1,5-Naphthalenedisulfonic acid, 2-[2-[8-[[4-chloro-6-[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]-1-hydroxy-3,6-disulfo-2-naphthalenyl]diazenyl]-, sodium salt (1:5)

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 2020 to September 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 25 June 2018

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

402-420-3

EC Name:

-

Cas Number:

89157-03-9

Molecular formula:

C31H19ClN7Na5O19S6

IUPAC Name:

pentasodium 5-{[4-chloro-6-({4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}amino)-1,3,5-triazin-2-yl]amino}-3-[2-(1,5-disulfonatonaphthalen-2-yl)diazen-1-yl]-4-hydroxynaphthalene-2,7-disulfonate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley SD rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco)
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: 94-125 g for males and 97-110 g for females
- Fasting period before study: No
- Housing: up to 5 of one sex to a cage, in clear polysulfone solid bottomed cages
- Diet (e.g. ad libitum): 4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy) ad libitum throughout the study, except at the end of Week 13 of treatment before blood and urine collection.
- Water (e.g. ad libitum): ad libitum to each cage via water bottles, except at the end of Week 13 of treatment before blood and urine collection.
- Acclimation period: 19 days before the start of treatment

DETAILS OF FOOD AND WATER QUALITY: certified by regular analysis
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet. Records of analysis of water, diet and bedding material are kept on file at the test facility.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C±2°C
- Humidity (%): 55%±15%
- Air changes (per hr): 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): artificial light for 12 hours each day.

IN-LIFE DATES: From: 06 May To: 08 September 2020

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route was selected as it is a possible route of exposure of the test item in man.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The required amount of test item was dissolved in the vehicle.

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the preparations prepared onWeeks 1 and 13 were analysed to check the concentration.
Results of the analyseswere within the acceptability limits stated in the protocol
for concentration (90-110%). Results of the analyses were within the acceptability limits
stated in ERBC SOPs for solution concentration (90-110%)

Duration of treatment / exposure:

All animals were dosed for a minimum of 13 consecutive weeks.

Frequency of treatment:

All animals were dosed once a day, 7 days a week.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

Each main group comprised 10 male and 10 female rats. Control and high dose groups
included 5 additional animals per sex to be sacrificed after 4 weeks of recovery.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were selected i based on information from a sub-acute toxicity study in rats
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: yes
- Rationale for selecting satellite groups: recovery or effects
- Post-exposure recovery period in satellite groups: 4 weeks
- Section schedule rationale (if not random): random
- Other:

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Mortality, Clinical signs
- Time schedule: 3 times a week
- Observations of the cage tray (data were not tabulated in the report but are archived with the raw data of the study)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before study start and at least once per week during the study
- Assessment in an open arena: changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern), changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 12 and Week 4 of recovery
- Dose groups that were examined: All
- Battery of functions tested: sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli), assessment of grip strength, motor activity

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of allocation to treatment group, first day of treatment, weekly thereafter, prior to necropsy

FOOD CONSUMPTION:
- Time schedule for examinations: weekly
- Food consumed by each cage of rats was recorded, the group mean daily intake per rat was calculated

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to study start and Week 13
- Dose groups that were examined: All animals at study start, high dose and control groups during Week 13

HAEMATOLOGY: Yes
- Time schedule for collection of blood: last week of treatment and recovery and during necropsy for coagulation
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells, Platelets), Coagulation, Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: last week of treatment and recovery
- Animals fasted: Yes
- How many animals: all
- Parameters examined:, Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Blood urea nitrogen, Creatinine, Bile acids, Glucose, Triglycerides, Inorganic phosphorus, Total bilirubin, Total cholesterol, HDL, LDL, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride

PLASMA/SERUM HORMONES/LIPIDS: Yes
- Time of blood sample collection: last week of treatment and recovery
- Animals fasted: Yes
- How many animals: all

URINALYSIS: Yes
- Time schedule for collection of urine: last week of treatment and recovery
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined:, Appearance, Volume (manually recorded), Specific gravity, pH, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Blood, Epithelial cells, Leucocytes, Erythrocytes, Crystals, Spermatozoa and precursors, Other abnormal components

IMMUNOLOGY: No

OTHER:
Estrous cycle
At the end of the study, just prior to necropsy, vaginal smears were taken from all surviving female animals, and the estrous cycle phase recorded.

