency was superior to control during the second and third week of treatment but this was considered to reflect the recovery of body weight gain following initial body weight losses during Week 1 and therefore did not represent an adverse effect of treatment. Superior food conversion efficiency was also apparent during the first week of the recovery period, but such a finding is unlikely to represent an adverse effect of treatment and therefore was considered to be of no toxicological significance.

WATER CONSUMPTION

- At 100/60/50 mg/kg bw/day, water consumption for males during the recovery period was generally lower than control but values were consistent with that observed during the preceding treatment period. The differences were considered to reflect the lower body weight of these animals (compared with control) and relatively high water intake for the recovery control animals rather than any latent treatment related effect on water intake.

- Notable differences in water consumption, compared to control, were occasionally observed for both sexes at 10 mg/kg bw/day and males at 30 mg/kg bw/day throughout the treatment period. Similar differences in water consumption were also occasionally observed for females at 30 mg/kg bw/day from the fourth day of dosing throughout the treatment period. In the absence of any consistent pattern to these differences in water intake, they were considered to be incidental and reflect normal biological variation.

HEMATOLOGY

- For males at 100/60/50 mg/kg bw/day, at the end of the two week treatment free recovery period, lower numbers of eosinophils and higher numbers of reticulocytes attained statistical significance compared to control but all individual values were within the historical control range. In the absence of any similar effects for males at the end of the treatment period, these findings were considered to be either incidental or to reflect ongoing

recovery from previous insult during the treatment period and were considered not to represent an adverse effect of treatment.

- At 10 mg/kg bw/day, lower mean cell hemoglobin for males at the end of the treatment period attained statistical significance when compared with control but all individual values for these treated animals were within the historical control range. As lower levels were also observed at higher dosages within this study, an association with treatment cannot be discounted, however, in isolation and in the absence of any supporting histopathological changes this finding was considered not to represent an adverse effect of treatment.

BLOOD CHEMISTRY

- For males at 10 mg/kg bw/day, higher levels of total cholesterol and total billirubin at the end of the treatment period attained statistical significance when compared with control but all individual values were within the historical control range. As higher levels for these parameters were also observed at higher dosages within this study, an association with treatment cannot be discounted, however, in isolation and in the absence of any

supporting histopathological changes these findings were considered not to represent an adverse effect of treatment.

- For males receiving 10 mg/kg bw/day, lower albumin/globulin ratio compared to control was observed at the end of the treatment period; all individual values were within the historical control range. There was no accompanying statistically significant differences apparent for total protein or albumin and, in the absence of any similar effects at higher dosage, this finding was considered to be incidental and unrelated to treatment.

- For females at 10 mg/kg bw/day, lower albumin and albumin/globulin ratio at the end of the treatment period attained statistical significance when compared with control. There was no accompanying statistically significant difference from control observed for total protein and all individual values for these parameters were within the historical control range. As lower levels for these parameters were also observed at higher dosages within this study, an association with treatment cannot be discounted, however, in isolation and in the absence of any supporting histopathological changes these findings were considered not to represent an adverse effect of treatment.

- For males at 100/60/50 mg/kg bw/day, higher levels of sodium, potassium and chloride and lower levels of total protein at the end of the treatment free recovery period attained statistical significance when compared with control; however the majority or all of the individual values for these parameters were within the historical control range. There were no statistically significant differences from control for these parameters for males at

the end of the treatment period and, while that these parameters may have been influenced by on-going recovery from previous insult during the treatment period, they were considered not to represent an adverse effect of treatment.

- For females at 100/60/50 mg/kg bw/day, levels of alkaline phosphatase and aspartate aminotransferase at the end of the treatment free recovery period were statistically significantly higher than control but all individual values were within the historical control range. While that these parameters may have been influenced by on-going recovery from previous insult during the treatment period, they were considered not to represent an adverse effect of treatment.

