Octasodium 2-(8-(4-chloro-6-(3-((4-chloro-6-(3,6-disulfonato-2-(1,5-disulfonatonaphthalen-2-ylazo)-1-hydroxynaphthalen-8-ylamino)-1,3,5-triazin-2-yl)aminomethyl)phenylamino)-1,3,5-triazin-2-ylamino)-3,6-disulfonato-1-hydroxynaphthalen-2-ylazo)naphthalene-1,5-disulfonate

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:

October 1991 to December 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted in compliance with GLP. No test standard is referenced within the study report. The methodology utilised is equivalent to OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1992

Report date:

1992

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

No test guideline is referenced within the study report. The methodology utilised is equivalent to OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents).

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Octasodium 2-(8-(4-chloro-6-(3-((4-chloro-6-(3,6-disulfonato-2-(1,5-disulfonatonaphthalen-2-ylazo)-1-hydroxynaphthalen-8-ylamino)-1,3,5-triazin-2-yl)aminomethyl)phenylamino)-1,3,5-triazin-2-ylamino)-3,6-disulfonato-1-hydroxynaphthalen-2-ylazo)naphthalene-1,5-disulfonate

EC Number:

413-550-5

EC Name:

Octasodium 2-(8-(4-chloro-6-(3-((4-chloro-6-(3,6-disulfonato-2-(1,5-disulfonatonaphthalen-2-ylazo)-1-hydroxynaphthalen-8-ylamino)-1,3,5-triazin-2-yl)aminomethyl)phenylamino)-1,3,5-triazin-2-ylamino)-3,6-disulfonato-1-hydroxynaphthalen-2-ylazo)naphthalene-1,5-disulfonate

Cas Number:

142068-96-0

Molecular formula:

Hill formula: C53H28Cl2N14Na8O26S8

IUPAC Name:

octasodium 5-[(4-chloro-6-{[4-({[4-chloro-6-({7-[2-(1,5-disulfonatonaphthalen-2-yl)diazen-1-yl]-8-hydroxy-3,6-disulfonatonaphthalen-1-yl}amino)-1,3,5-triazin-2-yl]amino}methyl)phenyl]amino}-1,3,5-triazin-2-yl)amino]-3-[2-(1,5-disulfonatonaphthalen-2-yl)diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate

Details on test material:

Substance H112339 was supplied by ICI Specialties as a dark brown powder. A single batch of Substance H112339 was used throughout the study (NBY 405/74) and was assigned the CTL reference number Y07719/001. The test substance was reported to have a water content of 10.5% w/w. The purity of Substance H112339 in the test substance was 71*6% w/w (certificate of analysis ASG Ref. No. 9108127, dated 8/8/91). From informationsupplied by the Sponsor, the test substance was stable for the duration of the study.

The sample was stored in a ventilated cupboard in the CTL Central Dispensary facility at ambient temperature.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd
- Age at study initiation: approximately 4 weeks old
- Weight at study initiation: Mean - Males: 161.2g, Females: 133.2g
- Fasting period before study: No data
- Housing: multiple rat cages five per cage.
- Diet (e.g. ad libitum): CT1, ad libitum
- Water (e.g. ad libitum): filter-sterilised mains water, via an automatic watering system, ad libitum
- Acclimation period: No data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40-70%
- Air changes (per hr): 25-30
- Photoperiod (hrs dark / hrs light): Twelve hour periods of light were cycled with twelve hour periods of darkness

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionised

Details on oral exposure:

The dosing solutions were prepared by the Central Dispensary at CTL. The control article and vehicle for the test substance was deionised water, CTL reference number Y04517/Q15, For each dose level an appropriate amount of water was added when required to a weighed amount of test substance, adjusted for the water content of 10.5% w/w. Each preparation was thoroughly mixed and subdivided into aliquots. The control article was also dispensed into aliquots. Fresh aliquots were used for each day of the study. Further preparations were made at appropriate intervals throughout the study.