Sacrifice and pathology:

Euthanasia
Animals that had completed the scheduled test period were killed by exsanguination under isoflurane anaesthesia. All animals, including those found dead,were subjected to necropsy, supervised by a pathologist, as detailed below.

Necropsy
From all animals completing the scheduled test period, the following organs: Adrenal glands; Brain (cerebrum, cerebellum, medulla/pons); Epididymides; Heart; Kidneys; Liver; Ovaries; Pituitary gland; Seminal vesicles; Spleen; Testes; Thymus (where present); Thyroid gland; Uterus – cervix, were dissected free of fat and weighed. The ratios of organ weight to body weight were
calculated for each animal.

Tissues fixed and preserved
Samples of: Abnormalities, Adrenal glands, Aorta, Bone marrow (from sternum), Brain (cerebrum, cerebellum, medulla/pons), Caecum, Coagulating glands, Colon, Duodenum, Epididymides, Eyes, Femur with joint, Heart, Ileum, Jejunum (including Peyer’s patches), KidneyLiver, Lungs (including mainstem bronchi), Lymph nodes – cervical, Lymph nodes – mesenteric, Mammary gland (Males and Females), Oesophagus, Ovaries, Oviducts, Pancreas, Parathyroid glands, Pituitary gland, Prostate gland, Rectum, Salivary glands, Sciatic nerve, Seminal vesicles, Skeletal muscle,
Skin, Spinal column, Spinal cord (cervical, thoracic, lumbar), Spleen, Stomach, Testes, Thymus (where present), Thyroid gland, Trachea, Urinary bladder, Uterus – cervix, Vagina, were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves, Harderian glands, testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol).

Histopathological examination
The tissues required for histopathological examination are:
Abnormalities
Adrenal glands
Aorta
Bone marrow (from sternum)
Brain (cerebrum, cerebellum, medulla/pons)
Caecum
Coagulating glands
Colon
Duodenum
Epididymides
Heart
Ileum
Jejunum (including Peyer’s patches)
Kidneys
Liver
Lungs (including mainstem bronchi)
Lymph nodes – cervical
Lymph nodes – mesenteric
Mammary gland (Males and Females)
Oesophagus
Ovaries
Oviducts
Pancreas
Parathyroid glands
Pituitary gland
Prostate gland
Rectum
Salivary glands
Sciatic nerve
Seminal vesicles
Skin
Spinal cord (cervical, thoracic, lumbar)
Spleen
Stomach
Testes
Thymus (where present)
Thyroid gland
Trachea
Urinary bladder
Uterus – cervix
Vagina

Optional endpoint(s):

Optional endpoints: Yes

Statistics:

Standard deviations were calculated as considered appropriate. For continuous variables
the significance of the differences amongst groups was assessed by analysis of variance.
Differences between each treated group and the control group were assessed by Dunnett’s
test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s
test before Dunnett’s test. If the data were found to be inhomogeneous aModified t test
(Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from the
actual values in the computer without rounding off. Statistical analysis of histopathological
finding was carried out by means of a nonparametric Kolmogorov-Smirnov test.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

red staining of the tail: all males and females at 300 and 1000 mg/kg body weight/day from the second week of treatment period onward

Mortality:

mortality observed, non-treatment-related

Description (incidence):

- one male from the low dose group (no. 42) was found dead on Day 86. No clinical signs were observed in this animal. At macroscopic observation, pink colour of trachea, oesophagus, stomach (glandular and non glandular region) and pancreas were seen. Histopathology war not possible, as most organs were autolytic. Presumably, this was related to misdosing.
- one female from the high dose group (no. 79) was found dead on Day 15. Clinical signs were limited to red staining of the tail. At macroscopic observation, dark colour of lungs, dark red fluid of thoracic cavity and red staining of the muzzle were seen. At microscopic observation, marked alveolar and interstitial haemorrhage of lungs were found. Death of this animal was attributed to
accidental trauma most likely due to misgavage.

