Orthosulfamuron

Endpoint summary

Description of key information

Study 1

IR5878 administrated by oral route (by capsule) to rats for 4 consecutive weeks at the dose of 100, 500, 2500 and 12500 ppm was well tolerated. Minimal changes were seen, predominantly for animals which received 2500 or 12500 ppm, in bodyweight gain, food consumption, haematology and blood chemistry parameters and spleen weight.

Dietary concentrations of 250, 1500 and 9000 ppm have been selected for the subsequent 13-week study.

The NOAEL was considered to be 500 ppm (equivalent to 61 mg/kg/day in males and 60 mg/kg/day in females).

Study 2

IR5878 administrated via the diet to mice for 4 consecutive weeks at doses of 50, 250, 1250 and 6000 ppm was well tolerated. Minimal changes were observed for males receiving 6000 ppm in bodyweight gain, food conversion efficiency and liver weights. Females were not clearly affected by treatment. Increased liver weights, observed in males at this dose level, is a common adaptive response to the administration of a xenobiotic at high concentrations and, in absence of any associated histopathological findings, was considered not to be of toxicological importance.

Dietary concentrations of 250, 1250 and 6000 ppm have been selected for the subsequent 13-week study.

The NOAEL was considered to be 6000 ppm (equivalent to 997 mg/kg/day in males and 1332 g/kg/day in females).

Study 3

The doses to be administered in a 4-week study in Beagle dogs have been set at 150, 450 and 1000 mg/kg/day.

Study 4

IR5878 administrated by oral route (by capsule) to dogs for 4 consecutive weeks at the dose of 150, 450 and 1000 mg/kg/day induced a few adaptive changes in liver only at the highest dose, in one female.

In consideration of the above changes, NOAEL of the study is of 1000 mg/kg/day and NOEL is of 450 mg/kg/day.

Study 5

Administration of IR5878 to Han Wistar rats for 13 weeks via the diet at concentrations of 250, 1500 and 9000 ppm was well tolerated with no over signs of toxicity. The few changes that were noted showed partial or full recovery 4 weeks after cessation of treatment.

The NOAEL can be considered equal to 1500 ppm (equivalent to 113 mg/kg/day in males and 131 g/kg/day in females), as the changes observed were minimal in degree.

Study 6

Administration of IR5878 to CD-1 mice for 13 weeks via the diet at concentrations of 250, 1250 and 6000 ppm was well tolerated. Non specific changes were observed in males receiving 6000 ppm in bodyweight gain, food conversion efficiency and liver weights. Females were not affected by treatment. None of the findings observed in the study preclude administration of dietary levels of up to 6000 ppm in a subsequent carcinogenicity study. The NOAEL was considered to be 6000 ppm (equivalent to 865 mg/kg/day in males and 1096 mg/kg/day in females).

Study 7

IR5878 administered by oral route (by capsules) to dogs for 13 consecutive weeks at the doses of 1000, 450 and 150 mg/kg/day induced some non specific changes in haematological, blood chemistry parameters and organ weight (liver weight increase) in the high- and mid-dose group. In consideration of the above results, the dose of 150 mg/kg/day was considered the NOAEL. Also the dose of 1000 mg/kg/day did not induce overt toxicity.

Study 8

IR5878 administered by oral route (by capsules) to dogs for 52 consecutive weeks at the doses of 1000, 300 and 75 mg/kg/day induced toxicity at 1000 mg/kg/day to the liver, being the main target organ. In addition, there was evidence from this study that the adrenals and the haemapoetic system were also target organs of IR5878. Despite toxicity being evident at 1000 mg/kg/day, the majority of the animals tolerated 52-weeks of treatment.

Evidence of minor liver toxicity at 300 mg/kg/day induced the choice of the dose of 75 mg/kg/day as NOAEL (No Observed Adverse Effect Level).

Study 9 Dermal toxicity

A daily 6-hour semi-occluded dermal application of IR5878 to CD rats for 4 weeks was well-tolerated, producing no toxicologically significant change or any clear response at the dermal application site.

The NOAEL (No Observed Adverse Effect Level) in this study was considered to be 1000 mg/kg/day.

Study 10 Inhalation toxicity

This was not considered relevant since Orthosulfamuron is not volatile.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2000

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)

Deviations:

yes

Remarks:

Deviations: on the first day of treatment males of the low and mid dose levels received a higher dose than that foreseen (i.e. 250 mg/kg bw and 500 mg/kg bw instead of 150 mg/kg bw and 450 mg/kg bw, respectively)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test material:
IR5878
Batch number: FCF/T/168-00 (ex 20525/03/9)
Purity: 98.54 ± 0.51 %

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals:
Species and Strain: Male and Female Beagle dogs
Age: 7 months
Body weight: 5.53 ÷ 8.38 kg
Housing: limited-access dog facility. Individual pens.
Feed: diet “Altromin H”, produced by A. Rieper, Vandoies (BZ), Italy. The diet was supplemented by the Producer with vitamins and trace elements. The diet was distributed once a day. Water was distributed ad libitum.

Environmental conditions:
Temperature and humidity measured during the study were 19 ± 2°C and 55 ± 10%, respectively.
Light: artificial lighting with a 12-hour circadian cycle (07:00 – 19:00).

Route of administration:

oral: capsule

Details on route of administration:

Groups of three Beagle dogs/sex were given by gelatine capsules doses of 0 (empty capsules), 150, 450 or 1000 mg/kg/day.

Vehicle:

other: Gelatine

Details on oral exposure:

The purpose of the study was to evaluate the effects of repeated administrations of IR5878 to male and female Beagle dogs. IR5878 was administrated by oral route once a day per 7 days a week, for 4 consecutive weeks to 12 males and to 12 females of Beagle dog. Groups of three Beagle dogs/sex were given by gelatine capsules doses of 0 (empty capsules), 150, 450 or 1000 mg/kg/day. These dosages were determined in a preliminary tolerability study, which had the purpose of assessing the toxic effects resulting from the oral administration of IR5878 to Beagle dogs and to determine the maximum tolerated dose to be used in this study (see point 05.03.01/03).

Duration of treatment / exposure:

. IR5878 was administrated by oral route once a day per 7 days a week, for 4 consecutive weeks to 12 males and to 12 females of Beagle dog.

Frequency of treatment:

. IR5878 was administrated by oral route once a day per 7 days a week, for 4 consecutive weeks to 12 males and to 12 females of Beagle dog.

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

IR5878 was administrated by oral route once a day per 7 days a week, for 4 consecutive weeks to 12 males and to 12 females of Beagle dog. Groups of three Beagle dogs/sex were given by gelatine capsules doses of 0 (empty capsules), 150, 450 or 1000 mg/kg/day.

Observations and examinations performed and frequency:

Clinical observations were carried out daily, body weight and food consumption measurements were recorded weekly during treatment, ophthalmoscopy and laboratory investigations (haematology, blood chemistry and urinalysis) were carried out before the treatment and at study termination.

Sacrifice and pathology:

All the animals were sacrificed at the end of the dosing period for pathology examinations (organ weights and histopathology).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weight: IR5878 induced a slight increase in mean liver weights (absolute and relative) in highest dose females, when compared to controls. This change did not reach statistical significance, and was due to an outlier individual liver weight (female No. 3168). No other treatment-related changes were observed.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Histology: treatment induced in the liver of the outlier female No. 3168 slightly hypertrophy of the centrilobular hepatocytes. As no degenerative or inflammatory effects were observed, hypertrophy could be considered as a metabolic adaptive change of the liver, rather than a toxic effect. No other treatment-related histological modifications were seen.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

gross pathology

haematology

mortality

ophthalmological examination

urinalysis

Key result

Dose descriptor:

NOEL

Effect level:

>= 450 mg/kg bw/day (actual dose received)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Findings

Clinical observation

Mortality: no animals died during the study.

Clinical signs: no clinical signs were observed during the study.

Body weight and food consumption: no effects on body weight or on food consumption were noted at any dose.