Dosing solutions were shaken before dosing. Rats were dosed by oral gavage, at l.0ml/l00g according to their daily individual bodyweights. Rats were dosed sequentially within group order at approximately the same time each day.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A sample of each solution was analysed prior to dosing to verify the achieved concentration of Substance H112339 in water. In addition, prior to the start of dosing, the stability of Substance HI12339 in water, under the conditions of storage used, was determined.

Samples were diluted with deionised water to give solutions containing approximately 22 ug/ml of the test substance. The absorbances of the diluted solutions were measured on a spectrophotometer at 540 nm and the concentration calculated by reference to a single calibration standard.

The absorbances of the sample and the standard solutions were measured in quartz cells against a reference cell containing deionised water, using a Philips PU8800 spectrophotometer.

Duration of treatment / exposure:

Test duration: 28 days. A recovery group dosed at 1000 mg/kg/day was observed for a recovery period of a further 14 days.

Frequency of treatment:

Dosing regime: 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0 (control), 50, 250 or 1000mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Group Dose Level of Substance H112339 (mg/kg/day) Identities of Rats
Males : Females
1 0 1-5 31-35
2 0 (recovery) 6-10 36-40
3 50 11-15 41-45
4 250 16-20 46-50
5 1000 21-25 51-55
6 1000 (recovery) 26-30 56-60

Control animals:

yes

Details on study design:

The study was divided into two single sex replicates (randomised blocks). Each replicate consisted of six cages of rats, one per treatment group.
During the initial acclimatisation period, the rats were allocated randomly to cages by a method designed to minimise any intergroup differences in bodyweight. Animals not required for the study were discarded.

Positive control:

No

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS:
Prior to the start of the study all rats were examined to ensure that they were physically normal and exhibited normal activity. Daily clinical observations were recorded immediately prior to dosing. Each rat was also observed at least once daily, post-dosing, during the study.

DETAILED CLINICAL OBSERVATIONS:
Detailed clinical observations, including the finding of no abnormalities detected, were recorded at the same time that bodyweights were recorded. Any rats requiring euthanasia were killed and subjected to a post, mortem examination.

BODY WEIGHT:
The bodyweight of each rat was recorded daily, immediately before dosing on days 1 to 28. In addition, rats in groups 1, 3, 4 and 5 were weighed prior to termination on day 29 and rats in groups 2 and 6 were weighed on days 29, 36 and prior to termination on day 43.

FOOD CONSUMPTION:
Food consumption for each cage of animals was recorded at intervals from day 1 throughout the study for each cage until termination on days 29 or 43. Food consumption was calculated weekly. Food consumption for females (replicate 2) was not recorded on day 1, in error. Food consumption recording for these animals commenced on day 2.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY:
At termination on day 29 or day 43, all surviving rats were bled by cardiac puncture and samples collected in tubes containing EDTA as an anticoagulant. The following parameters were determined: haemoglobin, total white cell count, red cell count, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, haematocrit and platelet count. All these values were measured using a Technicon HI (Bayer Diagnostics PLC). Further samples of blood were collected in tubes containing 0.11M trisodium citrate as an anticoagulant and prothrombin and kaolin-cephalin times were measured on a 'Coag-a-mate' (Organon Teknika), In addition, blood films were prepared from all animals and submitted for Romanowsky staining. A white cell differential count and examination of red cell morphology were performed on control and high dose animals. Bone marrow smears were taken at necropsy and submitted for Romanowsky staining but were not examined.

CLINICAL CHEMISTRY:
At termination on day 29 or day 43, all surviving rats were bled by cardiac puncture and samples collected into tubes containing lithium heparin as an anticoagulant.

Samples were submitted for measurement of the following plasma parameters: urea, creatinine, glucose, albumin, total protein, cholesterol, triglycerides, total bilirubin, sodium, potassium, chloride, calcium, phosphorus (as phosphate) and alkaline phosphatase, alanine transaminase, creatine kinase, aspartate transaminase and gamma-glutamyl transferase activities. . All these parameters were measured on a KONE SPECIFIC analyser.

URINALYSIS:
Individual urine samples were collected from all rats in groups 1, 3, 4 and 5 on day 22/23, and from all rats in groups 2 and 6 on day 36/37. Samples were collected over a period of 16-18 hours during which the rats were individually housed in metabolism cages (North Kent Plastics Ltd) and denied access to food and water.