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:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Slight increases of urea and creatinine in females dosed at 300 and 1000 mg/kg body weight/day and in males receiving 1000 mg/kg body weight/day (creatinine only) were seen.
Due to the slight severity and the absence of renal morphological changes, these findings were considered to be not adverse.

A dose-related plasma colouration (pink to fuchsia) was recorded in all treated samples. This was due to the presence of test substance and/or its metabolites in the blood.

Endocrine findings:

no effects observed

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

A dose-related urinary colouration (pink to fuchsia) was recorded in all treated samples.
This was due to the presence of test substance and/or its metabolites in the urine. In the
absence of other findings, this change was not considered to be adverse. This effect was no longer seen in recovery animals.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

A slight, but statistically significant increase in relative kidneys weight was recorded in males treated at 1000 and 300 mg/kg body weight/day and females treated at 1000 mg/kg body weight/day. Absolute mean kidneys weight were minimally increased in males treated at 1000 mg/kg body weight/day

At the end of the recovery phase, no changes were noted in treated males indicating the reversibility of this effect, in females relative kidney weights were minimally increased.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Dark and/or pink discolouration of most tissues and organs was seen in most high dose animals of both sexes, including urogenital tract (i.e. kidneys, urinary bladder, testes, prostate, vagina and uterus), gastrointestinal tract (i.e. glandular and non glandular region of the stomach, duodenum,
ileum and rectum), trachea, cervical lymph nodes, mesenteric lymph nodes and on the external surface of the body and tail, when compared to controls. This dark and/or pink discolouration was also observed in kidneys of all males and most of the females of the mid-dose group, and, although with a lower incidence, in low and/or mid- dose male and/or female rats in some organs and tissues such as testes, gastro-intestinal tract (stomach and rectum) and tail. This finding was considered likely due to the colour of the test item.

After a four-week recovery period, the macroscopic change consisting in dark and/or pink discolouration was still present in most animals of both sexes previously dosed at 1000 mg/kg body weight/day; however in a reduced number of tissues and organs compared to those observed at the end of the treatment period, indicating the reversibility of this effect.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Tubular cell vacuolation of the cortex of the kidneys, characterised by the presence of dark red (test item like) material in the vacuoles, was observed in all mid- and high dose treated animals of both sexes. The severity of the renal finding was minimal to moderate in high dose animals and minimal in mid-dose animals of both sexes.
No renal morphological changes were associated with the intra-cellular deposition of the test item in the tubular cells of the cortex.

After 4 weeks of recovery period, deposition of the test material in vacuoles of tubular cells in kidneys was still present, but to a much lower degree, indicating reversibility of this effect.

Since at histopathological evaluation no renal morphological changes were associated with
the intra-cellular deposition of the test item in the tubular cells of the cortex and the slight changes in clinical chemistry parameters were not considered to be suggestive of organ injury at 300 and 1000 mg/kg body weight/day, the treatment-related change in kidneys was not considered adverse.

Other effects:

no effects observed

Description (incidence and severity):

Staging of spermatogenic cycle
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; the qualitative testis staging did not indicate any abnormalities in the integrity of the various cell types present within the different stages of the spermatogenic cycle (Stages I- XIV) in all control and treated males of the high dose.

Estrous cycle
Normal physiology of the estrous cycle (estrous, metestrous, diestrous and proestrous) was noted in control and treated high dose females. No morphological changes were detected when compared to each “estrous phase” in the ovaries, uterus/cervix and vagina.