URINALYSIS

- One recovery female at 100/60/50 mg/kg bw/day showed protein in the urine at the end of the treatment-free recovery period. One control recovery male showed hemoglobin in the urine at the end of the recovery period. These isolated findings were considered to be of no toxicological significance and not to indicate an adverse effect of treatment.

NECROPSY

- At 100/60/50 mg/kg bw/day, one female showed an enlarged and fluid fill right uterine horn; no similar findings were apparent for the uterus at the end of the two week recovery period. There was no statistically significant effect on uterus weights at the end of the treatment period and, in the absence of any evidence of histopathological change for this organ at the end of treatment, this finding was considered to be of no toxicological significance.

- One male at 10 mg/kg bw/day showed small epididymides and testes at the end of the treatment period. There was no statistically significant effect on testes weights during the study and no similar findings were apparent for the testes at higher dosages at the end of treatment. In the absence of any evidence of histopathological change for this organ at the highest dosage level at the end of treatment, this finding was considered to be incidental and unrelated to treatment.

- One male at 100/60/50 mg/kg bw/day showed small testes at the end of the two week recovery period. There was no statistically significant effect on testes weights during the study and, in the absence of any evidence of histopathological change for this organ at the end of the treatment period, this finding was considered to be incidental and unrelated to treatment.

ORGAN WEIGHTS

- For females at 30 and 100/60/50 mg/kg bw/day, absolute and body weight relative thyroid weights at the end of the treatment period were lower than control, with differences attaining statistical significance. All individual values for these treated animals were within the historical control range while values for 2/5 control females exceeded this historical range. In the absence of any evidence of histopathological change for this organ, these observed differences were considered to reflect high control values rather than any adverse effect of treatment.

- For females at 100/60/50 mg/kg bw/day, absolute and body weight relative weights for the uterus were lower than control at the end of the recovery period, with differences attaining statistical significance. All individual values for these treated animals were within the historical control range and were no significant differences from control apparent at the end of the treatment period. In the absence of any evidence of histopathological change for this organ, these observed differences were considered to be incidental and unrelated to treatment.

- At 100/60/50 mg/kg bw/day mean absolute and body weight relative heart weights for males at the end of treatment were statistically significantly higher than control. Although all body weight relative values exceeded the historical control range, only 2/5 absolute values exceeded this historical range. In the absence of any histopathological change these differences were considered to be a consequence of the marked effect on body weight at this dosage rather than a toxicologically significant effect on the heart and of no toxicological significance. Recovery females also showed statistically significantly higher heart weight but, in the absence of any similar increase in heart weights or evidence of histopathological change at the end of treatment, this finding was considered to be incidental and not to represent an adverse effect of treatment.

HISTOPATHOLOGY

- Erythocytosis was present at a minimal level in the mesenteric lymph nodes of 2/5 males and 1/5 females at 30 mg/kg bw/day and 2/5 females at 100/60/50 mg/kg bw/day at the end of the treatment period and 2/5 males and 1/5 females at 100/60/50 mg/kg bw/day at the end of recovery period. The incidence and severity of this finding for these animals were considered to be within control limits therefore this finding for these animals was considered to be of no toxicological significance.

- Hematopoiesis in the spleen was also observed for 2/5 males and 1/5 females at 30 mg/kg bw/day at the end of treatment and 1/5 males and 1/5 females at 100/60/50 mg/kg bw/day at the end of the recovery period, but was considered to be within normal control limits and therefore was of no toxicological significance.

- For remaining organs, normal variations in a number of background findings were noted but no consistent variations were apparent that could be attributed to treatment.

Applicant's summary and conclusion

Conclusions:

Based on the results of this study the No Observed Adverse Effect Level (NOAEL) of the test item when administered to the rat over twenty-eight consecutive days was considered to be 10 mg/kg bw/day.

Executive summary:

GUIDELINE

The study was designed to investigate the systemic toxicity of the test item and was compatible with the OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008) and Commission Directive 96/54/EC (Method B7). The study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

 

METHODS…….