The volume and colour of each sample were recorded and the pH measured with a Corning 109 pH meter. The specific gravity was measured using an Atago refractometer. Glucose, ketones, urobilinogen and blood (semi-quantitative using BM-Test-7 strips, BCL) and protein (using a modified Ponceau S method) were also measured.

In addition, an aliquot of each urine sample was centrifuged, stained with Sedicolor (Dr Molter GMBH) and examined under a microscope for the identification of the components of the urine sediment

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

All rats were killed by exsanguination using cardiac puncture under terminal anaesthesia with halothane BP (FLUOTHANE, ICI Pharmaceuticals, Macclesfield, Cheshire, UK) vapour and subjected to a post mortem examination. Testes, adrenal glands, kidneys, liver and brain were weighed. Paired organs were weighed together. The following tissues were removed:

Adrenal gland, aorta, bladder, bone (femur including stifle joint), bone marrow (femur), brain, caecum, cervical lymph node, cervix, colon, duodenum, epididymis, eye, Harderian gland, heart, ileum, jejunum, kidney, liver, lung, mammary gland (inguinal - female only), mesenteric lymph node, nasal passages, oesophagus, oral cavity, ovary, pancreas, parathyroid gland, pituitary gland, prostate gland, rectum, salivary gland, sciatic nerve, seminal vesicle, skin (right flank), spinal cord, spleen, sternum, stomach, testis, thymus, thyroid gland, trachea, uterus, voluntary muscle and any macroscopically abnormal tissue.

All tissues were fixed in 10% neutral buffered formol saline except for testis, epididymis, skin and mammary gland which were fixed in Bouin's fluid, and eye and Harderian gland which were fixed in Davidson's solution. The nasal cavities of all rats were perfused with 10% neutral buffered formol saline.

Tissues for histology were routinely processed, embedded in paraffin wax, and 5p,m thick sections were cut and then stained with haematoxylin and eosin. All sections were examined by light microscopy.

Adrenal gland, heart, kidney, liver, spleen and any macroscopically abnormal tissues from all groups were routinely embedded in paraffin wax. The remaining tissues were stored. 5p,m sections of all processed tissues from control rats and rats receiving lOOOmg/kg/day at terminal kill, and from any intercurrently killed animal, and kidney, liver, spleen and macroscopically abnormal tissue from rats receiving 50 or 250mg/kg/day at terminal kill, together with kidney, liver and spleen from control rats and rats receiving l000mg/kg/day at recovery kill, were' cut and stained with haematoxylin and eosin. All sections were examined by light microscopy.

Other examinations:

None

Statistics:

Bodyweights were considered by analysis of covariance on initial (day 1) bodyweight. separately for males and females.

Haematology and blood and urine clinical chemistry were considered by analysis of variance. With the exception of urine protein, for which the sexes showed markedly differing variances, male and female data were analysed together and the results examined to determine whether any differences between control and treated groups were consistent between sexes.

Organ weights were considered by analysis of variance and analysis of covariance on final bodyweight, separately for males and females.

All analyses were carried out separately for main study and recovery animals.

All analyses were carried out using the GLM procedure in 5AS (1985). Least square means for each group were calculated using the LSMEAN option in SAS PROC GLM. Unbiased estimates of differences from control were provided by the difference between each treatment group least square mean and the control group least square mean. Differences from control were tested statistically by comparing each treatment group least square mean with the control group least square mean using a two-sided Student's t-test, based on the error mean square in the analysis.

The differences from control based on the analysis of bodyweight adjusted for initial weight are also presented graphically. The centre of each bar represents the mean percentage difference between control and treated group least square means, and the top and bottom of each bar represent the upper and lower 95% confidence limits for this difference. If the bar does not cross the zero difference line at a particular time, there is a statistically significant difference between the control and treated group at that time. For ease of reference, lines have been added to the plot to show differences of ±10%.

