Cuprate(4-), [C-(aminosulfonyl)-C-[[[2-[[4-chloro-6-[(2,5-disulfophenyl)amino]-1,3,5-triazin-2-yl]amino]ethyl]amino]sulfonyl]-29H,31H-phthalocyanine-C,C-disulfonato(6-)-N29,N30,N31,N32]-, sodium salts

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:

10 June 1993 to 22 July 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

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)

Version / remarks:

The OECO Guidelines for Testing of Chemicals No. 407 "repeated dose oral toxicity - rodent 28-day or 14-day study".

Deviations:

not specified

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Method B7 of EEC Commission Directive 92/69/EEC.

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Cuprate(4-), [C-(aminosulfonyl)-C-[[[2-[[4-chloro-6-[(2,5-disulfophenyl)amino]-1,3,5-triazin-2-yl]amino]ethyl]amino]sulfonyl]-29H,31H-phthalocyanine-C,C-disulfonato(6-)-N29,N30,N31,N32]-, sodium salts

EC Number:

689-188-9

Cas Number:

149343-84-0

Molecular formula:

C32H24N12O8S4Cu to C76H56N28O32S12Cl4Na8Cu

IUPAC Name:

Cuprate(4-), [C-(aminosulfonyl)-C-[[[2-[[4-chloro-6-[(2,5-disulfophenyl)amino]-1,3,5-triazin-2-yl]amino]ethyl]amino]sulfonyl]-29H,31H-phthalocyanine-C,C-disulfonato(6-)-N29,N30,N31,N32]-, sodium salts

Test material form:

solid: particulate/powder

Details on test material:

Identification: JPR Blue 100
Description: Blue powder
Chemical Name: Cuprate(2-),[2-[[4-chloro-6-[[2-[(29H,31H-phthalocyaninylsulphonyl)amino]ethyl]amino]-1,3,5-triazin-2-yl]amino]-1,4-benzenedisulphonato(4-)-N29,N30,N31,N32]-mono(or bis)aminosulphonyl mono (or di)sulpho derivs., sodium salts.
Lot Number: 303001
Purity: 95.2%
Major Impurities: H2o, NaCl, Na2SO4
Date Received: 30 March 1993
Container: Opaque plastic jar x 7
Storage conditions: Room temperature

Specific details on test material used for the study:

No further details specified in the study report.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Speices/strain selected in accordance with the test guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

A sufficient number of male and female Sprague-Dawley CD strain rats were obtained from Charles River (U.K.) Limited, Manston, Kent. 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 the treatment the males weighed 137 to 167 g, and the females weighed 128 to 167 g, and were approximately five to six weeks old.
The animals were housed in groups of five by sex in polypropylene grid floor cages suspended over trays lined with absorbent paper. The animals were allowed free access to food, except during urine collection when food was withdrawn overnight.
A pelleted diet (Rat and Mouse SQC Expanded Diet No. 1, Special Diet Services Limited, Witham, Essex, U.K.) was used. The animals were allowed free access to mains water from polycarbonate bottles attached to the cage. The diet and drinking water were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Safepharm 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 the mean temperatures and humidities were included in the study records.
During the study the room temperature was maintained within the range of 18 – 24 °C and relative humidity of 39 - 75%. On several occasions the room temperature or humidity fell below protocol limits but this was considered not to have affected the purpose or integrity of the study.
The animals were randomly allocated to dose groups using random letter tables and the group mean bodyweights were then determined to ensure similarity between the dose 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 these laboratories. Colour coded cage labels were used to assist recognition of dose groups.

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The test material was administered daily, for twenty-eight consecutive days, by gavage using a metal cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 ml/kg/day of distilled water.

Vehicle:

water

Details on oral exposure:

For the purpose of this study the test material was prepared at the appropriate concentrations as a solution in distilled water. The stability and homogeneity of the test material formulations were determined by Safepharm Analytical Laboratory. Formulations were therefore prepared weekly and stored at approximately 4 °C in the dark.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples were taken of each test material formulation and were analysed for concentration of JPR Blue 100 at Safepharm Analytical Laboratory.
The results indicate that the prepared formulations were within ± 9% of the nominal concentration.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

15 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

150 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Six groups, each of ten rats (five males and five females) were dosed

Control animals:

yes, concurrent vehicle

Details on study design:

The dose levels were chosen based on the results of a range-finding study. The oral route was selected by the study sponsor as a possible route of human exposure and the results of the study are believed to be of value in predicting the likely toxicity of the test material to man.
The test material was administered daily, for twenty-eight consecutive days, by gavage using a metal cannula attached to a disposable plastic syringe. Control animals were treated in an identical manner with 5 ml/kg/day of distilled water. Animals from satellite groups 5 and 6 were maintained for a further 14 days treatment-free period following termination of treatment.
The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

Positive control:

Not required

Examinations

Observations and examinations performed and frequency:

Clinical Signs
All animals were examined for overt signs of toxicity, ill-health or behavioural change immediately before dosing and one and five hours after dosing during the working week. On Day 28, dosing was delayed slightly due to the blood sampling procedure and the final observation was carried out as late in the working day as possible. Animals were observed immediately before dosing and one hour after dosing at weekends. During the treatment-free period, satellite group animals were observed twice daily, morning (am) and afternoon (pm). Satellite group animals were observed once daily at weekends. All observations were recorded.

Bodyweight
Individual bodyweights were recorded on day 0 (the day before the start of treatment) and on days 7, 14, 21 and 28 and, in the case of satellite group animals, on days 35 and 42. Bodyweights were also recorded at necropsy.

Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study.

Water Consumption
Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

Laboratory Investigations
Haematological and blood chemical investigations were performed on all animals from groups 1 to 4 at the end of the treatment period.
Parameters showing possible treatment-related changes in these animals were examined in satellite group animals at the end of the treatment-free period. Blood samples were obtained from the lateral tail vein. A few repeat samples were also obtained prior to necropsy by cardiac puncture. Animals were not fasted prior to sampling.
Urinalysis was performed on all animals from groups 1 to 4 during week 4 and on animals from groups 5 and 6 during the final week of the study. Urine samples were collected over a period of approximately sixteen hours by housing the rats in metabolism cages.
Animals were maintained under conditions of normal hydration during collection but without access to food.

Haematology
The following parameters were measured on blood collected into tubes containing potassium EDTA anti-coagulant:
Haematocrit (Hct); Haemoglobin (Hb); Erythrocyte count (RBC); Total leucocyte count (WBC); Differential leucocyte count; Platelet count (PLT); Erythrocyte indices (mean corpuscular haemoglobin (MCH); mean corpuscular volume (MCV); mean corpuscular haemoglobin concentration (MCHC)); Methaemoglobin; Reticulocyte count
Clotting time (CT) was assessed by 'Hepato Quick' time using samples collected into sodium citrate solution (0.11 mol/l).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anticoagulant:
Blood urea; Total protein; Albumin; Albumin/ globulin ratio (by calculation); Sodium; Potassium; Chloride; Calcium; Inorganic phosphorus; Alkaline phosphatase (AP); Alanine aminotransferase (ALAT); Aspartate aminotransferase (ASAT); Glucose; Gamma glutamyl transpeptidase (ƴGT); Triglycerides; Total cholesterol; Total bilirubin; Creatinine

Urinalysis
The following parameters were measured on freshly collected urine.
Volume; Specific gravity; pH; Protein; Glucose; Ketones; Bilirubin; Urobilinogen; Reducing substances; Blood

Sacrifice and pathology:

Pathology
On completion of the dosing period or, in the case of satellite group animals, at the end of the treatment-free period; all animals were killed by intravenous overdose of sodium pentobarbitone (Sagatal 60 mg/ml; May and Baker Limited, Oagenham, Essex, UK) followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Organ Weights
The following organs from animals that were killed at the end of the study, dissected free from fat, were weighed before fixation:
Adrenals; Brain; Gonads; Heart; Kidneys; Liver; Pituitary; Spleen

Histopathology
Samples of the following tissues were removed from all animals and preserved in 10% buffered formalin.
Adrenals; Aorta (thoracic); Bone & Bone Marrow (femur); Brain; Caecum; Colon; Duodenum; Eyes; Gross lesions; Heart; Ileum; Jejunum; Kidneys; Liver; Lungs; Lymph nodes (cervical and mesenteric); Muscle (skeletal); Oesophagus; Ovaries; Pancreas; Pituitary; Prostate; Rectum; Salivary glands; Sciatic nerve; Seminal vesicles; Skin (hind limb); Spleen; Stomach; Testes; Thymus; Thyroid/parathyroid; Trachea; Urinary bladder; Uterus

All tissues were despatched to Experimental Pathology Services, Willow Court, Netherwood Road, Rotherwas, Hereford, U.K. for processing. The following preserved tissues from all test and control group animals (groups 1 to 6) were prepared as paraffin blocks, sectioned at nominal thickness of 5 um and stained with haematoxylin and eosin.
Adrenals; Spleen; Heart; Kidneys; Liver; Testes; Lymph nodes (cervical and mesenteric)
Macroscopically observed lesions were also processed.