Ophthalmic examination: no eye changes were seen in any dog.

 

Laboratory investigations

Haematology: no changes attributable to oral treatment with IR5878 were noted at any dose tested.

Blood chemistry: no treatment-related changes were seen in any animal at any dose tested.

Urinalysis: urine examination did not reveal treatment-related modifications at any dose.

 

Post mortemexaminations

Organ weight: IR5878 induced a slight increase in mean liver weights (absolute and relative) in highest dose females, when compared to controls. This change did not reach statistical significance, and was due to an outlier individual liver weight (female No. 3168). No other treatment-related changes were observed.

 

Table 5.3-10   4-week dietary study in dogs: absolute and relative liver weights(individual female No. 3168 values)

	Males				Females
	0           mg/kg bw/d	150        mg/kg bw/d	450        mg/kg bw/d	1000       mg/kg bw/d	0           mg/kg bw/d	150        mg/kg bw/d	450        mg/kg bw/d	1000       mg/kg bw/d
Absolute (g)	325.16	318.02	363.56	349.80	292.92	252.28	279.80	350.86(395.50)
Relative (%)	3.39	3.64	3.99	3.71	3.73	3.29	3.53	4.05      (5.04)

 

Gross pathology: no treatment-related changes were seen.

Histology: treatment induced in the liver of the outlier female No. 3168 slightly hypertrophy of the centrilobular hepatocytes. As no degenerative or inflammatory effects were observed, hypertrophy could be considered as a metabolic adaptive change of the liver, rather than a toxic effect. No other treatment-related histological modifications were seen.

Conclusions:

IR5878 administrated by oral route (by capsule) to dogs for 4 consecutive weeks at the dose of 150, 450 and 1000 mg/kg/day induced a few adaptive changes in liver only at the highest dose, in one female.
In consideration of the above changes, NOAEL of the study is of 1000 mg/kg/day and NOEL is of 450 mg/kg/day.

Executive summary:

In order to evaluate the effects of repeated administrations of IR5878 to male and female Beagle dogs, IR5878 was administrated by oral route once a day per 7 days a week, for 4 consecutive weeks to 12 males and to 12 females of Beagle dog. Groups of three Beagle dogs/sex were given by gelatine capsules doses of 0 (empty capsules), 150, 450 or 1000 mg/kg/day.

IR5878 induced a few adaptive changes in liver only at the highest dose, in one female. In consideration of the above changes, NOAEL of the study is of 1000 mg/kg/day and NOEL is of 450 mg/kg/day.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Principles of method if other than guideline:

Method:
The objective of the study was to assess the systemic toxic potential of IR5878 and to aid the selection of dose levels for an associated combined chronic toxicity and carcinogenity study (see point 05.05.01/01). The reversibility of any treatment-related effects was assessed during a 4-week recovery period.
Groups of 10 male and 10 female Han Wistar rats received IR5878, via the diet, at concentrations of 0, 250, 1500 or 9000 ppm for 13 weeks. A further 5 male and 5 female rats were assigned to the Control and high concentration groups for a 4-week recovery period following the 13-week treatment period.
Throughout the study the animals were observed daily for general clinical signs. A more detailed physical examination was performed weekly. Bodyweights and food consumption were recorded at day 0, weekly during the treatment period and at study termination. Functional observational battery tests were performed before treatment commenced, weekly during the treatment period and during week 4 of the recovery period. Ophthalmoscopy examinations were performed pre-treatment and during week 13. Haematology, blood chemistry and urinalysis investigations were performed during week 13 of treatment and during week 4 of recovery.
On completion of treatment and recovery periods all the animals were sacrificed and examined macroscopically; selected organs were weighted and tissues were processed for microscopic evaluation.

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test material:
IR5878
Batch number: FCF/T/172-00 (ex 20525/03/8)
Purity: 97.99 ± 0.39 %

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals:
Species and Strain: Male and Female Han Wistar rats
Age: 26 to 30 days (37 to 41 days when treatment started)
Body weight: 90 ÷ 107 g (males); 77 ÷ 88 g (females)
Housing: 5 animals/sex/cage.
Feed: animals were allowed free access, except when urine was being collected and overnight before routine blood sampling, to a powdered rodent diet, Rat and Mouse No. 1 Maintenance Diet from Special Diets Services Ltd, Witham, Essex, England.

Environmental conditions:
Temperature and humidity measured during the study were 19 ÷ 23°C and 40 ÷ 70%, respectively.
Light: artificial lighting with a 12-hour cycle (07:00 – 19:00).

Route of administration:

oral: feed

Details on route of administration:

Groups of 10 male and 10 female Han Wistar rats received IR5878, via the diet, at concentrations of 0, 250, 1500 or 9000 ppm for 13 weeks. A further 5 male and 5 female rats were assigned to the Control and high concentration groups for a 4-week recovery period following the 13-week treatment period.

Details on oral exposure:

Groups of 10 male and 10 female Han Wistar rats received IR5878, via the diet, at concentrations of 0, 250, 1500 or 9000 ppm for 13 weeks. A further 5 male and 5 female rats were assigned to the Control and high concentration groups for a 4-week recovery period following the 13-week treatment period.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical determinations on the diets
The mean concentrations of IR5878 in test diet formulations analysed during the study (weeks 1, 6 and 12) were within ±5% of nominal concentrations, confirming accurate formulation.

Duration of treatment / exposure:

Groups of 10 male and 10 female Han Wistar rats received IR5878, via the diet, at concentrations of 0, 250, 1500 or 9000 ppm for 13 weeks. A further 5 male and 5 female rats were assigned to the Control and high concentration groups for a 4-week recovery period following the 13-week treatment period.

Frequency of treatment:

Groups of 10 male and 10 female Han Wistar rats received IR5878, via the diet, at concentrations of 0, 250, 1500 or 9000 ppm for 13 weeks. A further 5 male and 5 female rats were assigned to the Control and high concentration groups for a 4-week recovery period following the 13-week treatment period.

Dose / conc.:

0 ppm

Dose / conc.:

250 ppm

Dose / conc.:

1 500 ppm

Dose / conc.:

9 000 ppm

No. of animals per sex per dose:

Groups of 10 male and 10 female Han Wistar rats received IR5878, via the diet, at concentrations of 0, 250, 1500 or 9000 ppm for 13 weeks. A further 5 male and 5 female rats were assigned to the Control and high concentration groups for a 4-week recovery period following the 13-week treatment period.

Control animals:

yes

Details on study design:

Method:
The objective of the study was to assess the systemic toxic potential of IR5878 and to aid the selection of dose levels for an associated combined chronic toxicity and carcinogenity study (see point 05.05.01/01). The reversibility of any treatment-related effects was assessed during a 4-week recovery period.
Groups of 10 male and 10 female Han Wistar rats received IR5878, via the diet, at concentrations of 0, 250, 1500 or 9000 ppm for 13 weeks. A further 5 male and 5 female rats were assigned to the Control and high concentration groups for a 4-week recovery period following the 13-week treatment period.
Throughout the study the animals were observed daily for general clinical signs. A more detailed physical examination was performed weekly. Bodyweights and food consumption were recorded at day 0, weekly during the treatment period and at study termination. Functional observational battery tests were performed before treatment commenced, weekly during the treatment period and during week 4 of the recovery period. Ophthalmoscopy examinations were performed pre-treatment and during week 13. Haematology, blood chemistry and urinalysis investigations were performed during week 13 of treatment and during week 4 of recovery.
On completion of treatment and recovery periods all the animals were sacrificed and examined macroscopically; selected organs were weighted and tissues were processed for microscopic evaluation.

Observations and examinations performed and frequency:

Throughout the study the animals were observed daily for general clinical signs. A more detailed physical examination was performed weekly. Bodyweights and food consumption were recorded at day 0, weekly during the treatment period and at study termination. Functional observational battery tests were performed before treatment commenced, weekly during the treatment period and during week 4 of the recovery period. Ophthalmoscopy examinations were performed pre-treatment and during week 13. Haematology, blood chemistry and urinalysis investigations were performed during week 13 of treatment and during week 4 of recovery.
On completion of treatment and recovery periods all the animals were sacrificed and examined macroscopically; selected organs were weighted and tissues were processed for microscopic evaluation.