Results and discussion

Results of examinations

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

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Considered incidental

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance colouration

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Considered incidental

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Test substance colouration

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Liver & kidneys

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
There were no compound-related deaths during the study

Purple staining of the tail and production of purple coloured faeces and/or urine, was noted for animals receiving 1000mg/kg/day throughout the dosing period. Throughout the recovery period, staining of the tail was still apparent for animals from the 1000mg/kg/day group, although purple coloured faeces and urine were not passed by animals from this group from day 30 (i.e. day 2 of recovery). Staining of the tail was also seen for some animals receiving 250mg/kg/day during the last week of the dosing period. Staining of the tail and production of coloured faeces or urine was considered to be related to the highly coloured nature of the test material. Data relating to the production of purple coloured faeces or urine is included in the Individual Animal Data Supplement to this report.

Hunched posture and piloerection were also recorded for one female (No, 60) from the 1000mg/kg/day group on the last day of the recovery period.

There were no other clinical observations noted during the study that were considered to be compound-related.


BODY WEIGHT AND WEIGHT GAIN
During weeks 1 to 4 of treatment, there were no effects on bodyweight that could be attributed to administration of Substance H112339. During the 14-day recovery period, group mean bodyweight, for animals in the 1000mg/kg/day group, was slightly lower than that of the concurrent control group, especially for males. However, these differences did not attain statistical significance.

Markedly reduced bodyweight was recorded for one female (No, 60) from the 1000mg/kg/day group during the last week of the recovery period.

The loss of bodyweight on day 23 for all main group animals was considered to be due to the collection of urine samples on that day.

FOOD CONSUMPTION
During weeks 1 to 4 of treatment, there were no effects on food consumption that could be attributed to administration of Substance H112339. During the last week of the recovery period, food consumption for females from the l000mg/kg/day group was slightly reduced.

HAEMATOLOGY
Slight reductions in group mean haemoglobin, haematocrit and red cell count were noted for females receiving 1000mg/kg/day at terminal kill, in comparison with controls. These changes were also present for females from the l000mg/kg/day group at recovery kill. These changes were not apparent for males receiving 1000mg/kg/day, or for males or females at 50 or 250mg/kg/day, at terminal or recovery kill.

All other intergroup differences were minor and/or sporadic and are considered incidental to treatment with Substance HI12339.

CLINICAL CHEMISTRY
Pink colouration of the plasma was noted for animals receiving 1000mg/kg/day at terminal kill and was considered to be a result of the highly coloured nature of the test material. This was not apparent following completion of the recovery period.

Mean plasma alanine transaminase activities for males receiving 250 or 1000mg/kg/day, and for females at 1000mg/kg/day, were statistically significantly lower than the corresponding control values at terminal kill. Mean plasma alkaline phosphatase activities for males and females receiving 1000mg/kg/day were also statistically significantly lower than the control values at terminal kill. These changes were not apparent for animals from the 1000mg/kg/day at recovery kill.

At recovery kill, the plasma sample from one female (No. 60) from the 1000mg/kg/day group, was observed as being grey in colour, with raised urea, creatinine, total protein and potassium levels, increased alkaline phosphatase activity and reduced alanine transaminase activity.

Other statistically significant differences between control and test groups were considered to be incidental and of no toxicological importance. The statistically significant increase in mean plasma bilirubin levels seen at terminal kill for males and females receiving 1000mg/kg/day, in comparison with the controls, was considered to be due to interference from the highly coloured nature of the test material and to be of no toxicological importance.

URINALYSIS
Test substance colouration of the urine was noted for all animals receiving 250 or 1000mg/kg/day in week 4, with urine colours of light orange at 250mg/kg/day and orange to dark red at 1000mg/kg/day being recorded. The effect was so marked at 1000mg/kg/day that all the qualitative tests were rendered unreadable. In the recovery period (week 6), the urine from all animals in the 1000mg/kg/day group was still coloured pale orange to dark red and all urines were cloudy. The intensity of colouration at week 6 was not as great as that in week 4 and did not prevent all qualitative tests from being performed.

Group mean urinary pH for females receiving 250 or 1000mg/kg/day during week 4, and for females at 1000mg/kg/day during week 6, was statistically significantly higher than that of the corresponding controls. In addition, group mean urinary protein levels for females at 1000mg/kg/day were higher than control values in weeks 4 and 6.