Initially, high dose microscopic examination was performed on control and tissues (groups 1 and 4) only but since there were indications of treatment-related changes in the spleen, kidneys and mesenteric lymph nodes examination was subsequently extended to include sections of these tissues from all animals in the remaining dose groups.

Other examinations:

Not specified

Statistics:

Data were processed to give group mean values and standard deviations where appropriate.
Absolute and relative organ weights, haematological and blood chemical data were analysed by one way analysis of variance incorporating 'F-max' test for homogeneity of variance. Data showing heterogeneous variances were analysed using Kruskal Wallis non-parametric analysis of variance and Mann Whitney U-Test.
Probability values were calculated as:
p < 0.001 ***
p < 0.01 **
p < 0.05 *
p ≥ 0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinically observable signs of toxicity were confined to high dose animals. Pallor of the extremities was first observed on day 8 and, from day 17 onwards, all high dose and satellite high dose animals of either sex appeared pale in comparison with controls. Pallor of the extremities persisted throughout the fourteen day recovery period following cessation of treatment although it was noticeably diminished for most animals towards the end of the study.
Intermediate and low dose animals showed no clinically observable signs of toxicity during the study.
Incidents of blue staining of the fur and, occasionally, around the mouth by the test material were observed during the study whilst dark faeces were noted for all animals treated with the test material from day 2 onwards. These are common findings when a coloured test material is administered to rats by gavage and are of no toxicological significance. Red/brown staining of the snout was recorded for one intermediate dose female on the final two days of dosing but, in isolation, was considered not to be indicative of test material toxicity.

Mortality:

no mortality observed

Description (incidence):

There were no deaths during the study.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

All animals showed normally expected bodyweight development during the study. Animals treated with the test material showed similar bodyweight gains to controls.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no adverse effect on food consumption during the study.

Food efficiency:

no effects observed

Description (incidence and severity):

Food efficiency in animals treated with the test material was comparable with that seen in controls.

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

no effects observed

Description (incidence and severity):

Daily visual inspection of water bottles failed to reveal any overt intergroup difference in water consumption.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Intermediate and high dose females showed a slight but statistically significant reduction in haemoglobin concentration compared with controls. None of the individual values were abnormally low for rats of the strain and age used in this study (normal range; 10.1 16.9 g/dl) and there was no associated reduction in erythrocyte numbers but, in view of the microscopic splenic changes identified for high dose males, a treatment-related effect cannot be entirely ruled out. In addition, haemoglobin concentration remained slightly lowered in satellite high dose females following a further fourteen days without treatment. Again, none of the individual values were outside the normally expected range for this parameter but, since there were no residual microscopic splenic changes in satellite high dose males, this reduction is of dubious toxicological significance.
No treatment-related effects were detected for intermediate and high dose males or for low dose animals of either sex.
High dose males showed a slight but statistically significant reduction in mean corpuscular haemoglobin concentration compared with controls but, in the absence of any effect on the directly measured parameters, this was probably of no toxicological importance. High dose males also showed a slight reduction in clotting time but as none of the individual values were abnormally low for rats of the strain and age used in this study (normal range : 21 - 39 sees) this was considered not to be toxicologically significant. The remaining statistically significant intergroup differences, a reduction in female haematocrit and a reduced male lymphocyte count, were confined to intermediate dose group animals. These changes were not dose-related and, as such, were considered not to be treatment-related .

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related effects were detected in the blood chemical parameters measured.
Intermediate and high dose females showed a statistically significant increase in plasma potassium concentration compared with control values but none of the individual values were outside the normally expected range for rats of the strain and age used in this study (normal range; 2.95- 6.27 mmol/l) and, in the absence of any dose-relationship, this increase was of no toxicological importance. Plasma cholesterol was slightly elevated in high dose males but, in the absence of any histopathological evidence of hepatic change, this of no toxicological following a further increase was considered to be fortuitous and significance. Both high dose females and, fourteen days without treatment, satellite high dose females showed a reduced plasma alanine aminotransferase concentration. A reduction in this parameter is, however, of no toxicological importance. The remaining statistically significant intergroup differences, an increase in male alkaline phosphatase together with an increase in female bilirubin and a reduction in female inorganic phosphorus, were confined to intermediate dose group animals. These were not dose-related and, as such, were considered not to be treatment-related.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

A marked haemoglobinuria was detected for all high dose animals towards the end of the dosing period and, for all satellite high dose animals, towards the end of the recovery period following a further twelve days without treatment. Such changes are often seen when a highly coloured test material is administered by gavage and may result from the presence of test material and/or its metabolite(s) in the urine. In this instance, however, a treatment-related effect cannot be entirely ruled out in view of the reduced haemoglobin levels seen for females at this dose level.
No treatment-related effects were detected at the remaining dose levels.
Urine micturated by high dose and, to a lesser extent, intermediate dose animals was coloured blue/green by the test material and/or its metabolite(s). This is a common finding when a coloured test material is administered to rats, by gavage, and is of no toxicological importance although the discolouration of the urine masked many of the semi-quantitative analyses performed at the high dose.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

High dose animals of either sex showed a slight but statistically significant increase in relative spleen weight compared to controls. Absolute spleen weight was also elevated for these animals although statistical significance was not achieved for the females. The majority of the individual values were within the normally expected ranges for rats of the strain and age used in this study (normal ranges; male absolute spleen weight, 0.5294 - 1.2132 g, female absolute spleen weight 0.4236 - 0.9049 g, male relative spleen weight 0.1536 - 0.3470%, female relative spleen weight 0.2000 - 0.3896%) but, in view of the morphological splenic changes seen histopathologically, a treatment-related effect cannot be ruled out at this dose level. Absolute spleen weight was slightly elevated for satellite high dose females at the end of the fourteen day recovery period but no such intergroup difference existed when expressed relative to bodyweight and this increase was, therefore, considered not to be toxicologically significant. High dose females showed a statistically significant increase in kidney weight, both absolute and relative to bodyweight, compared to controls. Again, none of the individual values were abnormally high for rats of the strain and age used in this study (normal ranges; absolute kidneys weight 1.3735 - 1.9828 g, relative kidney weight 0.6022 - 0.8579%) but, in view of the renal changes detected histopathologically, a treatment related effect again cannot be ruled out. No such effect again was apparent for satellite high dose females following a further fourteen days without treatment.
No treatment-related organ weight changes were apparent at the remaining dose levels.
High dose females showed a slight but statistically significant increase in liver weight, relative to bodyweight, compared to controls. None of the individual values were outside the normally expected range for this parameter (normal range; 3.2075 - 4. 5719%) and, in the absence of any histopathological evidence of hepatic change, this increase was considered to be fortuitous and of no toxicological statistically importance. Satellite high dose males showed a significant reduction in pituitary weight, both absolute and relative to bodyweight, but in the absence of a similar finding at the end of the twenty-eight day dosing period, such a reduction was considered not to be toxicologically significant. The remaining statistically significant intergroup differences, an increase in absolute brain and pituitary weight in intermediate dose males and females respectively together with a reduction in relative kidney weight for low dose males, were not dose-related and, as such, were considered not to be treatment-related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No toxicologically significant macroscopic abnormalities were observed at terminal kill.
Extensive blue staining of the viscera, particularly the kidneys and mesenteric lymph nodes, was observed for all high dose animals at necropsy. Blue staining was confined to the kidneys and mesenteric lymph nodes at the intermediate dose level. Staining of the viscera by the test material was also observed for all satellite high dose animals at terminal kill following a further fourteen days without treatment. Such staining is common when a coloured test material is administered to rats, by gavage, and is not attributable to test material toxicity.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related changes were observed in the spleen, kidneys, and mesenteric lymph nodes:
SPLEEN: An increased severity of extramedullary haemopoiesis was observed for male rats dosed at 1000 mg active ingredient/ kg/ day.
A similar effect was not convincingly observed for male rats in the remaining treatment groups and the severity of extramedullary haemopoiesis amongst satellite 1000 mg active ingredient/kg/day rats was no different from that reported for satellite control rats following an additional fourteen days without treatment.
KIDNEYS: Tubular epithelial degeneration and accumulations of blue pigment were observed in the renal tubules of rats of either sex receiving 1000 mg active ingredient/ kg/day. In addition, female rats dosed at 1000 mg active ingredient/ kg/day demonstrated an increased severity of tubular basophilia in association with treatment. Rats from the remaining treatment groups were unaffected. Tubular degeneration was no longer evident amongst satellite 1000 mg active ingredient/kg/day male rats, and was reduced in severity for female rats after an additional fourteen days without treatment. For male rats, the incidence and severity of tubular basophilia were increased following the fourteen day recovery period, the condition remaining unaffected for satellite group female rats. The extent and severity of pigment accumulations were not affected for either sex following an additional fourteen days without treatment.
MESENTERIC LYMPH NODES: Accumulations of blue pigment and foamy macrophages were observed in relation to treatment for male and for female rats dosed at 1000 mg active ingredient/ kg/day. The presence of either condition was unaffected for satellite group animals following an additional fourteen days without treatment.
All remaining morphological changes were those commonly observed in laboratory maintained rats of the age and strain employed and, since there were no differences in incidence or severity between control and treatment groups, all were considered to be without toxicological significance.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Details on results:

The oral administration of JPR Blue 100 by gavage for a period of twenty eight consecutive days resulted in toxicologically significant changes at dose levels of 150 and 1000 mg active ingredient/kg/day.
Repeated administration of the test material at 1000 mg active ingredient/kg/day resulted in an elevated female kidney weight. This was probably associated with the renal changes detected histopathologically although these microscopic changes were not confined to females. Examination of kidney sections from high dose animals of either sex revealed accumulations of blue pigment together with tubular epithelial degeneration. An increased severity of basophilia/groups of dilated tubules was also identified for females alone at this dose level. Despite these microscopic abnormalities and the slight increase in kidney weight no convincing detrimental effect on renal function was detected following repeated administration of this test material.
Both histopathological and macroscopic findings showed that test material accumulated not only in the kidneys but also in the mesenteric lymph nodes. It is not surprising that particulate test material accumulated in the mesenteric lymph nodes at the two highest dose levels examined and that scavenging macrophages were associated with such agglomerates at the high dose. Any particles of test material too large to be directly absorbed from the gastro-intestinal tract into the blood may enter the lymphatic system. Such particles are eventually transported to lymph nodes where macrophages tend to prevent their general dissemination throughout the body by phagocytosis. The presence of scavenging macrophages in lymph nodes therefore represents the normal physiological process for removal of foreign particulate matter and, in itself, is not indicative of test material toxicity. Despite the action of scavenging macrophages at the highest dose level, the blue discolouration of the remaining viscera, seen at the highest dose level at necropsy, suggests that some of the test material was also disseminated throughout the remainder of the body.
An elevated spleen weight, detected for high dose animals of either sex, was probably associated with the increased splenic extramedullary haemopoiesis identified histopathologically at this dose level, although this latter finding was confined to males. In addition, a slight albeit unconvincing reduction in haemoglobin concentration was detected for the females whilst both males and females showed a marked haemoglobinuria. Pallor of the extremities was also observed for animals of either sex during the study. Such findings suggest that an anaemia has developed at the highest dose level examined although the nature of this anaemia and its' aetiology are unclear. The pallor and the marked haemoglobinuria are particularly difficult to explain in the absence of a more demonstrable effect on either the red blood cell count or the haemoglobin concentration. Pallor is usually only observed when there is a substantial anaemia whilst the degree of haemoglobinuria seen in this study would normally lead to a more marked reduction in haemoglobin concentration, even allowing for the normal compensatory erythropoietic mechanisms. In the absence of a convincing reduction in haemoglobin levels, it is possible that the haemoglobinuria seen at the high dose is an artefact and that a false positive result has been generated by the presence of the test material and/ or its' metabolite(s) in the urine. Furthermore, the pallor may also have been an artefact since systemic staining of the skin by the blue test material would give the animals a pale appearance. Much of the evidence indicating an anaemia therefore remains equivocal or, in the case of haemoglobin levels, unconvincing although the splenic changes indicate that some underlying disturbance of the erythropoietic system may have occurred at the high dose.
Several possible adverse effects of treatment persisted during the recovery period. Animals remained pale in comparison with controls whilst a marked haemoglobinuria was still apparent following a further fourteen days without treatment. Haematological determinations again failed to support the existance of an anaemia however, with the slight reduction in female haemoglobin concentration seen at the end of the recovery period considered to be spurious.
Nevertheless, several of the microscopic abnormalities identified at the end of the twenty-eight day dosing period, including tubular basophilia and the presence of blue pigment in the kidneys and mesenteric lymph nodes, were still visible in satellite high dose animals following a further fourteen day treatment -free period. Indeed, tubular basophilia developed in satellite high dose males during this recovery period indicating a continuing cellular response to the persistent accumulation of test material and/or its' metabolite(s).
Effects of treatment at the intermediate dose level were confined to a slight reduction in female haemoglobin concentration. Although this reduction was unconvincing and was not accompanied by any further changes to indicate an anaemia, a treatment-related effect could not be entirely ruled out in view of the changes seen at the highest dose level examined. This effect was not indicative of a serious threat to the health of the animals however.
No toxicologically significant changes were detected either for intermediate dose males or at the low dose level.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (nominal)

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

haematology

Target system / organ toxicity

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

150 mg/kg bw/day (nominal)

Any other information on results incl. tables

SUMMARY INCIDENCE OF DAILY CLINICAL OBSERVATIONS - MALES

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 1			DAY: 2			DAY: 3			DAY: 4			DAY: 5			DAY: 6			DAY: 7
			Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h
1	0 (Control)	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
2	15▪	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
3	150▪	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
4	1000▪	Fur stained blue by test material No abnormalities detected	0   5	0   5	0   5	0   5	1   4	1   4	1   4	5   0	*   *	1   4	2   3	*   *	0   5	2   3	1   4	1   4	2   3	2   3	0   5	2   3	0   5

Pre = immediately before dosing                   1h = 1 hour after dosing                    5h = 5 hours after dosing

* = 5 hour observation not performed at weekend                      ▪ = dark faeces from day 2 to day 7

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 8			DAY: 9			DAY: 10			DAY: 11			DAY: 12			DAY: 13			DAY: 14
			Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h
1	0 (Control)	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
2	15▪	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
3	150▪	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
4	1000▪	Pallor of the extremities Fur stained blue by test material No abnormalities detected	0 0   5	0 4   1	0 3   2	0 0   5	0 5   0	0 0   5	0 0   5	0 1   4	* *   *	1 0   4	1 0   4	* *   *	1 0   4	1 0   4	1 2   2	1 0   4	1 0   4	1 0   4	1 0   4	1 0   4	1 0   4

Pre = immediately before dosing                   1h = 1 hour after dosing                    5h = 5 hours after dosing

* = 5 hour observation not performed at weekend                      ▪ = dark faeces from day 8 to day 14

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 15			DAY: 16			DAY: 17			DAY: 18			DAY: 19			DAY: 20			DAY: 21
			Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h
1	0 (Control)	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
2	15▪	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
3	150▪	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
4	1000▪	Pallor of the extremities Fur stained blue by test material No abnormalities detected	1 0   4	1 1   3	1 1   3	1 0   4	1 0   4	1 0   4	5 0   0	5 0   0	* *   *	5 0   0	5 0   0	* *   *	5 0   0	5 0   0	5 0   0	5 0   0	5 0   0	5 0   0	5 0   0	5 5   0	5 5   0

Pre = immediately before dosing                   1h = 1 hour after dosing                    5h = 5 hours after dosing

* = 5 hour observation not performed at weekend                      ▪ = dark faeces from day 15 to day 21

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 22			DAY: 23			DAY: 24			DAY: 25			DAY: 26			DAY: 27			DAY: 28
			Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	4h
1	0 (Control)	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
2	15▪	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
3	150▪	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
4	1000▪	Pallor of the extremities Fur stained blue by test material	5 0	5 2	5 0	5 0	5 3	5 2	5 0	5 3	* *	5 0	5 0	* *	5 0	5 1	5 1	5 0	5 0	5 0	5 0	5 3	5 3