Details on results:

One low dose male (100mg/kg body weight/day) and one high dose female (1000mg/kg
body weight/day) were found dead on Days 86 and 15 of dosing, respectively.
The cause of death was not established for the low dose male; the high dose female died for
an accidental trauma.
No relevant clinical signs were recorded during the study. No signs indicating neurotoxic
effects were seen during the in vivo phase of the study.
Body weight, food consumption and estrous cycle were not affected by treatment. No
lesions were recorded at ophthalmological examination.
No treatment-related changes were recorded at the end of treatment and recovery periods
in haematological, coagulation and urinalysis parameters. No treatment-related changes
were recorded for thyroid hormones.
Storage of dye particles (such as cytoplasmic vacuolation, characterized by the presence
of dark red, test item like, vacuoles in renal tubule epithelium of the cortex) were seen in
the kidneys of mid- and high dose animals, resulting in a higher kidney weight and a slight
increase in creatinine and/or urea. Since at histopathological evaluation no renal morphological
changes were associated with the intra-cellular deposition and accumulation of
the test item in the tubular cells of the cortex and the slight changes in clinical chemistry
parameters were not considered to be suggestive of organ injury, the treatment-related
change in kidneys was not considered adverse.
A statistically significant increase in the kidneys weight was still seen in the females at the
end of recovery but not in males, indicating the reversibility of the effects, supported by the
reversibility of the effects in clinical chemistry and organ weights.
Dark and/or pink discolouration of most tissues and organs was observed in most animals
of both sexes dosed at 300 and 1000mg/kg body weight/day at post mortem macroscopic
examination. This was still present in animals of both sexes at the end of the recovery,
however, in a reduced number of tissues and organs compared to those observed at the
end of the treatment period, indicating the reversibility of this effect.
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle
and to the integrity of the various cell types within the different stages; regular layering in
the germinal epithelium was noted in all control and treated males.
Normal physiology of the estrous cycle (estrous, metestrous, diestrous and proestrous)
was noted in control and treated females. No morphological changes were detected, when
compared to each “estrous phase” in the ovaries, uterus/cervix and vagina.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

No treatment-related adverse effects were seen trhoughout the study. The No Observed Adverse Effect Level (NOAEL) for this study is 1000 mg/kg body weight/day.

Executive summary:

The toxicity of Reactive Red 239, in rats after oral administration for 13 weeks and recovery from any treatment-related effects during a recovery period of 4 weeks, were investigated in this study. Three groups, each of 10 male and 10 female Sprague Dawley rats, received the test item by gavage at dosages of 100, 300 and 1000 mg/kg body weight/day for 13 consecutive weeks. A fourth similarly constituted group received the vehicle alone (softened water) and acted as a control. Five additional animals for each sex were included in the control and high dose groups for recovery assessment.

The following investigations were performed: daily clinical signs, weekly detailed clinical signs (removal from cage and open field observations), evaluation of sensory reactivity to stimuli, grip strength and motor activity, bodyweight, food consumption, ophthalmoscopy, clinical pathology investigations (including thyroid hormones determination), terminal body weight, organ weights, macroscopic observations, histopathological examination.

Two cases of premature death, one male from the low-dose group (100 mg/kg body weight/day) and one female from the high dose group (1000 mg/kg body weight/day) were found dead on Days 86 and 15 of dosing, respectively. No relevant clinical signs were observed in these animals. The cause of death was not established for the low dose male; the high dose female died for an accidental trauma.

No signs of toxicological relevance were observed during the study. Slight to marked red staining and red faeces were observed in the cages of mid- and high dose animals (dosed at 300 and 1000 mg/kg body weight/day). Weekly detailed clinical signs observation of animals at removal from the cage and in an open arena did not reveal changes attributable to the test item. Neurotoxicity assessment revealed no relevant changes between treated and control animals, at functional tests (sensory reactivity, landing foot splay, grip strength). No treatment-related differences were noted in the motor activity measurements.