Sacrifice and pathology:

On completion of treatment and recovery periods all the animals were sacrificed and examined macroscopically; selected organs were weighted and tissues were processed for microscopic evaluation.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Signs and mortality: there were no treatment-related deaths. A slightly high incidence of dorsal hairloss was observed for animals receiving 9000 ppm. There were no other clinical signs related to treatment.

Mortality:

no mortality observed

Description (incidence):

Signs and mortality: there were no treatment-related deaths

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weight and food consumption: bodyweight gain, food consumption and food conversion efficiency were considered to be unaffected by treatment.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Ophthalmic examination: there were no ophthalmic findings related to treatment

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematology: haematology investigations during week 13 of treatment revealed slightly low total white blood cell and lymphocyte counts for males and females receiving 250, 1500 or 9000 ppm and slightly low packed cell volume (haematocrit), haemoglobin concentration and red blood cell count for females receiving 9000 ppm. All these changes showed recovery following cessation of treatment.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood chemistry: blood chemistry investigations during week 13 of treatment revealed some effects (low phosphorus levels and albumin) in males at any dose; other parameters were altered only at the highest dose tested (9000 ppm). Females appeared to be less sensitive and only the levels of phosphorus was significantly affected at 1500 and 9000 ppm. During week 4 of recovery, most alterations reached complete recovery, except for phosphorus levels for females and creatinine levels which were still statistically different from control values.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Urinalysis: urinalysis investigations during week 13 of treatment revealed slightly low pH and slightly high specific gravity for males receiving 9000 ppm. These changes were not apparent during week 4 of the recovery period. Females were not affected by the treatment.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weight: liver and kidney weights were slightly high after 13 weeks of treatments for males which received 9000 ppm when compared with those of the Control. These changes were no more present after 4 weeks of recovery. There were no effects on organ weights in females.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Gross pathology: there were no macroscopic findings on completion of the treatment or recovery periods which were considered to be related to treatment with IR5878.

Neuropathological findings:

no effects observed

Description (incidence and severity):

Functional observation battery: there was no evidence of neurotoxicity during functional observational battery assessments.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic pathology: an increased incidence of hepatocyte hypertrophy of the centrilobular region of the liver was seen after 13 weeks of treatment for males which received 1500 or 9000 ppm. The change was not observed in females. Examination of the liver from animals killed at the end of the recovery period showed complete recovery from the effects of treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 500 ppm

Based on:

act. ingr.

Sex:

male

Basis for effect level:

organ weights and organ / body weight ratios

urinalysis

other: blood chemistry (e.g. protein levels, phosphorous levels, creatine levels, albumin/globulin ratios)

Critical effects observed:

not specified

Lowest effective dose / conc.:

9 000 ppm

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Critical effects observed:

not specified

Lowest effective dose / conc.:

9 000 ppm

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Findings

Analytical determinations on the diets

The mean concentrations of IR5878 in test diet formulations analysed during the study (weeks 1, 6 and 12) were within ±5% of nominal concentrations, confirming accurate formulation.

 

Clinical observation

Signs and mortality: there were no treatment-related deaths. A slightly high incidence of dorsal hairloss was observed for animals receiving 9000 ppm. There were no other clinical signs related to treatment.

Body weight and food consumption: bodyweight gain, food consumption and food conversion efficiency were considered to be unaffected by treatment.

Achieved dosage: group mean achieved dosages were 19, 113 and 706 mg/kg/day for males and 22, 131 and 773 mg/kg/day for females, receiving 250, 1500 and 9000 mg IR5878/kg diet.

 

Table 5.3-11:  Achieved dosage (mg/kg/day)

	0 ppm	250 ppm	1500 ppm	9000 ppm
Males	-	19	113	706
Females	-	22	131	773

 

Ophthalmic examination: there were no ophthalmic findings related to treatment.

Functional observation battery: there was no evidence of neurotoxicity during functional observational battery assessments.

 

Laboratory investigations

Haematology: haematology investigations during week 13 of treatment revealed slightly low total white blood cell and lymphocyte counts for males and females receiving 250, 1500 or 9000 ppm and slightly low packed cell volume (haematocrit), haemoglobin concentration and red blood cell count for females receiving 9000 ppm. All these changes showed recovery following cessation of treatment.

 

Table 5.3-12:  Haematology at the end of the treatment(at the end of recovery period)

Parameter	Males				Females
	0        ppm(recovery)	250       ppm	1500          ppm	9000      ppm(recovery)	0        ppm(recovery)	250       ppm	1500          ppm	9000      ppm(recovery)
Haematocrit (L/L)	0.456	0.462	0.449	0.454	0.429(0.433)	0.425	0.438	0.412*(0.419)
Haemoglobin conc. (g/dL)	15.8	16.0	15.8	15.6	15.0(15.0)	14.8	15.4	14.4*(14.5)
Red blood cell (x10^12/L)	8.62	8.73	8.51	8.58	7.90(7.74)	7.66	7.95	7.44*(7.55)
Total white cell (x10^9/L)	8.46(7.57)	6.99*	6.85**	6.70**(7.10)	5.52(4.10)	4.66*	4.50*	3.99**(5.08)
Lymphocyte (x10^9/L)	6.42(5.74)	5.39*	5.27*	4.77**(5.34)	4.45(3.27)	3.61*	3.36**	3.06**(3.90)
* p<0.05 and ** p<0.01

 

Blood chemistry: blood chemistry investigations during week 13 of treatment revealed some effects (low phosphorus levels and albumin) in males at any dose; other parameters were altered only at the highest dose tested (9000 ppm). Females appeared to be less sensitive and only the levels of phosphorus was significantly affected at 1500 and 9000 ppm. During week 4 of recovery, most alterations reached complete recovery, except for phosphorus levels for females and creatinine levels which were still statistically different from control values.

 

Table 5.3-13:  Blood chemistry at the end of the treatment(at the end of recovery period)

Parameter	Males				Females
	0        ppm(recovery)	250       ppm	1500          ppm	9000      ppm(recovery)	0        ppm(recovery)	250       ppm	1500          ppm	9000      ppm(recovery)
Alkaline phosphatase (U/L)	180   (182)	175	164	156    (159)	91       (64)	90	84	70       (80)
Aspartate amino-transferase (U/L)	69      (61)	69	63	59*     (54)	67	72	65	68
Creatinine (umol/L)	45       (47)	47	47	49*  (53**)	55	52	56	53
Triglycerides (mmol/L)	1.41  (0.82)	1.30	1.37	0.96*  (0.61)	0.38	0.51	0.43	0.54
Phosphorus (mmol/L)	1.95  (1.92)	1.74**	1.63**	1.71**  (1.92)	1.59   (1.37)	1.41	1.28*	1.34* (1.04*)
Total protein (g/L)	66       (64)	65	65	70**   (66)	70	69	72	71
Albumin (g/L)	38       (34)	36*	36*	38       (35)	42	41	42	41
Albumin/globulin ratio	1.33   (1.15)	1.29	1.27	1.21*  (1.14)	1.45	1.43	1.38	1.40
* p<0.05 and ** p<0.01

 

Urinalysis: urinalysis investigations during week 13 of treatment revealed slightly low pH and slightly high specific gravity for males receiving 9000 ppm. These changes were not apparent during week 4 of the recovery period. Females were not affected by the treatment.

 

 

Table 5.3-14:  Urinalysis at the end of the treatment(at the end of recovery period)

Parameter	Males
	0 ppm(recovery)	250 ppm	1500 ppm	9000 ppm(recovery)
pH	6.7(6.7)	6.6	6.6	6.1**(7.0)
Specific gravity	1039(1058)	1036	1039	1045*(1037)
* p<0.05 and ** p<0.01

 

Post mortemexaminations

Organ weight: liver and kidney weights were slightly high after 13 weeks of treatments for males which received 9000 ppm when compared with those of the Control. These changes were no more present after 4 weeks of recovery. There were no effects on organ weights in females.