The qualitative tests showed no trends or effects at week 4. However, at week 6 all animals from the 1000mg/kg/day group had blood present in the urine, with females more severely affected. This was consistent with the cloudy appearance of the urine.

Cloudy urine, with high levels of blood and urinary protein was produced by one female (No. 60) from the 1000mg/kg/day group in week 6.

Urine Sediments: Large numbers of renal epithelial cells were present in the urine sediments from all animals receiving 1000mg/kg/day, in week 4, and in the urine sediments from all animals in the 1000mg/kg/day group at week 6. Large numbers of red blood cells were also present in the urine sediments from one female (No 38) from the control group and one female (No, 60) from the 1000mg/kg/day group in week 6.

ORGAN WEIGHTS
At terminal kill, group mean kidney weights, absolute and adjusted for initial bodyweight, for males and females receiving 1000mg/kg/day and for males only at 250mg/kg/day, were statistically significantly higher than the corresponding control values. At the recovery'kill, mean kidney weight, absolute and adjusted for initial bodyweight, for females from the 1000mg/kg/day group only, was statistically significantly higher than the control value. Kidney weight for two males from the 1000mg/kg/day recovery group was also higher than that of the concurrent controls.

In addition, at recovery kill, group mean liver weight, adjusted for initial bodyweight, for males from the 1000mg/kg/day group was statistically significantly higher than that of the concurrent control group. Group mean brain weight for males from the 1000mg/kg/day group was also higher than the control value, at recovery kill. However, this was considered to be due to the lower bodyweight of these animals, in comparison to concurrent controls, and to be unrelated to administration of Substance H112339.

Other intergroup differences noted for the remaining treated groups were considered to be incidental and unrelated to treatment.

High individual kidney, liver and adrenal gland weights were recorded for one female (No. 60) from the 1000mg/kg/day group at recovery kill.

GROSS PATHOLOGY
Pink discolouration of most tissues from all animals receiving 1000mg/kg/day was seen at terminal and recovery kill. Pink staining was also present at 250mg/kg/day but was less prominent, being confined to staining of the tail of 5 males and stomach of one male and one female at terminal kill only. This was considered to be due to the highly coloured nature of the test material..

At recovery kill, one female (No. 60) from the 1000mg/kg/day group showed a range of abnormalities, including enlarged kidneys with pelvic dilatation, and pale areas in one kidney, dilated ureters, hard calculi in the bladder, pale bone marrow and enlarged adrenal glands and para-aortic lymph node.

There were no other changes considered to be related to treatment. One male (No. 10) from the control recovery group, which waskilled for humane reasons on day 28 had large red areas on the lungs,' froth in the trachea and pin-point red areas on the thymus. These findings are consistent with misdosing.


HISTOPATHOLOGY: NON-NEOPLASTIC
Treatment-related changes were present in the kidneys and liver.

At terminal kill, increased hyaline droplet formation was present in the proximal tubules of the kidneys of all males receiving 1000mg/kg/day. In females at terminal kill, there was a dose-related increase in the severity of degeneration of the proximal tubules of the kidneys at 250 or 1000mg/kg/day. This degeneration was characterisedflby increased basophilia (distinct from the minimal focal tubular basophilia noted for other groups of animals) and nuclearity of the tubular cells, especially in the straight portions, and hyaline droplet formation in the tubular cells of the convoluted portions. In addition, minimal, or slight interstitial mononuclear cell infiltration of the kidneys was apparent in all females at 1000mg/kg/day, and was considered to be a secondary change following degeneration of the tubules.

In the recovery rats, there was increased hyaline droplet formation, degeneration of the proximal convoluted tubules and interstitial mononuclear cell infiltration in the kidneys of all males from the 1000mg/kg/day group. In females at recovery kill, renal proximal tubular degeneration in all animals from the 1000mg/kg/day group, and interstitial mononuclear cell infiltration in 4 animals from this group, was noted. The features of the renal tubular degeneration in males and females at recovery kill were similar to those present in the females at terminal kill, which suggested that, for male rats in particular the onset of the lesion is delayed and for female rats the severity of the renal lesion had not reduced over the.recovery period.