Pre = immediately before dosing                   1h = 1 hour after dosing                    5h = 5 hours after dosing

* = 5 hour observation not performed at weekend                      ▪ = dark faeces from day 22 to day 28                           4h = 4 hours after dosing

 

SUMMARY INCIDENCE OF DAILY CLINICAL OBSERVATIONS - FEMALES

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 1			DAY: 2			DAY: 3			DAY: 4			DAY: 5			DAY: 6			DAY: 7
			Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h
1	0 (Control)	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
2	15▪	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
3	150▪	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
4	1000▪	Fur stained blue by test material No abnormalities detected	0   5	5   0	5   0	1   4	1   4	1   4	1   4	5   0	*   *	2   3	5   0	*   *	1   4	5   0	1   4	2   3	5   0	5   0	2   3	5   0	4   1

Pre = immediately before dosing                   1h = 1 hour after dosing                    5h = 5 hours after dosing

* = 5 hour observation not performed at weekend                      ▪ = dark faeces from day 2 to day 7

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 8			DAY: 9			DAY: 10			DAY: 11			DAY: 12			DAY: 13			DAY: 14
			Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h
1	0 (Control)	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
2	15▪	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
3	150▪	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
4	1000▪	Pallor of the extremities Fur stained blue by test material No abnormalities detected	0 0   5	0 5   0	0 5   0	0 2   3	0 5   0	0 3   2	0 0   5	0 5   0	* *   *	5 0   0	5 0   0	* *   *	5 0   0	5 2   0	5 5   0	5 0   0	5 0   0	5 0   0	5 0   0	5 2   0	5 1   0

Pre = immediately before dosing                   1h = 1 hour after dosing                    5h = 5 hours after dosing

* = 5 hour observation not performed at weekend                      ▪ = dark faeces from day 8 to day 14

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 15			DAY: 16			DAY: 17			DAY: 18			DAY: 19			DAY: 20			DAY: 21
			Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h
1	0 (Control)	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
2	15▪	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
3	150▪	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
4	1000▪	Pallor of the extremities Fur stained blue by test material	5 1	5 1	5 0	5 0	5 4	5 2	5 0	5 5	* *	5 0	5 2	* *	5 0	5 1	5 1	5 0	5 2	5 3	5 0	5 4	5 4

Pre = immediately before dosing                   1h = 1 hour after dosing                    5h = 5 hours after dosing

* = 5 hour observation not performed at weekend                      ▪ = dark faeces from day 15 to day 21

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 22			DAY: 23			DAY: 24			DAY: 25			DAY: 26			DAY: 27			DAY: 28
			Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h
1	0 (Control)	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
2	15▪	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
3	150▪	Red/brown staining around snout No abnormalities detected	0   5	0   5	0   5	0   5	0   5	0   5	0   5	0   5	*   *	0   5	0   5	*   *	0   5	0   5	0   5	1   4	1   4	1   4	1   4	1   4	0   5
4	1000▪	Pallor of the extremities Fur stained blue by test material	5 0	5 5	5 0	5 0	5 5	5 5	5 5	5 5	* *	5 5	5 1	* *	5 0	5 2	5 2	5 2	5 3	5 1	5 1	5 5	5 5

Pre = immediately before dosing                   1h = 1 hour after dosing                    5h = 5 hours after dosing

* = 5 hour observation not performed at weekend                      ▪ = dark faeces from day 22 to day 28           4 h = 4 hours after dosing

 

SUMMARY INCIDENCE OF DAILY CLINICAL OBSERVATIONS – SATELLITE GROUPS - MALES

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 1			DAY: 2			DAY: 3			DAY: 4			DAY: 5			DAY: 6			DAY: 7
			Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h
5	0 (Control)	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
6	1000▪	Fur stained blue by test material No abnormalities detected	0   5	2   3	2   3	0   5	0   5	0   5	0   5	5   0	*   *	3   2	5   0	*   *	0   5	3   2	2   3	0   5	5   0	5   0	0   5	4   1	3   2

Pre = immediately before dosing                   1h = 1 hour after dosing                    5h = 5 hours after dosing

* = 5 hour observation not performed at weekend                      ▪ = dark faeces from day 2 to day 7

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 8			DAY: 9			DAY: 10			DAY: 11			DAY: 12			DAY: 13			DAY: 14
			Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h
5	0 (Control)	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
6	1000▪	Pallor of the extremities Fur stained blue by test material No abnormalities detected	2 0   3	2 0   3	2 1   2	2 0   3	2 0   3	2 0   3	2 0   3	2 0   3	* *   *	1 0   4	1 0   4	* *   *	1 0   4	1 3   1	1 5   0	1 0   4	1 1   3	1 1   3	1 0   4	4 0   1	4 0   1

Pre = immediately before dosing                   1h = 1 hour after dosing                    5h = 5 hours after dosing

* = 5 hour observation not performed at weekend                      ▪ = dark faeces from day 8 to day 14

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 15			DAY: 16			DAY: 17			DAY: 18			DAY: 19			DAY: 20			DAY: 21
			Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h
5	0 (Control)	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
6	1000▪	Pallor of the extremities Fur stained blue by test material No abnormalities detected	4 0   1	4 1   1	4 4   1	4 0   1	4 0   1	4 0   1	5 0   0	5 1   0	* *   *	5 0   0	5 0   0	* *   *	5 0   0	5 0   0	5 0   0	5 0   0	5 0   0	5 0   0	5 0   0	5 5   0	5 5   0

Pre = immediately before dosing                   1h = 1 hour after dosing                    5h = 5 hours after dosing

* = 5 hour observation not performed at weekend                      ▪ = dark faeces from day 15 to day 21

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 22			DAY: 23			DAY: 24			DAY: 25			DAY: 26			DAY: 27			DAY: 28
			Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	4h
5	0 (Control)	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
6	1000▪	Pallor of the extremities Fur stained blue by test material	5 0	5 4	5 0	5 0	5 5	5 5	5 0	5 5	* *	5 0	5 0	* *	5 0	5 5	5 5	5 0	5 0	5 0	5 0	5 5	5 5

Pre = immediately before dosing                   1h = 1 hour after dosing                    5h = 5 hours after dosing

* = 5 hour observation not performed at weekend                      ▪ = dark faeces from day 22 to day 28                           4h = 4 hours after dosing

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 29		DAY: 30			DAY: 31		DAY: 32		DAY: 33		DAY: 34		DAY: 35
			Am	Pm	Am	Pm	Am		Pm	Am	Pm	Am	Pm	Am	Pm	Am	Pm
5	0 (Control)	No abnormalities detected	5	5	5	5	5		*	5	*	5	5	5	*	5	5
6	1000▪	Pallor of the extremities	5	5	5	5	5		*	5	*	5	5	5	5	5	5

* = afternoon observation not performed at weekend                ▪ = dark faeces from day 29 to day 30

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 36		DAY: 37			DAY: 38		DAY: 39		DAY: 40		DAY: 41		DAY: 42
			Am	Pm	Am	Pm	Am		Pm	Am	Pm	Am	Pm	Am	Pm	Am	Pm
5	0 (Control)	No abnormalities detected	5	5	5	5	5		*	5	*	5	5	5	*	5	5
6	1000	Pallor of the extremities Slight pallor of the extremities No abnormalities detected	5 0   0	5 0   0	5 0   0	5 0   0	5 0   0		* *   *	5 0   0	* *   *	1 4   0	1 4   0	1 4   0	1 3   1	1 3   1	1 2   2

* = afternoon observation not performed at weekend

 

INCIDENCE OF DAILY CLINICAL OBSERVATIONS – SATELLITE GROUPS - FEMALES

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 1			DAY: 2			DAY: 3			DAY: 4			DAY: 5			DAY: 6			DAY: 7
			Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h
5	0 (Control)	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
6	1000▪	Fur stained blue by test material Mouth stained blue by test material No abnormalities detected	0   0   5	0   0   5	0   0   5	0   0   5	0   0   5	0   0   5	2   0   3	5   0   0	*   *   *	3   0   2	5   0   0	*   *   *	0   0   5	2   0   3	0   0   5	1   0   4	4   0   1	4   0   1	1   0   4	5   1   0	5   1   0

Pre = immediately before dosing                   1h = 1 hour after dosing                    5h = 5 hours after dosing

* = 5 hour observation not performed at weekend                      ▪ = dark faeces from day 2 to day 7