 

Table 5.3-15:  Absolute and relative organ weights at the end of the treatment(at the end of recovery period)

Parameter	Males
	0 ppm(recovery)	250 ppm	1500 ppm	9000 ppm(recovery)
LIVER Absolute (g) Relative (%)	12.15(13.46) 3.293(3.493)	12.21 3.302	11.57 3.247	13.53(13.93) 3.860**(3.320)
KIDNEY Absolute (g) Relative (%)	1.99(2.08) 0.542(0.542)	2.01 0.547	1.92 0.541	2.04(2.17) 0.583*(0.518)
* p<0.05 and ** p<0.01

 

Gross pathology: there were no macroscopic findings on completion of the treatment or recovery periods which were considered to be related to treatment with IR5878.

Microscopic pathology: an increased incidence of hepatocyte hypertrophy of the centrilobular region of the liver was seen after 13 weeks of treatment for males which received 1500 or 9000 ppm. The change was not observed in females. Examination of the liver from animals killed at the end of the recovery period showed complete recovery from the effects of treatment.

 

Table 5.3-16:  Microscopic pathology at the end of the treatment(at the end of recovery period)

Parameter	Males
	0 ppm(recovery)	250 ppm	1500 ppm	9000 ppm(recovery)
LIVER             examined	10(5)	10	10	10(5)
Hepatocyte hypertrophy	0(0)	0	1	7**(0)
** p<0.01

 

Conclusions:

Conclusions
Administration of IR5878 to Han Wistar rats for 13 weeks via the diet at concentrations of 250, 1500 and 9000 ppm was well tolerated with no over signs of toxicity. The few changes that were noted showed partial or full recovery 4 weeks after cessation of treatment.
The NOAEL can be considered equal to 1500 ppm (equivalent to 113 mg/kg/day in males and 131 g/kg/day in females), as the changes observed were minimal in degree.

Executive summary:

Executive summary

In order to assess the systemic toxic potential of IR5878 and to aid the selection of dose levels for an associated combined chronic toxicity and carcinogenity study, groups of 10 male and 10 female Han Wistar rats received IR5878, via the diet, at concentrations of 0, 250, 1500 or 9000 ppm for 13 weeks. A further 5 male and 5 female rats were assigned to the Control and high concentration groups for a 4-week recovery period following the 13-week treatment period.

IR5878 was well tolerated with no overall signs of toxicity. The few changes that were noted showed partial or full recovery 4 weeks after cessation of treatment. The NOAEL can be considered equal to 1500 ppm (equivalent to 113 mg/kg/day in males and 131 g/kg/day in females), as the changes observed were minimal in degree.

 

Reference 3

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3150 (90-Day Oral Toxicity in Non-rodents)

Deviations:

no

Principles of method if other than guideline:

Method:
The purpose of the study was to evaluate the effects of repeated administrations of IR5878 to Beagle dogs.
IR5878 was administrated by oral route using gelatine capsules as vehicle once a day, seven days a week, for 13 consecutive weeks, to male and female Beagle dogs. Groups of 4 animals/sex received IR5878 at the doses of 0, 150, 450 or 1000 mg/kg/day.
Clinical observations were performed daily; bodyweight and food consumption measurements were conducted prior the starting day, at day 0, weekly during the treatment and at study termination; ophthalmoscopy was carried out before starting the treatment and at study termination; laboratory investigations (haematology, blood chemistry and urinalysis) were carried out pre-test (week -2) at week 7 and at the end of the treatment period (week 13).
All the animals were killed at the end of the dosing period for pathology investigations (organ weight, gross pathology and histology).

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test material:
IR5878
Batch number: FCF/T/181-01 (ex 20525/03/3)
Purity: 98.01 ± 0.33 %

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals:
Species and Strain: Male and Female Beagle dogs
Age: 7 to 8 months (8 to 9 months when treatment started)
Body weight: 7.41 ÷ 11.24 kg (9.30 ÷ 10.90 kg (males) and 7.75 ÷ 8.90 kg (females) when treatment started)
Housing: limited-access dog facility. Individual pens.
Feed: diet “Altromin H”, produced by A. Rieper, Vandoies (BZ), Italy. The diet was supplemented by the Producer with vitamins and trace elements. The diet was distributed once a day. Water was distributed ad libitum.

Environmental conditions:
Temperature and humidity measured during the study were 19 ± 2°C and 55 ± 10%, respectively.
Light: artificial lighting with a 12-hour circadian cycle (07:00 – 19:00).

Route of administration:

oral: capsule

Details on route of administration:

IR5878 was administrated by oral route using gelatine capsules as vehicle once a day, seven days a week, for 13 consecutive weeks, to male and female Beagle dogs. Groups of 4 animals/sex received IR5878 at the doses of 0, 150, 450 or 1000 mg/kg/day.

Vehicle:

other: Gelatine capsule

Details on oral exposure:

IR5878 was administrated by oral route using gelatine capsules as vehicle once a day, seven days a week, for 13 consecutive weeks, to male and female Beagle dogs. Groups of 4 animals/sex received IR5878 at the doses of 0, 150, 450 or 1000 mg/kg/day.

Duration of treatment / exposure:

IR5878 was administrated by oral route using gelatine capsules as vehicle once a day, seven days a week, for 13 consecutive weeks, to male and female Beagle dogs. Groups of 4 animals/sex received IR5878 at the doses of 0, 150, 450 or 1000 mg/kg/day.

Frequency of treatment:

IR5878 was administrated by oral route using gelatine capsules as vehicle once a day, seven days a week, for 13 consecutive weeks, to male and female Beagle dogs. Groups of 4 animals/sex received IR5878 at the doses of 0, 150, 450 or 1000 mg/kg/day.

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

150 mg/kg bw/day (nominal)

Dose / conc.:

450 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Groups of 4 animals/sex received IR5878 at the doses of 0, 150, 450 or 1000 mg/kg/day.

Details on study design:

The purpose of the study was to evaluate the effects of repeated administrations of IR5878 to Beagle dogs.
IR5878 was administrated by oral route using gelatine capsules as vehicle once a day, seven days a week, for 13 consecutive weeks, to male and female Beagle dogs. Groups of 4 animals/sex received IR5878 at the doses of 0, 150, 450 or 1000 mg/kg/day.
Clinical observations were performed daily; bodyweight and food consumption measurements were conducted prior the starting day, at day 0, weekly during the treatment and at study termination; ophthalmoscopy was carried out before starting the treatment and at study termination; laboratory investigations (haematology, blood chemistry and urinalysis) were carried out pre-test (week -2) at week 7 and at the end of the treatment period (week 13).
All the animals were killed at the end of the dosing period for pathology investigations (organ weight, gross pathology and histology).

Observations and examinations performed and frequency:

Clinical observations were performed daily; bodyweight and food consumption measurements were conducted prior the starting day, at day 0, weekly during the treatment and at study termination; ophthalmoscopy was carried out before starting the treatment and at study termination; laboratory investigations (haematology, blood chemistry and urinalysis) were carried out pre-test (week -2) at week 7 and at the end of the treatment period (week 13).
All the animals were killed at the end of the dosing period for pathology investigations (organ weight, gross pathology and histology).

Sacrifice and pathology:

All the animals were killed at the end of the dosing period for pathology investigations (organ weight, gross pathology and histology).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Bodyweight: bodyweight was unaffected by oral treatment with IR5878 in males treated with the low dose and in females treated with the low and the intermediate doses. In males treated with the intermediate and high doses and in females treated with the high dose, slight bodyweight decrease was recorded from week 3-4 (males) or from week 11 (females) onward.