The test item was administered by gavage to three groups, each of five male and five female Wistar Han:RccHan:WIST strain rats, for an intended period of twenty-eight consecutive days, at dose levels of 10, 30 and 100 mg/kg bw/day. Animals at 10 and 30 mg/kg bw/day successfully completed this treatment period. For animals initially at 100 mg/kg bw/day, treatment was reduced to 60 mg/kg bw/day on Day 9 of the study with dosing suspended for males and females on Day 12 and Day 11 respectively. Dosing recommenced for both sexes on Day 13 at 50 mg/kg bw/day and continued to Day 28. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP) over the same treatment period as animals at 10 and 30 mg/kg bw/day. Two recovery groups, each of five males and five females, were treated with the high dose (100/60/50 mg/kg bw/day) or the vehicle alone as previously indicated and then maintained without treatment for a further fourteen days. Clinical signs, body weight change, food and water consumption were monitored during the study. Hematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period. All animals were subjected to gross necropsy examination and histopathological examination of selected tissues was performed.

 

RESULTS

Mortality: There were no unscheduled deaths on the study.

Clinical observations: At 100/60 mg/kg bw/day, seven males and all females showed hunched posture with four females also showing piloerection on Day 11. Hunched posture was subsequently apparent for one female on Day 13 and another female on Days 13 and 17 at 100/60/50 mg/kg bw/day. No such effects were detected in animals of either sex treated with 30 or 10 mg/kg bw/day.

Behavioural assessment: Behavioural assessment did not indicate any obvious adverse effects of treatment at 10, 30 or 100/60/50 mg/kg bw/day.

Functional performance tests: Assessment of functional performance did not indicate any obvious adverse effects of treatment at 10, 30 or 100/60/50 mg/kg bw/day.

Sensory reactivity assessments: Sensory reactivity assessment did not indicate any obvious adverse effects of treatment at 10, 30 or 100/60/50 mg/kg bw/day.

Body weight: Treatment at 100 mg/kg bw/day was associated with body weight loss for both sexes during the first week of treatment and body weight gain of males was lower than control during Week 2. For females at 30 mg/kg bw/day, body weight gain was lower than control during the first week of treatment. No such effects were detected in males treated with 30 mg/kg bw/day or animals of either sex treated with 10 mg/kg bw/day.

Food consumption: Treatment at 100/60/50 mg/kg bw/day was associated with lower food consumption compared to control for both sexes during the first week of treatment, which persisted, to a lesser extent during Week 2. For females at 30 mg/kg bw/day, food consumption was lower than control during the first week of treatment. No such effects were detected in males treated with 30 mg/kg bw/day or animals of either sex treated with 10 mg/kg bw/day.

Food conversion efficiency: Treatment at 100/60/50 mg/kg bw/day was associated with inferior food conversion efficiency compared to control for both sexes during the first week of treatment. No such effects were detected in animals of either sex treated with 30 or 10 mg/kg bw/day.

Water consumption: At 100/60/50 mg/kg bw/day males showed higher consumption during Days 1-16, compared to control. For females, higher water consumption was apparent during Days 1-9, with lower water intake observed on Days 9-10. Water consumption was again higher than control during Days 11-13 (when dosing was suspended) and generally remained higher than control for the remaining treatment period and the treatment-free recovery period. For females at 30 mg/kg bw/day, water consumption tended to be higher than control during the first three days of treatment. No such effects were detected in males treated with 30 mg/kg bw/day or animals of either sex treated with 10 mg/kg bw/day.