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 8			DAY: 9			DAY: 10			DAY: 11			DAY: 12			DAY: 13			DAY: 14
			Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h
5	0 (Control)	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
6	1000▪	Pallor of the extremities Fur stained blue by test material No abnormalities detected	0 1   4	0 1   4	0 1   4	0 0   5	0 5   0	0 5   0	0 4   1	0 5   0	* *   *	2 1   3	2 1   3	* *   *	2 0   3	2 2   1	2 5   0	2 0   3	2 1   3	2 1   3	2 0   3	5 1   0	5 1   0

Pre = immediately before dosing                   1h = 1 hour after dosing                    5h = 5 hours after dosing

* = 5 hour observation not performed at weekend                      ▪ = dark faeces from day 8 to day 14

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 15			DAY: 16			DAY: 17			DAY: 18			DAY: 19			DAY: 20			DAY: 21
			Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h
5	0 (Control)	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
6	1000▪	Pallor of the extremities Fur stained blue by test material	5 1	5 3	5 5	5 0	5 1	5 1	5 0	5 3	* *	5 0	5 4	* *	5 0	5 2	5 2	5 0	5 2	5 3	5 0	5 5	5 5

Pre = immediately before dosing                   1h = 1 hour after dosing                    5h = 5 hours after dosing

* = 5 hour observation not performed at weekend                      ▪ = dark faeces from day 15 to day 21

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 22			DAY: 23			DAY: 24			DAY: 25			DAY: 26			DAY: 27			DAY: 28
			Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	5h	Pre	1h	4h
5	0 (Control)	No abnormalities detected	5	5	5	5	5	5	5	5	*	5	5	*	5	5	5	5	5	5	5	5	5
6	1000▪	Pallor of the extremities Fur stained blue by test material	5 2	5 3	5 0	5 0	5 5	5 5	5 3	5 5	* *	5 2	5 2	* *	5 0	5 4	5 5	5 2	5 5	5 3	5 3	5 5	5 5

Pre = immediately before dosing                   1h = 1 hour after dosing                    5h = 5 hours after dosing

* = 5 hour observation not performed at weekend                      ▪ = dark faeces from day 2 to day 7                4h = 4 hours after dosing

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 29		DAY: 30			DAY: 31		DAY: 32		DAY: 33		DAY: 34		DAY: 35
			Am	Pm	Am	Pm	Am		Pm	Am	Pm	Am	Pm	Am	Pm	Am	Pm
5	0 (Control)	No abnormalities detected	5	5	5	5	5		*	5	*	5	5	5	*	5	5
6	1000▪	Pallor of the extremities Fur stained blue by test material	5 2	5 2	5 0	5 0	5 0		* *	5 0	* *	5 0	5 0	5 0	5 0	5 0	5 0

* = afternoon observation not performed at weekend                ▪ = dark faeces from day 29 to day 30

GROUP NUMBER	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day	CLINICAL OBSERVATIONS	NUMBER SHOWING EFFECTS AT OBSERVATION
			DAY: 36		DAY: 37			DAY: 38		DAY: 39		DAY: 40		DAY: 41		DAY: 42
			Am	Pm	Am	Pm	Am		Pm	Am	Pm	Am	Pm	Am	Pm	Am	Pm
5	0 (Control)	No abnormalities detected	5	5	5	5	5		*	5	*	5	5	5	*	5	5
6	1000	Pallor of the extremities Slight pallor of the extremities No abnormalities detected	5 0   0	5 0   0	5 0   0	5 0   0	5 0   0		* *   *	5 0   0	* *   *	2 2   1	2 2   1	2 2   1	2 2   1	2 2   1	2 2   1

* = afternoon observation not performed at weekend

 

GROUP MEAN WEEKLY BODYWEIGHTS AND STANDARD DEVIATIONS (SD) – MALES

	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day		Bodyweight (g) at day
			0	7	14	21	28	35	42
1	Control	Mean SD	160 6	216 13	273 17	320 23	345 26	- -	- -
2	15	Mean SD	158 9	221 13	282 15	335 15	363 16	- -	- -
3	150	Mean SD	150 7	209 10	264 13	312 15	339 20	- -	- -
4	1000	Mean SD	152 12	212 16	265 21	319 26	350 33	- -	- -
5	S. Control	Mean SD	157 10	219 16	278 18	320 17	355 26	380 28	397 27
6	1000	Mean SD	151 6	202 9	249 11	289 17	323 11	349 20	366 26

- = not applicable

 

GROUP MEAN WEEKLY BODYWEIGHTS AND STANDARD DEVIATIONS (SD) – FEMALES

	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day		Bodyweight (g) at day
			0	7	14	21	28	35	42
1	Control	Mean SD	146 8	180 6	208 10	235 10	247 11	- -	- -
2	15	Mean SD	144 3	172 6	200 6	224 12	236 12	- -	- -
3	150	Mean SD	143 15	171 20	195 26	218 29	231 26	- -	- -
4	1000	Mean SD	147 4	181 11	206 15	232 19	243 21	- -	- -
5	S. Control	Mean SD	142 5	176 7	203 12	223 12	241 12	260 14	265 11
6	1000	Mean SD	140 7	174 6	203 9	227 12	247 13	262 13	270 14

- = not applicable

 

GROUP MEAN HAEMATOLOGICAL VALUES AND STANDARD DEVIATIONS (SD) – MALES

	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day		Hb (g/dl)	RBC (10^12/l)	Hct (%)	MCH (pg)	MCV (fl)	MCHC (g/dl)	WBC (10^9/l)	Meth (%)
1	Control	Mean SD	15.3 0.5	7.82 0.13	44.4 2.1	19.6 0.5	56.7 2.2	34.5 0.5	15.4 3.4	0.69 0.29
2	15	Mean SD	15.6 1.0	7.92 0.29	45.6 3.0	19.7 0.8	57.5 2.5	34.3 0.3	15.2 3.5	0.75 0.46
3	150	Mean SD	15.6 0.6	7.90 0.32	45.1 1.8	19.8 0.6	57.1 1.7	34.6 0.2	12.6	0.87 0.40
4	1000	Mean SD	15.1 0.4	7.78 0.22	44.8 0.8	19.4 1.0	57.6 1.9	33.7* 0.9	15.2 2.4	0.81 0.20
5	S. control	Mean SD	15.7 0.5	- -	- -	- -	- -	- -	- -	- -
6	1000	Mean SD	15.5 0.7	- -	- -	- -	- -	- -	- -	- -
			DIFFERENTIAL (10^9/l)					CT (secs)	PLT (10^9/l)	Retic (%)
			Neut	Lymph	Mono	Eos	Bas
1	Control	Mean SD	1.16 0.56	14.25 3.08	0.04 0.08	0.00 0.00	0.00 0.00	31 2	1341 159	4 3
2	15	Mean SD	1.83 1.79	13.28 1.91	0.04 0.09	0.05 0.07	0.00 0.00	31 1	1255 145	2 1
3	150	Mean SD	2.03 2.22	10.51* 1.18	0.02 0.05	0.08 0.08	0.00 0.00	30 1	1320 194	2 1
4	1000	Mean SD	2.05 0.35	13.03 2.29	0.09 0.15	0.05 0.07	0.00 0.00	29* 1	1338 108	3 1
5	S. control	Mean SD	- -	- -	- -	- -	- -	29 2	- -	- -
6	1000	Mean SD	- -	- -	- -	- -	- -	28 2	- -	- -

* = significantly different from corresponding control group value p <0.05

- = not evaluated

 