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

Haematology: IR5878 given by oral route did not result in any haematological change either in males or in females at the week 6 investigation. At the end of the study no changes were noted in females, while in males from all groups slight decreases, not dose-related, occurred in red blood cells and in haematocrit and haemoglobin values.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Blood chemistry: no test item-related changes were noted in animals treated with the low dose at investigations carried out at weeks 6 and 13 of the study. At the analysis carried out at the middle of the study, IR5878 oral administration resulted in an increase in alkaline phosphatase serum levels in both sexes of the two higher doses (statistically significant in both doses males and in the highest dose females). Confined to the high dose, both sexes also showed an increase, not statistically significant, in total cholesterol levels. At the final investigation, the statistically significant increase in alkaline phosphatase serum levels in both sexes was again observed at the highest dose level only. Mean values of animals of group 3 appeared to recover (females) or tended to a decrease (males). Other spotted, not-related changes were recorded, but they were considered not relevant and not related to the treatment.

Urinalysis findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weights: absolute (females only) or relative (both sexes) liver weights were slightly increased, with statistical significance attained, following treatment with 1000 mg/kg bw/day of IR5878. A trend towards liver weight increase was also observed in the intermediate dose females and in males of the intermediate and low doses; however individual values were within the limits of the background data of the performing laboratory, and histology did not show any treatment-related modification.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Histology: no treatment-related modifications were observed. Histologic modification was observed only at the high dose where hypertrophy of hepatocytes of the centrilobular area was seen in the liver. The histologic appearance and absence of associated degenerative changes were consistent with an adaptive response to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 150 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

clinical biochemistry

haematology

organ weights and organ / body weight ratios

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

haematopoietic

Organ:

blood

Treatment related:

yes

Conclusions:

IR5878 administered by oral route (by capsules) to dogs for 13 consecutive weeks at the doses of 1000, 450 and 150 mg/kg/day induced some non specific changes in haematological, blood chemistry parameters and organ weight (liver weight increase) in the high- and mid-dose group. In consideration of the above results, the dose of 150 mg/kg/day was considered the NOAEL. Also the dose of 1000 mg/kg/day did not induce overt toxicity.

Executive summary:

In order to evaluate the effects of repeated administrations of IR5878 to Beagle dogs, IR5878 was administrated by oral route using gelatine capsules as vehicle once a day, seven days a week, for 13 consecutive weeks, to male and female Beagle dogs. Groups of 4 animals/sex received IR5878 at the doses of 0, 150, 450 or 1000 mg/kg/day.

IR5878 induced some non specific changes in haematological, blood chemistry parameters and organ weight (liver weight increase) in the high- and mid-dose group. In consideration of the above results, the dose of 150 mg/kg/day was considered the NOAEL. Also the dose of 1000 mg/kg/day did not induce overt toxicity.

Reference 4

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.4100 (Chronic Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: European Commission Directive 88/302/EEC; 92/89/EEC; 96/54/EC-B26 JMAFF 8147 (November 24, 2000)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test material:
IR5878
Batch number: G 009/02
Purity: 98.56 ± 0.19 %

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals:
Species and Strain: Male and Female Beagle dogs
Age: 20 to 24 weeks
Body weight: 7.1 ÷ 8.9 kg (males) and 6.6 ÷ 8.2 kg (females)
Housing: 2 animals in pair of the same sex and dosage group per pen.
Feed: 400 g of a standard dry pelleted diet (Diet A: Special Diets Services Ltd) daily. Water was freely available.

Environmental conditions:
Temperature measured during the study was 15 ÷ 24°C.
Light: 12 hours of light and 12 hours of dark.

Route of administration:

oral: capsule

Details on route of administration:

Groups of 4 male and 4 female Beagle dogs received IR5878 by oral capsule administration, at concentrations of 0, 75, 300 or 1000 mg/kg/day for 52 weeks. These dosages were selected based on a 13-week preliminary study (see point 05.03.03/01).

Duration of treatment / exposure:

Groups of 4 male and 4 female Beagle dogs received IR5878 by oral capsule administration, at concentrations of 0, 75, 300 or 1000 mg/kg/day for 52 weeks. These dosages were selected based on a 13-week preliminary study (see point 05.03.03/01).

Frequency of treatment:

Groups of 4 male and 4 female Beagle dogs received IR5878 by oral capsule administration, at concentrations of 0, 75, 300 or 1000 mg/kg/day for 52 weeks. These dosages were selected based on a 13-week preliminary study (see point 05.03.03/01).

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

75 mg/kg bw/day (nominal)

Dose / conc.:

300 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Groups of 4 male and 4 female Beagle dogs received IR5878 by oral capsule administration, at concentrations of 0, 75, 300 or 1000 mg/kg/day for 52 weeks. These dosages were selected based on a 13-week preliminary study (see point 05.03.03/01).

Observations and examinations performed and frequency:

Clinical observation were carried out daily; bodyweight and food consumption measurements were recorded at day 0, weekly during the treatment and before necropsy; before starting and at week 52 ophthalmological examination was performed; laboratory investigations (haematology, blood chemistry and urinalysis) were carried out before treatment commenced, at week 13, 26 and 52. All the animals were sacrified at the end of the dosing period for macroscopic and microscopic pathology investigations.

Sacrifice and pathology:

All the animals were sacrified at the end of the dosing period for macroscopic and microscopic pathology investigations.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs
Loss of appetite, mainly in females, and pale faeces were notes only in animals receiving 1000 mg/kg/day.

Mortality:

mortality observed, treatment-related

Description (incidence):

Female animal 992, receiving 1000 mg/kg/day, was sacrificed during week 40, after an eight week recovery period, due to bodyweight loss and elevated biochemical parameters in week 32, indicating severe liver dysfunction.

Decedent animal
From approximately week 26 one female receiving 1000 mg/kg/day showed large weight loss and some altered biochemistry values. Thus treatment of this animal ceased after 32 weeks of dose administration. The animal was allowed an 8 week recovery period during which time it showed recovery from the in-life treatment-related effects. In week 40 the animal was sacrificed.
During treatment haematological parameters were similar to those of the other females treated at 1000 mg/kg/day, but at the end of recovery period all altered parameters recovered.
The blood chemistry parameters of this animal showed similar alterations seen for females treated at 1000 mg/kg/day, but during recovery period the values gradually returned to expected ranges.
Organ weights recorded at sacrifice were within the expected background range.
The macroscopic examination showed pale kidneys, pale liver and the presence of a pituitary cyst. Slight prominent pigmented Kupffer, and minimal adrenal cortical vacuolation (zona fasciculata and reticularis) were noted at histopathology.
It was not possible to determine a specific cause of deterioration of health status for this decedent animal receiving 1000 mg/kg/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall group mean bodyweight gain was lower for both males and females receiving 1000 mg/kg/day.
Group mean bodyweight gain was also marginally lower than controls for males receiving 75 or 300 mg/kg/day. However the decrease was not statistically significant and no dosage-relationship was apparent for the inter-group differences among these males.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Lower average weekly food intake was recorded for females receiving 1000 mg/kg/day.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

The ophthalmoscopic findings were within normal limits for animals of this age, breed and strain and unrelated to treatment with IR5878.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Lower group mean haematocrit (Htc), erythrocytes (RBC) and haemoglobin (Hb) were noted in all animals treated at 1000 mg/kg/day from week 13 for females and from week 26 for males. Higher group mean platelet counts were evident for both sexes receiving 1000 mg/kg/day from week 13.
Higher reticulocyte counts, slightly higher mean cell haemoglobin (MCH) and mean cell volume (MCV) values were recorded in females receiving 1000 mg/kg/day.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Higher group mean alkaline phosphatase (ALP) values were noted for dogs receiving 1000 mg/kg bw/day from week 13, and to a lesser extent for animals receiving 300 mg/kg/day. Dosage-related lower group mean aspartate amino-transferase (AST) values were noted for females receiving 1000 and 300 mg/kg/day in week 13 and 26. In week 52 the values of AST were still lower, but without apparent dosage-relationship. Male and female dogs receiving 1000 mg/kg/day from week 13 showed lower values for total protein, albumin and A/G ratio. Lower calcium values were recorded from week 13 for dogs treated at 1000 mg/kg/day; in week 26 alteration in calcium levels were found also in dogs treated with the lower dose, but the effect was no more evident at week 52. Dose-related lower creatinine values were noted in males receiving 1000 or 300 mg/kg/day, starting from week 13.