Hematology: At 100/60/50 mg/kg bw/day, erythrocyte count, hemoglobin, mean cell hemoglobin, and mean cell hemoglobin concentration for both sexes and hemacrit and mean cell volume for males were lower than control. Additionally, for males at 100/60/50 mg/kg bw/day, neutophils and total leukocyte counts were higher than control. At 30 mg/kg bw/day, mean hemoglobin for females, mean cell hemoglobin and mean cell hemoglobin concentration for both sexes and mean cell volume for males were lower than control at the end of the treatment period. At 100/60/50 mg/kg bw/day, hemoglobin and hematocrit for males and mean cell haemoglobin concentration for both sexes at the end of the treatment-free recovery period were lower than control. No toxicologically significant effects were detected in animals of either sex treated with 10 mg/kg bw/day.

Blood chemistry: At 100/60/50 mg/kg bw/day, alanine aminotransferase, total cholesterol, total billirubin, and bile acids were higher than control and triglyceride levels were lower than control for both sexes at the end of the treatment period. Additionally for females, total protein, albumin and albumin/globulin ratio were also lower than control at the end of the treatment period. At 30 mg/kg bw/day, total cholesterol and total billirubin was higher than control for males at the end of the treatment period. For females at this dosage, bile acids were higher and total protein, albumin and albumin/globulin ratio were lower than control at the end of the treatment period. No toxicological significant effects were detected in animals of either sex at 10 mg/kg bw/day.

Urinalysis: Urinalysis assessments did not indicate any obvious adverse effects of treatment at 10, 30 or 100/60/50 mg/kg bw/day.

Necropsy: At 100/60/50 mg/kg bw/day, two males showed small prostate and seminal vesicles at the end of the treatment period. No toxicologically significant effects were detected in animals of either sex treated with 30 or 10 mg/kg bw/day or in recovery animals following fourteen days without treatment.

Organ weights: At 100/60/50 mg/kg bw/day, mean absolute and body weight relative kidney weights for both sexes at the end of treatment were higher than control. Following the treatment free recovery period, male absolute kidney weights were lower and male body weight relative values were higher than control. At 100/60/50 mg/kg bw/day, mean absolute and body weight relative liver weights were higher than control for both sexes at the end of treatment. Following the treatment free recovery period, absolute and body weight relative liver weights for males were lower than control. At 30 and 100/60/50 mg/kg bw/day, absolute and body weight relative spleen weights for both sexes at the end of treatment were higher than control. At 100/60/50 mg/kg bw/day, absolute and body weight relative prostate and seminal vesicles weights were lower than control for males at the end of treatment and at the end of treatment free recovery period.

Histopathology: At 100/60/50 mg/kg bw/day, retinal detachment was present in the eyes of both sexes at the end of the treatment and treatment-free recovery periods. At 30 mg/kg bw/day, females showed retinal detachment and males showed retinal degeneration at the end of treatment. At 30 and 100/60/50 mg/kg bw/day, histopathological examination of the liver revealed centrilobular hypertrophy, changes to the peri-portal region manifested as an increase in cytoplasmic rarefaction, periportal cellular change and periportal dilation (bile duct and vascular) for both sexes at the end of treatment period and at 100/60/50 mg/kg bw/day for both sexes at the end of the treatment-free recovery period. Increased hematopoiesis was also evident for both sexes at 100/60/50 mg/kg bw/day at the end of treatment and recovery periods and for one male receiving 30 mg/kg bw/day at the end of treatment. Additionally, there was also an increase in the number of animals with brown pigment in the periportal area at 100/60/50 mg/kg bw/day at the end of recovery period. At 100/60/50 mg/kg bw/day, erythrocytosis in the mesenteric lymph nodes was observed for males at the end of treatment. At 100/60/50 mg/kg bw/day, increased amounts of haematopoiesis was observed in the spleen for both sexes at the end of treatment. At 100/60/50 mg/kg bw/day, reduced secretion in the prostate and seminal vesicles was observed for 3/5 non-recovery and 2/5 recovery males. No such effects were detected in animals of either sex treated at 10 mg/kg bw/day.

 

CONCLUSION

Based on the results of this study the No Observed Adverse Effect Level (NOAEL) of the test item when administered to the rat over twenty-eight consecutive days was considered to be 10 mg/kg bw/day.