GROUP MEAN HAEMATOLOGICAL VALUES AND STANDARD DEVIATIONS (SD) – FEMALES

	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day		Hb (g/dl)	RBC (10^12/l)	Hct (%)	MCH (pg)	MCV (fl)	MCHC (g/dl)	WBC (10^9/l)	Meth (%)
1	Control	Mean SD	15.2 0.3	7.74 0.18	44.5 1.2	19.6 0.5	57.6 1.8	34.0 0.6	9.9 2.3	0.31 0.42
2	15	Mean SD	15.0 0.5	7.88 0.36	44.5 0.5	19.0 0.7	56.6 2.9	33.6 1.1	11.1 3.2	1.11 0.89
3	150	Mean SD	14.5* 0.3	7.54 0.23	42.9* 0.6	19.2 0.8	56.9 2.3	33.7 0.7	10.2 2.0	1.00 0.88
4	1000	Mean SD	14.6* 0.5	7.78 0.08	43.4 1.2	18.8 0.8	55.9 1.6	33.6 0.9	9.6 1.1	0.64 0.67
5	S. control	Mean SD	15.5 0.2	- -	- -	- -	- -	- -	- -	- -
6	1000	Mean SD	14.6** 0.5	- -	- -	- -	- -	- -	- -	- -
			DIFFERENTIAL (10^9/l)					CT (secs)	PLT (10^9/l)	Retic (%)
			Neut	Lymph	Mono	Eos	Bas
1	Control	Mean SD	0.67 0.32	9.23 2.13	0.00 0.00	0.05 0.10	0.00 0.00	30 3	1276 103	2 1
2	15	Mean SD	0.99 1.13	10.08 2.80	0.02 0.05	0.01 0.03	0.00 0.00	31 1	1306 180	3 1
3	150	Mean SD	1.00 0.67	9.13 1.36	0.04 0.06	0.04 0.06	0.00 0.00	31 1	1214 73	3 2
4	1000	Mean SD	0.79 0.37	8.66 1.10	0.02 0.05	0.08 0.14	0.00 0.00	32 2	1268 254	2 1
5	S. control	Mean SD	- -	- -	- -	- -	- -	28 1	- -	- -
6	1000	Mean SD	- -	- -	- -	- -	- -	28 1	- -	- -

* = significantly different from corresponding control group value p <0.05

** = significantly different from corresponding control group value p <0.01

- = not evaluated

 

GROUP MEAN BLOOD CHEMICAL VALUES AND STANDARD DEVIATIONS (SD) – MALES

	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day		UREA (mg/dl)	GLUCOSE (mg/dl)	TOT.PROT (g/dl)	ALBUMIN (g/dl)	A/G ratio	Na+ (mmol/l)	K+ (mmol/l)	Cl- (mmol/l)	Ca++ (mmol/l)
1	Control	Mean SD	30 5	149 13	6.79 0.41	3.94 0.19	1.39 0.10	133 2	4.41 0.16	97 2	2.46 0.06
2	15	Mean SD	30 4	156 15	6.73 0.30	3.93 0.19	1.40 0.07	133 2	4.43 0.41	98 2	2.46 0.06
3	150	Mean SD	29 4	149 7	6.89 0.36	4.00 0.16	1.39 0.09	134 2	4.36 0.26	96 2	2.47 0.07
4	1000	Mean SD	31 1	150 10	6.79 0.38	3.98 0.23	1.42 0.07	133 3	4.55 0.38	96 1	2.46 0.08
5	S. control	Mean SD	- -	- -	- -	- -	- -	- -	4.55 0.36	- -	- -
6	1000	Mean SD	- -	- -	- -	- -	- -	- -	4.60 0.29	- -	- -
			P (mmol/l)	ƴGT (IU/l)	ASAT (IU/l)	ALAT (IU/l)	AP (IU/l)	CREAT (mg/dl)	TRI (mg/dl)	Chol (mg/dl)	Bili (mg/dl)
1	Control	Mean SD	2.34 0.17	2 1	100 9	69 18	551 69	0.59 0.03	115 29	68 12	0.32 0.13
2	15	Mean SD	2.42 0.25	1 1	94 7	62 9	602 126	0.61 0.05	129 20	65 8	0.41 0.05
3	150	Mean SD	2.52 0.34	1 1	98 8	62 7	722* 135	0.60 0.04	127 56	70 7	0.40 0.8
4	1000	Mean SD	2.54 0.22	0 0	110 11	55 9	585 117	0.59 0.04	111 47	83* 15	0.33 0.11
5	S. control	Mean SD	- -	- -	- -	61 9	- -	- -	- -	80 13	- -
6	1000	Mean SD	- -	- -	- -	55 4	- -	- -	- -	93 41	- -

* = significantly different from corresponding control group value p <0.05

- = not evaluated

 

GROUP MEAN BLOOD CHEMICAL VALUES AND STANDARD DEVIATIONS (SD) – FEMALES

	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day		UREA (mg/dl)	GLUCOSE (mg/dl)	TOT.PROT (g/dl)	ALBUMIN (g/dl)	A/G ratio	Na+ (mmol/l)	K+ (mmol/l)	Cl- (mmol/l)	Ca++ (mmol/l)
1	Control	Mean SD	41 6	173 10	7.27 0.23	4.25 0.17	1.41 0.07	130 3	4.07 0.08	99 2	2.48 0.08
2	15	Mean SD	39 6	166 7	7.01 0.37	4.04 0.28	1.36 0.08	132 4	4.39 0.52	99 2	2.45 0.08
3	150	Mean SD	43 2	174 15	6.80 1.05	4.01 0.37	1.50 0.34	131 3	4.94** 0.34	99 2	2.50 0.08
4	1000	Mean SD	40 6	175 10	6.98 0.46	4.16 0.24	1.48 0.04	133 4	4.61* 0.35	98 2	2.39 0.07
5	S. control	Mean SD	- -	- -	- -	- -	- -	- -	4.78 0.26	- -	- -
6	1000	Mean SD	- -	- -	- -	- -	- -	- -	4.63 0.24	- -	- -
			P (mmol/l)	ƴGT (IU/l)	ASAT (IU/l)	ALAT (IU/l)	AP (IU/l)	CREAT (mg/dl)	TRI (mg/dl)	Chol (mg/dl)	Bili (mg/dl)
1	Control	Mean SD	2.19 0.26	1 1	99 5	73 19	483 64	0.70 0.01	49 12	76 18	0.35 0.04
2	15	Mean SD	2.16 0.23	1 1	114 17	68 8	495 127	0.68 0.05	46 11	79 27	0.38 0.07
3	150	Mean SD	1.85* 0.15	2 1	110 8	63 4	428 76	0.72 0.04	47 17	66 8	0.44* 0.05
4	1000	Mean SD	2.33 0.19	1 1	98 13	39*** 7	388 51	0.72 0.03	45 15	80 17	0.27 0.09
5	S. control	Mean SD	- -	- -	- -	62 17	- -	- -	- -	80 4	- -
6	1000	Mean SD	- -	- -	- -	40* 5	- -	- -	- -	83 11	- -

* = significantly different from corresponding control group value p <0.05

** = significantly different from corresponding control group value P <0.01

*** = significantly different from corresponding control group value p < 0.001

- = not evaluated

 

NECROPSY FINDINGS – SUMMARY INCIDENCE – MALES

	Group 1 Control	Group 2 15 mg Active Ingredient/kg/day	Group 3 150 mg Active Ingredient/kg/day	Group 4 1000 mg Active Ingredient/kg/day	Group 5 S. control	Group 6 1000 mg Active Ingredient/kg/day
Number of animals	5	5	5	5	5	5
Mesenteric Lymph Nodes
Blue discolouration	0	0	5	5	0	0
Kidneys
Blue discolouration	0	0	5	5	0	0
Stomach – Glandular Region
Blue discolouration	0	0	0	1	0	0
Remaining Viscera
Blue/slight blue discolouration	0	0	0	5	0	5

 

NECROPSY FINDINGS – SUMMARY INCIDENCE – FEMALES

	Group 1 Control	Group 2 15 mg Active Ingredient/kg/day	Group 3 150 mg Active Ingredient/kg/day	Group 4 1000 mg Active Ingredient/kg/day	Group 5 S. control	Group 6 1000 mg Active Ingredient/kg/day
Number of animals	5	5	5	5	5	5
Mesenteric Lymph Nodes
Blue discolouration	0	0	5	5	0	0
Kidneys
Blue discolouration	0	0	5	5	0	0
Lungs
Prominent blue discoloration – left lobe and right cranial lobe	0	0	0	2	0	0
Prominent blue discolouration – left lobe	0	0	0	2	0	0
Blue discolouration – anterior and middle lobe	0	0	0	0	0	1
Remaining Viscera
Blue/slight blue discolouration	0	0	0	5	0	5

 