Urinalysis findings:

no effects observed

Description (incidence and severity):

Loss of appetite, mainly in females

Behaviour (functional findings):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Group mean (relative to terminal body weight for females) and individual liver weights were higher for dogs of both sexes treated at 1000 mg/kg/day and for females receiving 300 mg/kg/day in comparison with controls. In the high dose group (1000 mg/kg/day9 a significant higher mean kidney weight (relative to terminal body weight) was found for females.
Individual and group mean prostate weights were lower for males treated at 1000 mg/kg/day, but the decrease was correlated to the lower body weight of these dogs, and considered of no toxicological importance in absence of any macroscopic or microscopic pathology findings.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Enlarged of the liver was noted in the majority of animals treated at 1000 mg/kg/day and in one male and two females receiving 300 mg/kg/day.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic pathology
Liver: generalised hepatocyte hypertrophy and prominent pigmented Kupffer cells were observed in dogs receiving 300 or 1000 mg/kg/day with a dosage-relationship. An increase in degree of periportal inflammatory cell infiltration was noted in dogs of both sexes treated at 1000 mg/kg/day, and to a lesser degree in animals receiving 300 mg/kg/day. In addition, increased incidence and degree of vascular/perivascular inflammatory cell infiltration in the centrilobular region was detected in males receiving 300 or 1000 mg/kg/day.
Gall bladder: epithelial ulceration with fibrosis and minimal epithelial hyperplasia were noted in one male receiving 1000 mg/kg/day; another male in the same group showed minimal epithelial hyperplasia and minimal inflammatory cell infiltration in the lamina propria. The dog showing the ulceration had the highest ALT and AST values pre-dose and thus it is possible that there was slight, sub-clinical, damage in the gall bladder prior to treatment.
Bone marrow sternum: increased in degree and incidence of cellularity of the marrow was identified in females treated at 1000 mg/kg/day.
Spleen: haemosiderosis was marginally increased in males receiving 300 or 1000 mg/kg/day.
Adrenal: cortical vacuolation of the zona fasciculata and reticularis was increased both in degree and incidence in males and only in degree in females receiving 1000 mg/kg/day.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 75 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical signs

haematology

organ weights and organ / body weight ratios

other: Blood chemistry (protein levels, creatine levels, etc), macropathology (liver), micropathology (liver)

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (nominal)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Findings

Mortality

Female animal 992, receiving 1000 mg/kg/day, was sacrificed during week 40, after an eight week recovery period, due to bodyweight loss and elevated biochemical parameters in week 32, indicating severe liver dysfunction.

 

Clinical signs

Loss of appetite, mainly in females, and pale faeces were notes only in animals receiving 1000 mg/kg/day.

 

Bodyweight

Overall group mean bodyweight gain was lower for both males and females receiving 1000 mg/kg/day.

Group mean bodyweight gain was also marginally lower than controls for males receiving 75 or 300 mg/kg/day. However the decrease was not statistically significant and no dosage-relationship was apparent for the inter-group differences among these males.

 

Table 5.3-25:  52-week dietary study in dogs: group mean bodyweight gain

	0 mg/kg/day	75 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
Males	6.1 kg	5.0 kg	4.9 kg	4.4 kg
Females	4.9 kg	5.8 kg	5.1 kg	3.2 kg*
* p≤0.05 (William’s test)

 

Food consumption

Lower average weekly food intake was recorded for females receiving 1000 mg/kg/day.

 

Table 5.3-26   52-week dietary study in dogs: mean weekly food consumption (g/animal)

	0 mg/kg/day	75 mg/kg/day	300 mg/kg/day	1000 mg/kg/day
Males	2801	2808	2751	2759
Females	2591	2736	2749	2389*
* p≤0.05 (William’s test)

 

Ophthalmic examination

The ophthalmoscopic findings were within normal limits for animals of this age, breed and strain and unrelated to treatment with IR5878.

 

Haematology

Lower group mean haematocrit (Htc), erythrocytes (RBC) and haemoglobin (Hb) were noted in all animals treated at 1000 mg/kg/day from week 13 for females and from week 26 for males.

Higher group mean platelet counts were evident for both sexes receiving 1000 mg/kg/day from week 13.

Higher reticulocyte counts, slightly higher mean cell haemoglobin (MCH) and mean cell volume (MCV) values were recorded in females receiving 1000 mg/kg/day.

 

Table 5.3-27   52-week dietary study in dogs: haematology – group mean values

Sex		Males				Females
Parameter	Time  (week)	Dosages (mg/kg/day)				Dosages (mg/kg/day)
		Control	75	300	1000	Control	75	300	1000
Htc (L/L)	Pre-dose	0.393	0.399	0.401	0.386	0.402	0.394	0.403	0.404
	13	0.391	0.399	0.402	0.398	0.427	0.417	0.428	0.401
	26	0.421	0.421	0.408	0.388	0.458	0.443	0.425	0.383**
	52	0.492	0.484	0.474	0.430**	0.496	0.497	0.474	0.440*
RBC (x1012 /L)	Pre-dose	5.64	5.84	5.84	5.74	5.92	5.79	5.95	5.90
	13	5.75	5.96	5.88	5.76	6.48	6.30	6.34	5.74*
	26	6.26	6.25	5.93	5.67	6.98	6.61	6.34*	5.56**
	52	7.10	7.05	6.71	6.13**	7.40	7.18	6.79	6.16**
Hb (g/dL)	Pre-dose	12.7	12.8	13.0	12.3	12.9	12.8	13.0	13.0
	13	13.2	13.4	13.7	13.4	14.4	14.2	14.7	13.6
	26	14.5	14.3	14.0	13.2	15.7	15.1	14.8	13.1**
	52	16.4	16.3	16.2	14.4**	16.9	16.8	16.0	14.6*
Retic (%)	Pre-dose	0.59	0.83	0.97	0.69	0.81	0.91	0.69	0.82
	13	0.48	0.74	0.89	0.80	0.62	0.60	0.55	0.96
	26	0.54	0.56	0.92	0.82	0.57	0.54	0.51	0.88
	52	0.73	0.75	1.09	0.88	0.87	1.08	0.60	1.74**
MCH (pg)	Pre-dose	22.6	21.9	22.3	21.5+	21.8	22.1	21.8	22.1
	13	23.0	22.5	23.3	23.3	22.3	22.6	23.3	23.7*
	26	23.2	23.0	23.6	23.3	22.5	22.8	23.4	23.6
	52	23.1	23.1	24.1	23.4	22.8	23.4	23.6	23.7
MCV (fL)	Pre-dose	69.8	68.3	68.8	67.2+	68.0	68.2	67.7	68.6
	13	68.1	67.0	68.4	69.2	66.1	66.3	67.7	69.9*
	26	67.3	67.4	68.7	68.5	65.7	67.0	67.3	69.1
	52	69.3	68.8	70.6	70.2	67.0	69.2	69.9	71.5*
Plt (x 109/L)	Pre-dose	310	324	371	380	352	353	334	302
	13	307	326	377	448**	326	335	350	417
	26	293	313	349*	413**	331	328	363	456*
	52	296	332	364	429**	375	370	369	491
+=p≤0.05 (Student’s t test) ** = p≤0.01 (William’s test) * = p≤0.05 (William’s test)

 

Blood chemistry

Higher group mean alkaline phosphatase (ALP) values were noted for dogs receiving 1000 mg/kg bw/day from week 13, and to a lesser extent for animals receiving 300 mg/kg/day. Dosage-related lower group mean aspartate amino-transferase (AST) values were noted for females receiving 1000 and 300 mg/kg/day in week 13 andweek 52 the values of AST were still lower, but without apparent dosage-relationship. Male and female dogs receiving 1000 mg/kg/day from week 13 showed lower values for total protein, albumin and A/G ratio. Lower calcium values were recorded from week 13 for dogs treated at 1000 mg/kg/day; in week 26 alteration in calcium levels were found also in dogs treated with the lower dose, but the effect was no more evident at week 52. Dose-related lower creatinine values were noted in males receiving 1000 or 300 mg/kg/day, starting from week 13.