Microscopic changes noted in the liver consisted of minimal or slight mononuclear cell infiltration of the periportal areas in 4 males and 4 females receiving 1000mg/kg/day at terminal kill and for all animals from the 1000mg/kg/day group at recovery kill.

In one female rat (No. 60) from the 1000mg/kg/day recovery group, there was marked cystitis, moderate pyelonephritis and associated bone marrow hyperplasia and para-aortic lymph node hyperplasia.

There were no other changes considered to be related to treatment, and no specific microscopic findings were present to account for the pink discolouration of tissues recorded macroscopically. Haemorrhage in the lungs and thymus, and oedema of the lungs, was apparent for the male (No. 10) from the control recovery group, which was killed for humane reasons on day 28. These findings are consistent with misdosing.

Effect levels

Dose descriptor:

NOEL

Effect level:

50 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: organ weights

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Tabulated data from the study is attached below.

Applicant's summary and conclusion

Conclusions:

Groups of six male and six female Crl:(WI)BR (Wistar) rats were dosed orally, by gavage, with Substance H112339 at dose levels of 0 (control), 50, 250 or 1000 mg/kg/day for 28 consecutive days. Two additional groups were similarly dosed with 0 or l000 mg/kg/day for 28 consecutive days and then retained without treatment for a further 14 days. Clinical observations, bodyweight and food consumption were recorded throughout the study. At termination, rats were given a full post mortem examination. Blood samples were taken for clinical chemistry and haematological examination. Urine samples were also submitted for clinical chemistry examination and tissues were taken for histopathological examination.

There were no treatment-related deaths or toxicologically significant effects on clinical condition, or food consumption. There were no treatment-related effects on bodyweight during weeks 1 to 4 of treatment. However, during the recovery period, group mean bodyweight for animals from the l000mg/kg/day group was slightly, but not statistically significantly, lower than that of the concurrent controls. Slight reductions in mean haemoglobin, haematocrit and red cell count were noted for females at 1000 mg/kg/day at terminal and recovery kill. Mean plasma alanine transaminase and/or alkaline phosphatase activities were also reduced for males and/or females at 250 and/or 1000 mg/kg/day at terminal kill, only.

Higher group mean urinary pH and/or protein levels for females at 250 and/or 1000mg/kg/day were noted in week 4 and/or week 6 (recovery week 2). In addition large quantities of renal epithelial cells were present in the urine sediments from all animals receiving 1000mg/kg/day in weeks 4 and 6.

Higher group mean kidney weights were noted for males and/or females at 250 and/or 1000mg/kg/day at terminal kill and for females, only, from the 1000mg/kg/day group at recovery kill. Kidney weights for two males from the l000 mg/kg/day group were also higher at recovery kill. Higher group mean liver weights far males from the 1000 mg/kg/day group were also apparent at recovery kill, only increased hyaline droplet formation in the proximal tubules of the kidneys of all males at 1000mg/kg/day, and a dose-related degeneration of the proximal tubules of all females at 250 or 1000mg/kg/day, was noted at terminal kill. At recovery kill, proximal tubule degeneration, similar to that seen for females at l000 mg/kg/day at terminal kill, was present in males and females from the 1000 mg/kg/day group.

Other histopathological changes considered to be related to treatment included minimal or.slight mononuclear cell infiltration of the periportal areas of the liver from animals receiving 1000 mg/kg/day, at both terminal and recovery kills.

Widespread pink/purple staining of tissues and colouration of plasma, urine and/or faeces was seen for animals receiving 250 and/or 1000mg/kg/day throughout the dosing and/or recovery periods and was considered to be due to the highly coloured nature of the test material and not due to any histopathological change.

The no-observable effect level for Substance HI12339, following daily oral administration to male and female rats for 28 days, is 50mg/kg/day.

Executive summary:

Study conducted in compliance with GLP. No test guidance is referenced within the study report.

Changes of toxicological importance were noted for animals received 250 or 1000mg/kg/day over a 28 day period, with the kidney and to a lesser extent the liver represnting the target organs for toxicity. The NOEL for the substance in the rat is therefore 50mg/kg/day for males and females following daily oral administration over a 28 day period.