GROUP MEAN ORGAN WEIGHTS AND STANDARD DEVIATIONS (S.D.) – MALES

	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day		Bodyweight at necropsy (g)	Organ weight (g)
				Adrenals	Brain	Gonads	Heart	Kidneys	Liver	Pituitary	Spleen
1	Control	Mean SD	345 28	0.0540 0.0107	1.9458 0.0696	4.2216 0.1926	1.2820 0.1244	2.4096 0.1499	12.6710 0.9168	0.0093 0.0018	0.7304 0.0854
2	15	Mean SD	361 16	0.0515 0.0054	1.9683 0.0752	4.4335 0.3618	1.2698 0.0873	2.3128 0.2049	13.1542 0.7680	0.0112 0.0016	0.8372 0.1524
3	150	Mean SD	340 17	0.0502 0.0015	2.0632* 0.0280	4.1900 0.1141	1.2740 0.1278	2.3067 0.1110	12.4066 0.3765	0.0106 0.0018	0.8508 0.1661
4	1000	Mean SD	349 31	0.0518 0.0076	1.9917 0.0900	4.0654 0.3842	1.2836 0.1484	2.3621 0.2400	12.9613 1.7774	0.0114 0.0019	0.9676* 0.1548
5	s. control	Mean SD	395 26	0.0496 0.0055	2.0686 0.0835	4.7977 0.2268	1.4087 0.2022	2.4986 0.1495	13.3215 1.2841	0.0108 0.0022	0.8783 0.0376
6	1000	Mean SD	364 24	0.0511 0.0037	1.9901 0.0498	4.5460 0.2288	1.3548 0.1332	2.5735 0.6276	12.5067 1.6458	0.0067* 0.0018	0.9023 0.0864

* = significantly different from corresponding control group value p < 0.05

 

GROUP MEAN ORGAN WEIGHTS AND STANDARD DEVIATIONS (S.D.) – FEMALES

	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day		Bodyweight at necropsy (g)	Organ weight (g)
				Adrenals	Brain	Gonads	Heart	Kidneys	Liver	Pituitary	Spleen
1	Control	Mean SD	244 12	0.0644 0.0072	1.8799 0.0598	0.1383 0.0154	1.0156 0.1271	1.6749 0.0560	8.6812 0.7903	0.0132 0.0013	0.6209 0.1586
2	15	Mean SD	236 12	0.0655 0.0062	1.9168 0.0691	0.1376 0.0274	1.0268 0.1783	1.7192 0.1001	8.1985 0.8395	0.0112 0.0044	0.7186 0.1176
3	150	Mean SD	228 26	0.0639 0.0071	1.8493 0.0107	0.1178 0.0093	0.9156 0.0940	1.5951 0.1785	7.6187 1.0893	0.0089* 0.0028	0.6542 0.1696
4	1000	Mean SD	242 20	0.0690 0.0098	1.8711 0.0552	0.1496 0.0154	0.9290 0.0827	1.8517* 0.1432	9.5640 1.6284	0.0139 0.0023	0.8125 0.1230
5	s. control	Mean SD	263 12	0.0636 0.0116	1.9188 0.1427	0.1485 0.0176	1.446 0.1485	1.7875 0.1600	8.6419 0.6997	0.0132 0.0052	0.6333 0.0703
6	1000	Mean SD	265 14	0.0724 0.0073	1.9689 0.0480	0.1660 0.0227	1.0976 0.1611	1.7120 0.1240	9.4530 0.9866	0.0120 0.0016	0.7465* 0.0700

* = significantly different from corresponding control group value p < 0.05

 

GROUP MEAN RELATIVE ORGAN WEIGHTS (% OF BODYWEIGHT) AND STANDARD DEVIATIONS (S.D.) – MALES

	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day		Bodyweight at necropsy (g)	Organ weight (g)
				Adrenals	Brain	Gonads	Heart	Kidneys	Liver	Pituitary	Spleen
1	Control	Mean SD	345 28	0.0158 0.0038	0.5667 0.0356	1.2288 0.0734	0.3728 0.0356	0.7006 0.0357	3.6807 0.1467	0.0027 0.0006	0.2132 0.0321
2	15	Mean SD	361 16	0.0144 0.0020	0.5466 0.0313	1.2286 0.0690	0.3527 0.0296	0.6408* 0.0387	3.6462 0.0650	0.0031 0.0004	0.2313 0.0349
3	150	Mean SD	340 17	0.0148 0.0008	0.6082 0.0361	1.2338 0.0384	0.3741 0.0210	0.6788 0.0208	3.6545 0.1672	0.0031 0.0005	0.2490 0.0374
4	1000	Mean SD	349 31	0.0149 0.0027	0.5721 0.0303	1.1649 0.0666	0.3677 0.0337	0.6767 0.0459	3.7050 0.2882	0.0033 0.0005	0.2762** 0.0306
5	s. control	Mean SD	395 26	0.0126 0.0013	0.5244 0.0224	1.2180 0.0975	0.3568 0.0497	0.6340 0.0493	3.3721 0.2664	0.0028 0.0007	0.2232 0.0200
6	1000	Mean SD	364 24	0.0141 0.0015	0.5480 0.0317	1.2542 0.1267	0.3738 0.0504	0.7070 0.1718	3.4379 0.4531	0.0018* 0.0005	0.2482 0.0234

* = significantly different from corresponding control group value p < 0.05

** = significantly different from corresponding control group value p < 0.01

 

GROUP MEAN RELATIVE ORGAN WEIGHTS (% OF BODYWEIGHT) AND STANDARD DEVIATIONS (S.D.) – FEMALES

	DOSE LEVEL OF ACTIVE INGREDIENT mg/kg/day		Bodyweight at necropsy (g)	Organ weight (g)
				Adrenals	Brain	Gonads	Heart	Kidneys	Liver	Pituitary	Spleen
1	Control	Mean SD	244 12	0.0265 0.0038	0.7727 0.0597	0.0568 0.0073	0.4156 0.0405	0.6870 0.0214	3.5567 0.2412	0.0054 0.0005	0.2530 0.0534
2	15	Mean SD	236 12	0.0277 0.0022	0.8115 0.0256	0.0579 0.0093	0.4328 0.0588	0.7277 0.0368	3.4622 0.2078	0.0047 0.0017	0.3049 0.0553
3	150	Mean SD	228 26	0.0281 0.0026	0.8181 0.0920	0.0519 0.0046	0.4025 0.0343	0.7024 0.0840	3.3283 0.1486	0.0038 0.0010	0.2860 0.0664
4	1000	Mean SD	242 20	0.0286 0.0044	0.7755 0.0473	0.0618 0.0035	0.3838 0.0220	0.7659* 0.0497	3.9325* 0.3873	0.0058 0.0014	0.3351* 0.0362
5	s. control	Mean SD	263 12	0.0241 0.0041	0.7297 0.0575	0.0656 0.0071	0.4345 0.0491	0.6782 0.0344	3.2792 0.1207	0.0050 0.0020	0.2406 0.0235
6	1000	Mean SD	265 14	0.0273 0.0020	0.7445 0.0531	0.0632 0.0062	0.4125 0.0430	0.6458 0.0386	3.5712 0.3903	0.0045 0.0004	0.2825 0.0343

* = significantly different from corresponding control group value p < 0.05

 

HISTOPATHOLOGICAL FINDINGS – SUMMARY INCIDENCE – MALES

	Group 1 Control	Group 2 15 mg Active Ingredient/kg/ day	Group 3 150 mg Active Ingredient/kg/ day	Group 4 1000 mg Active Ingredient/kg/ day	Group 5 s. control	Group 6 1000 mg Active Ingredient/kg/ day
Number of animals	5	5	5	5	5	5
	Heart
Focal myocarditis            No data            Absent            (minimal)            (slight)            (moderate)	0 1 3 0 1	5 0 0 0 0	5 0 0 0 0	0 0 4 1 0	5 0 0 0 0	5 0 0 0 0
	Kidneys
Groups of basophilic/ dilated tubules            Absent            (minimal)            (moderate)	4 1 0	3 2 0	3 2 0	3 2 0	5 0 0	1 3 1
Tubular epithelial degeneration            Absent            (minimal)            (slight)	5 0 0	5 0 0	5 0 0	0 2 3	5 0 0	5 0 0
Accumulation of blue pigment            Absent            Present	5 0	5 0	5 0	0 5	5 0	0 5
Cortical cyst            Absent            Present	4 1	5 0	5 0	5 0	5 0	5 0
Hydronephrosis            Absent            (minimal)	5 0	5 0	4 1	5 0	5 0	5 0
	Liver
Scattered mononuclear cell foci            No data            (minimal)            (slight)	0 4 1	5 0 0	5 0 0	0 5 0	5 0 0	5 0 0
	Mesenteric lymph nodes
Accumulation of blue pigment            Absent            Present	5 0	5 0	5 0	0 5	5 0	0 5
Accumulation of foamy macrophages            Absent            Present	5 0	5 0	5 0	1 4	5 0	0 5
	Spleen
Extramedullary haemopoiesis            (minimal)            (slight)	5 0	4 1	3 2	0 5	5 0	5 0
	Testes
Atrophy            No data            Absent            (minimal)	0 4 1	5 0 0	5 0 0	0 5 0	5 0 0	5 0 0
	Statistical Information
Mode of death            Terminal kill	5	5	5	5	5	5