 

Table 5.3-28   52-week dietary study in dogs: blood chemistry - group mean values

Sex		Males				Females
Parameter	Time  (week)	Dosages (mg/kg/day)				Dosages (mg/kg/day)
		Control	75	300	1000	Control	75	300	1000
ALP (U/L)	Pre-dose	161	183	177	149	140	158	144	122
	13	121	147	227*	598**	115	148	234	512**
	26	96	121	261**	657**	89	140	269	512**
	52	79	96	296**	582**	71	122	299*	301*
AST (U/L)	Pre-dose	32	35	31	38	37	35	35	36
	13	36	33	33	37	46	39	33*	29**
	26	43	46	62	50	49	47	34	33*
	52	46	44	52	47	48	42	34	39
Creatinine (μmol/L)	Pre-dose	58	56	56	57	59	55	59	58
	13	73	67	64*	60**	69	71	73	65
	26	82	75	68**	67**	74	72	73	66
	52	84	78	71*	66**	76	73	73	68
Total protein (g/L)	Pre-dose	53	54	52	51	52	54	53	53
	13	53	54	51	46**	56	56	52	50*
	26	54	55	50	44**	57	56	50*	50*
	52	60	57	55	51**	60	59	56	52 *
Albumin (g/L)	Pre-dose	25	26	25	23	26	25	25	25
	13	29	29	27	23**	30	31	29	25**
	26	30	30	26**	22**	32	31	28**	26**
	52	30	32	28	24**	32	31	30	25**
A/G (%)	Pre-dose	0.86	0.94	0.92	0.85	1.00	0.89	0.90	0.89
	13	1.18	1.18	1.11	0.98*	1.19	1.20	1.27	1.01*
	26	1.22	1.23	1.10	1.01**	1.32	1.23	1.21	1.08*
	52	1.02	1.23	1.03	0.92	1.17	1.10	1.14	0.96
Calcium (mmol/L)	Pre-dose	3.10	3.21	3.12	3.12	3.11	3.09	3.11	3.08
	13	3.03	2.97	2.92	2.80**	3.02	2.97	2.98	2.86**
	26	2.94	2.92	2.75*	2.75*	3.01	2.83*	2.80*	2.83**
	52	2.78	2.81	2.66	2.61*	2.82	2.76	2.75	2.62**
** = p≤0.01 (William’s test) * = p≤0.05 (William’s test)

 

Urinalysis

No treatment-related changes were observed.

 

Organ weights

Group mean (relative to terminal body weight for females) and individual liver weights were higher for dogs of both sexes treated at 1000 mg/kg/day and for females receiving 300 mg/kg/day in comparison with controls. In the high dose group (1000 mg/kg/day9 a significant higher mean kidney weight (relative to terminal body weight) was found for females.

Individual and group mean prostate weights were lower for males treated at 1000 mg/kg/day, but the decrease was correlated to the lower body weight of these dogs, and considered of no toxicological importance in absence of any macroscopic or microscopic pathology findings.

 

Table 5.3-29   52-week dietary study in dogs: organ weights – group mean values (g)

Sex		Males				Females
Parameter		Dosages (mg/kg/day)				Dosages (mg/kg/day)
		Control	75	300	1000	Control	75	300	1000
Liver weight	Absolute	412	437	467	617**	359	409	491	479
	Adjusted	-	-	-	-	359	389	486**	513**
Kidney weight	Absolute	62.2	65.6	62.2	72.0	52.3	50.6	54.8	57.9
	Adjusted	-	-	-	-	52.3	48.3	54.2	61.7**
Spleen weight	Absolute	120.3	127.5	89.9	73.4*	95.1	100.8	102.5	93.7
Prostate weight	Absolute	9.975	9.273	10.246	5.507*	-	-	-	-
**=p≤0.01 (Williams’ test) *=p≤0.05 (Williams’ test)

 

Macroscopic pathology

Enlarged of the liver was noted in the majority of animals treated at 1000 mg/kg/day and in one male and two females receiving 300 mg/kg/day.

 

Microscopic pathology

Liver: generalised hepatocyte hypertrophy and prominent pigmented Kupffer cells were observed in dogs receiving 300 or 1000 mg/kg/day with a dosage-relationship. An increase in degree of periportal inflammatory cell infiltration was noted in dogs of both sexes treated at 1000 mg/kg/day, and to a lesser degree in animals receiving 300 mg/kg/day. In addition, increased incidence and degree of vascular/perivascular inflammatory cell infiltration in the centrilobular region was detected in males receiving 300 or 1000 mg/kg/day.

Gall bladder: epithelial ulceration with fibrosis and minimal epithelial hyperplasia were noted in one male receiving 1000 mg/kg/day; another male in the same group showed minimal epithelial hyperplasia and minimal inflammatory cell infiltration in the lamina propria. The dog showing the ulceration had the highest ALT and AST values pre-dose and thus it is possible that there was slight, sub-clinical, damage in the gall bladder prior to treatment.

Bone marrow sternum: increased in degree and incidence of cellularity of the marrow was identified in females treated at 1000 mg/kg/day.

Spleen: haemosiderosis was marginally increased in males receiving 300 or 1000 mg/kg/day.

Adrenal: cortical vacuolation of the zona fasciculata and reticularis was increased both in degree and incidence in males and only in degree in females receiving 1000 mg/kg/day.

 

Table 5.3-30   52-week dietary study in dogs: micropathology data

Sex		Males				Females
Parameter		Dosages (mg/kg/day)				Dosages (mg/kg/day)
		Control	75	300	1000	Control	75	300	1000
Liver
Generalised hepathocyte hypertrophy	Minimal	0	0	1	0	0	0	2	0
	Slight	0	0	3	2	0	0	2	3
	Moderate	0	0	0	2	0	0	0	0
	Total	0	0	4*	4*	0	0	4*	3*
Prominent pigmented Kupffer cells	Slight	0	0	2	2	0	1	2	2
	Moderate	0	0	0	1	0	0	0	0
	Total	0	0	2	3	0	1	2	2
Periportal inflammatory cell infiltration	Minimal	0	0	2	0	0	1	1	0
	Slight	0	0	0	1	0	0	0	1
	Total	0	0	2	1	0	1	1	1
Vascular/perivascular inflammatory cell, centrilobular	Minimal	1	2	2	2	1	2	1	0
	Slight	0	0	1	1	0	0	0	1
	Moderate	0	0	0	0	1	0	0	0
	Total	1	2	3	3	2	2	1	1
Bone marrow
Increased cellularity  of marrow	Minimal	0	0	0	0	0	1	1	1
	Slight	0	0	0	0	1	0	0	2
	Total	0	0	0	0	1	1	1	3
Spleen
Haemosiderosis	Minimal	0	0	1	0	0	1	2	0
	Slight	0	1	1	3	1	1	2	1
	Moderate	0	0	0	0	1	1	0	0
	Total	0	1	2	3	2	3	4	1
Adrenals
Cortical vacuolation zona fasciculata/reticularis	Minimal	1	3	2	2	1	1	1	1
	Slight	0	0	0	0	2	0	0	1
	Moderate	0	0	0	2	0	0	0	1
	Total	1	3	2	4	3	1	1	3
* = p <0.05 (Fisher’s Exact Test)

 

Decedent animal

From approximately week 26 one female receiving 1000 mg/kg/day showed large weight loss and some altered biochemistry values. Thus treatment of this animal ceased after 32 weeks of dose administration. The animal was allowed an 8 week recovery period during which time it showed recovery from the in-life treatment-related effects. In week 40 the animal was sacrificed.