No changes were noted in mean body weight and mean body weight gain, during the treatment and recovery periods. Food consumption was not affected by treatment with the test item. No treatment-related findings were recorded during ophthalmological examinations of control and test-item treated animals. Also, were no treatment-related anomalies noted in the estrous cycle of treated females, when compared to controls.

During haematological evaluations, no treatment-related changes were recorded at the end of treatment and recovery periods in haematological and coagulation parameters. In clinical chemistry parameters, there were no changes that could be considered adverse at the end of treatment or recovery phases. Treatment-related, but not adverse, findings resulting test-item storage in vacuoles of the tubular cells of kidneys were represented by the increases of urea and creatinine in females dosed at 300 and 1000 mg/kg body weight/day and in males receiving 1000 mg/kg body weight/day (creatinine only). A dose-related plasma colouration (pink to fuchsia) was recorded in all treated samples. This was due to the presence of test substance and/or its metabolites in the blood. No relevant differences between control and treated animals were recorded at the end of recovery, confirming complete reversibility. Thyroid hormone determination (T3, T4 and TSH) revealed no treatment-related changes. No changes that could be considered adverse were observed at the end of treatment in urine parameters. A dose-related urinary colouration (pink to fuchsia) was recorded in all treated samples. No treatment-related changes were observed at the end of recovery, representing the reversibility of this effect.

No changes were observed in terminal body weight of treated animals of both sexes that completed the treatment and recovery period, when compared to the controls. A slight, but statistically significant, increase in relative and/or absolute mean kidneys weight was recorded in males dosed at 300 and 1000 mg/kg body weight/day and in females dosed at 1000 mg/kg body weight/day. This increase was accompanied by histopathological changes and therefore was considered to be treatment related. A minimal statistically significant increase was still seen in the kidneys of the females previously dosed at 1000 mg/kg body weight/day at the end of recovery, but not in males, indicating the reversibility of this effect.

Macroscopic observations found dark and/or pink discolouration of most tissues and organs in most mid- and high-dose animals of both sexes, including urogenital tract, gastrointestinal tract, trachea, cervical lymph nodes, mesenteric lymph nodes and on the external surface of the body and tail, when compared to controls. After 4 weeks of recovery, dark and/or pink discolouration was still seen in some organs or tissues, however in a reduced number of tissues and organs compared to those observed at the end of the treatment period, indicating the reversibility of this effect.

In microscopic examinations, tubular cell vacuolation were present in the kidneys of mid- (minimal degree) and high (minimal to moderate) dose animals of both sexes (300 and 1000 mg/kg body weight/day), representing a deposition of the test-item in these cells. After 4 weeks of recovery, histopathological changes of minimal degree were observed in the kidneys of all high dose animals; the lower severity of this effect indicated partial recovery. As the intra-cellular deposition of the test item in the tubular cells of the cortex did not cause renal morphological changes, they were considered to be not adverse.

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted in all control and treated males. Normal physiology of the estrous cycle (estrous, metestrous, diestrous and proestrous) was noted in control and treated females. No morphological changes were detected, when compared to each “estrous phase” in the ovaries, uterus/cervix and vagina.

Signs of effects related to treatment with Reactive Red 239, when administered by oral gavage for 13 consecutive weeks at the dosages of 100, 300 and 1000 mg/kg body weight/day were mainly caused due to the staining properties of the dye in animals from mid- and high-dose groups. No treatment-related changes were observed at the low dose of 100 mg/kg body weight/day. Histopathology revealed the deposition of the excreted test item (most likely due to re-resorption) in vacuoles of tubular cells of the cortex of kidneys. This reversible renal storage of the test item was accompanied by slight increases in urea and creatinine blood levels and also expressed by a slight increase in kidneys weights at dose levels of ≥300 mg/kg body weight/day. These changes did not cause tissue damage, were of a low magnitude and showed reversibility. Hence, the treatment-related changes in kidneys were not considered to be adverse. Based on these findings, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study is 1000 mg/kg body weight/day and the No Observed Effect Level (NOEL) is 100 mg/kg body weight/day.