 

HISTOPATHOLOGICAL FINDINGS – SUMMARY INCIDENCE – FEMALES

	Group 1 Control	Group 2 15 mg Active Ingredient/kg/ day	Group 3 150 mg Active Ingredient/kg/ day	Group 4 1000 mg Active Ingredient/kg/ day	Group 5 s. control	Group 6 1000 mg Active Ingredient/kg/ day
Number of animals	5	5	5	5	5	5
	Heart
Focal myocarditis            No data            Absent            (minimal)	0 1 4	5 0 0	5 0 0	0 2 3	5 0 0	5 0 0
	Kidneys
Groups of basophilic/ dilated tubules            Absent            (minimal)            (moderate)	4 1 0	4 1 0	4 1 0	2 1 2	4 1 0	4 1 0
Tubular epithelial degeneration            Absent            (minimal)            (slight)            (moderate)	5 0 0 0	5 0 0 0	5 0 0 0	0 0 2 3	5 0 0 0	0 5 0 0
Accumulation of blue pigment            Absent            Present	5 0	5 0	5 0	0 5	5 0	0 5
	Liver
Scattered mononuclear cell foci            No data            (minimal)            (slight)	0 4 1	5 0 0	5 0 0	0 4 1	5 0 0	5 0 0
	Mesenteric lymph nodes
Accumulation of blue pigment            Absent            Present	5 0	5 0	5 0	0 5	5 0	0 5
Accumulation of foamy macrophages            Absent            Present	5 0	5 0	5 0	1 4	5 0	0 5
	Spleen
Extramedullary haemopoiesis            (minimal)            (slight)	3 2	5 0	5 0	3 2	5 0	5 0
	Non-Protocol Organs
Lung            No evidence gross lesion            Perivascular/ peribronchiolar lymphoid aggregations (minimal)            Prominent accumulations of blue pigment            Groups of alveolar macorphages (minimal)	0   0       0     0	0   0       0     0	0   0       0     0	3   4       1     1	0   0       0     0	0   0       0     0
	Statistical Information
Mode of death            Terminal kill	5	5	5	5	5	5

 

Applicant's summary and conclusion

Conclusions:

Oral administration of the test material, JPR Blue 100, to rats for a period of twenty-eight consecutive days, by gavage, at dose levels of up to 1000 mg active ingredient/kg/day resulted in treatment-related changes at dose levels of 150 and 1000 mg active ingredient/kg/day for females and at 1000 mg active ingredient/kg/day only for males. No such changes were demonstrated in animals of either sex treated with 15 mg active ingredient/kg/day or for males treated with 150 mg active ingredient/kg/day and the "No Observed Effect Level" (NOEL) is therefore considered to be 15 mg active ingredient/kg/day for females and 150 mg active ingredient/kg/day for males.
The sole effect of treatment at 150 mg active ingredient/kg/day, a slight reduction in female haemoglobin concentration, was considered not to be indicative of serious damage to the health of the animals however and, in this respect, the "No Observed Adverse Effect level" (NOAEL) is considered to be 150 mg active ingredient/kg/day for animals of either sex.

Executive summary:

The test material was administered by gavage to three groups, each of five male and five female Sprague-Dawley CD strain rats, for twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg active ingredient/kg/day (incorporating a correction factor to allow for 95.2% purity). A control group of five males and five females was dosed with vehicle alone (distilled water). Two satellite groups, each of five males and five females were treated with the high dose (1000 mg active ingredient/kg/day) or the vehicle alone throughout the twenty-eight day study period and then maintained without treatment for a further fourteen days.

 

Clinical signs, bodyweight, food and water consumptions were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all main group animals during the final week of dosing. Parameters showing possible abnormalities were examined in satellite group animals at the end of the treatment-free period.

 

All animals were subjected to a gross necropsy examination together with histopathological evaluation of selected tissues.

 

The results are summarised as follows:

Mortality Data

There were no deaths during the study.

 

Clinical Observations

Clinically observable signs of toxicity were confined to high dose animals. Pallor of the extremities was first observed on day 8 and, from day 17 onwards, all high dose and satellite high dose animals of either sex appeared pale in comparison with controls. Pallor of the extremities persisted throughout the fourteen day recovery period following cessation of treatment although it was noticeably diminished for most animals towards the end of the study.

Intermediate and low dose animals showed no clinically observable signs of toxicity during the study.

 

Bodyweight

Animals treated with the test material showed similar bodyweight gains to controls during the study.

 

Food Consumption

There was no adverse effect on food consumption during the study.

 

Water Consumption

No overt intergroup differences were detected.

 

Haematology

Intermediate and high dose females showed a slight but statistically significant reduction in haemoglobin concentration compared with controls.

Haemoglobin concentration remained slightly reduced in satellite high dose females following a further fourteen days without treatment.

No treatment -related effects were detected for intermediate and high dose males or for low dose animals of either sex.

 

Blood Chemistry

No treatment-related effects were detected.

 

Urinalysis

A marked haemoglobinuria was detected for all high dose animals towards the end of the dosing period and, for all satellite high dose animals, at the end of the recovery period.

No treatment-related effects were detected at the remaining dose levels.

 

Necropsy

No toxicologically significant macroscopic abnormalities were observed at terminal kill.

 

Organ Weights

High dose animals of either sex showed a slight increase in absolute and relative spleen weight compared to controls. In addition, high dose females showed a statistically significant increase in kidney weight, both absolute and relative to bodyweight.

No treatment-related organ weight changes were apparent at the remaining dose levels or, following a further fourteen days without treatment, for satellite high dose animals.

 

Histopathology

Treatment-related changes were observed in the spleen, kidneys and mesenteric lymph nodes:

SPLEEN: An increased severity of extramedullary haemopoiesis was observed for male rats dosed at 1000 mg active ingredient/kg/day. A similar effect was not convincingly observed for male rats in the remaining treatment groups and the severity of extramedullary haemopoiesis amongst satellite

1000 mg active ingredient/kg/day rats was no different from that reported for satellite control rats following an additional fourteen days without treatment.

KIDNEYS: Tubular epithelial degeneration and accumulations of blue pigment were observed in the renal tubules of rats of either sex receiving 1000 mg active ingredient/kg/day. In addition, female rats dosed at 1000 mg active ingredient/kg/day demonstrated an increased severity of tubular basophilia in association with treatment. Rats from the remaining treatment groups were unaffected. Tubular degeneration was no longer evident amongst satellite 1000 mg active ingredient/kg/day male rats and was reduced in severity for female rats after an additional fourteen days without treatment. For male rats, the incidence and severity of tubular basophilia were increased following the fourteen day recovery period, the condition remaining unaffected for satellite group female rats. The extent and severity of pigment accumulations were not affected for either sex following an additional fourteen days without treatment.

MESENTERIC LYMPH NODES: Accumulations of blue pigment and foamy macrophages were observed in relation to treatment for male and for female rats dosed at 1000 mg active ingredient/kg/day. The presence of either condition was unaffected for satellite group animals following an additional fourteen days without treatment.

 

CONCLUSION

Oral administration of the test material, JPR Blue 100, to rats for a period of twenty-eight consecutive days, by gavage, at dose levels of up to 1000 mg active ingredient/kg/day resulted in treatment -related changes at dose levels of 150 and 1000 mg active ingredient/kg/day for females and at 1000 mg active ingredient/kg/day only for males. No such changes were demonstrated in animals of either sex treated with 15 mg active ingredient/kg/day or for males treated with 150 mg active ingredient/kg/day and the "No Observed Effect Level" (NOEL) is therefore considered to be 15 mg active ingredient/kg/day for females and 150 mg active ingredient/kg/day for males.

The sole effect of treatment at 150 mg active ingredient/kg/day, a slight reduction in female haemoglobin concentration, was considered not to be indicative of serious damage to the health of the animals however and, in this respect, the "No Observed Adverse Effect Level" (NOAEL) is considered to be 150 mg active ingredient/kg/day for animals of either sex.