During treatment haematological parameters were similar to those of the other females treated at 1000 mg/kg/day, but at the end of recovery period all altered parameters recovered.

The blood chemistry parameters of this animal showed similar alterations seen for females treated at 1000 mg/kg/day, but during recovery period the values gradually returned to expected ranges.

Organ weights recorded at sacrifice were within the expected background range.

The macroscopic examination showed pale kidneys, pale liver and the presence of a pituitary cyst. Slight prominent pigmented Kupffer, and minimal adrenal cortical vacuolation (zona fasciculata and reticularis) were noted at histopathology.

It was not possible to determine a specific cause of deterioration of health status for this decedent animal receiving 1000 mg/kg/day.

Conclusions:

IR5878 administered by oral route (by capsules) to dogs for 52 consecutive weeks at the doses of 1000, 300 and 75 mg/kg/day induced toxicity at 1000 mg/kg/day to the liver, being the main target organ. In addition, there was evidence from this study that the adrenals and the haemapoetic system were also target organs of IR5878. Despite toxicity being evident at 1000 mg/kg/day, the majority of the animals tolerated 52-weeks of treatment.
Evidence of minor liver toxicity at 300 mg/kg/day induced the choice of the dose of 75 mg/kg/day as NOAEL (No Observed Adverse Effect Level).

Executive summary:

In order to evaluate the effects of repeated administrations of IR5878 to Beagle dogs over a period of 52 weeks, groups of 4 male and 4 female Beagle dogs received IR5878 by oral capsule administration, at concentrations of 0, 75, 300 or 1000 mg/kg/day for 52 weeks.

IR5878 induced toxicity at 1000 mg/kg/day to the liver, being the main target organ. In addition, there was evidence from this study that the adrenals and the haemapoetic system were also target organs of IR5878. Despite toxicity being evident at 1000 mg/kg/day, the majority of the animals tolerated 52-weeks of treatment. Evidence of minor liver toxicity at 300 mg/kg/day induced the choice of the dose of 75 mg/kg/day as NOAEL.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

75 mg/kg bw/day

Study duration:

chronic

Species:

dog

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

Not required since IR5878 is not a volatile compound.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Quality of whole database:

Not required since IR5878 is not a volatile compound.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 410 (Repeated Dose Dermal Toxicity: 21/28-Day Study)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3200 (Repeated Dose Dermal Toxicity -21/28 Days)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.9 (Repeated Dose (28 Days) Toxicity (Dermal))

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: JMAFF 12 NohSan No. 8147, Guideline 2-1-10

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test material:
IR5878
Batch number: G 009/02
Purity: 98.56 ± 0.19 %

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals:
Species and Strain: Male and Female Crl:CD (SD)IGS BR rats
Age: 62 to 69 days
Body weight: 321 ÷ 367 g (males) and 213 ÷ 238 g (females)
Housing: one animal per cage of polycarbonate body with a stainless mesh lid.
Feed: free access to a standard rodent diet (Rat and Mouse No. 1 Maintenance Diet from Special Diets Services Ltd., Witham, Essex, England), except overnight before routine blood sampling. Potable water was freely available.

Environmental conditions:
Temperature and humidity measured during the study were 19 ÷ 23°C and 40 ÷ 70%, respectively.
Light: 12 hours of continuous light and 12 hours of continuous dark.

Type of coverage:

semiocclusive

Details on exposure:

One group of 10 male and 10 female Crl:CD (SD)IGS BR rats received IR5878 topically, by means of a 6-hour period of semi-occluded application, at a dosage of 1000 mg/kg/day for 4 consecutive weeks.
At the end of the daily treatment period the dressing was carefully removed and the exposed area was cleansed with saline and dabbed- dry with a disposable towel to avoid the oral ingestion during grooming.
The application site remained non-occluded until the next administration.
A similarly constituted control group received the vehicle (purified water) obtained by reverse osmosis, at the same volume-dosage (2 mL/kg bw) and at the same period of occlusion.

Duration of treatment / exposure:

One group of 10 male and 10 female Crl:CD (SD)IGS BR rats received IR5878 topically, by means of a 6-hour period of semi-occluded application, at a dosage of 1000 mg/kg/day for 4 consecutive weeks. At the end of the daily treatment period the dressing was carefully removed and the exposed area was cleansed with saline and dabbed- dry with a disposable towel to avoid the oral ingestion during grooming. The application site remained non-occluded until the next administration. A similarly constituted control group received the vehicle (purified water) obtained by reverse osmosis, at the same volume-dosage (2 mL/kg bw) and at the same period of occlusion.

Frequency of treatment:

One group of 10 male and 10 female Crl:CD (SD)IGS BR rats received IR5878 topically, by means of a 6-hour period of semi-occluded application, at a dosage of 1000 mg/kg/day for 4 consecutive weeks.

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

One group of 10 male and 10 female Crl:CD (SD)IGS BR rats received IR5878 topically, by means of a 6-hour period of semi-occluded application, at a dosage of 1000 mg/kg/day for 4 consecutive weeks

Control animals:

yes

Details on study design:

. A similarly constituted control group received the vehicle (purified water) obtained by reverse osmosis, at the same volume-dosage (2 mL/kg bw) and at the same period of occlusion.

Observations and examinations performed and frequency:

Clinical condition and dermal responses particularly in the application site were performed daily; bodyweight and food consumption were recoreded at day 0, weekly during the treatment and before necrospsy; ophthalmic examination was carried out before treatment commenced anf during week 4; haematology, blood chemistry, organ weight, macroscopic and microscopic pathology investigations were carried out according to the guideline indications.

Sacrifice and pathology:

Organ weights
Bodyweight-relative spleen weights were slightly increased for treated males. Females were not affected.


Macroscopic pathology
Macroscopic examination revealed an increased incidence of pale areas on the livers in treated males.

Microscopic pathology
There were no microscopic findings that were attributable to the administration of IR5878.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related systemic signs associated with dosing and no animals died prematurely. Changes at the dermal application site (slight to well-defined erythema, eschar-formation and exfoliation) occurred on occasion in a few treated animals, but it was not clearly attributable to treatment.

Dermal irritation:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related systemic signs associated with dosing and no animals died prematurely. Changes at the dermal application site (slight to well-defined erythema, eschar-formation and exfoliation) occurred on occasion in a few treated animals, but it was not clearly attributable to treatment.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Overall bodyweight gain was slightly increased for treated females, when compared to that of the control. Males were unaffected.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

effects observed, treatment-related

Description (incidence and severity):

Overall food conversion efficiency was slightly increased for treated females, when compared to that of the controls. This is attributed to the increased bodyweight gain in these animals. Males were unaffected.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Organ weights
Bodyweight-relative spleen weights were slightly increased for treated males. Females were not affected.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Macroscopic examination revealed an increased incidence of pale areas on the livers in treated males.

Histopathological findings: non-neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

dermal irritation

food consumption and compound intake

food efficiency

haematology

mortality

ophthalmological examination

organ weights and organ / body weight ratios

Key result

Critical effects observed:

no

Conclusions:

A daily 6-hour semi-occluded dermal application of IR5878 to CD rats for 4 weeks was well-tolerated, producing no toxicologically significant change or any clear response at the dermal application site.
The NOAEL (No Observed Adverse Effect Level) in this study was considered to be 1000 mg/kg/day.

Executive summary:

In order to assess the systemic toxic potential of IR5878 following dermal application (semi-occlusive 6 hours/day) to rats for four weeks, one group of 10 male and 10 female Crl:CD (SD)IGS BR rats received IR5878 topically, by means of a 6-hour period of semi-occluded application, at a dosage of 1000 mg/kg/day for 4 consecutive weeks. A similarly constituted control group received the vehicle (purified water) obtained by reverse osmosis, at the same volume-dosage (2 mL/kg bw) and at the same period of occlusion.

IR5878 was well-tolerated, producing no toxicologically significant change or any clear response at the dermal application site. The NOAEL in this study was considered to be 1000 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day