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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Start of experimental phase (Allocation of animals): 08 October 2020 End of experimental phase (Last day of necropsy): 10 February 2021 Study completion: 01 October 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guideline
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
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
-
EC Number:
474-870-9
EC Name:
-
Cas Number:
80156-97-4
Molecular formula:
Hill formula: C28H20ClN9Na4O16S5 CAS formula: C28H24ClN9O16S5.4Na
IUPAC Name:
tetrasodium 7-[(1E)-2-[2-(carbamoylamino)-4-{[4-chloro-6-({4-[2-(sulfonatooxy)ethanesulfonyl]phenyl}amino)-1,3,5-triazin-2-yl]amino}phenyl]diazen-1-yl]naphthalene-1,3,6-trisulfonate
Test material form:
solid: particulate/powder
Details on test material:
Batch No. LK2003715

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
110 Hsd: Sprague Dawley SD rats (55 males and 55 females, females nulliparous
and non pregnant), 27-29 days old and with body weight of approximately 75-99 g, were
ordered from Charles River Italia S.p.A., Calco (Lecco), Italy and supplied by Charles River
Germany, Sulzfeld, Germany.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: 91 - 106 g
- 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, prior to blood sampling.
- Water (e.g. ad libitum): ad libitum to each cage via water bottles.
- Acclimation period: 6 days prior to the start of treatment

DETAILS OF FOOD AND WATER QUALITY:

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: 24 September 2020 (animals arrival) To: 10 February 2021 (last day of necropsy)

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 Reactive Yellow 176 was dissolved in the vehicle.


- VEHICLE
- Justification for use and choice of vehicle (if other than water): NA
- Concentration in vehicle: 10, 30 and 100nmg/mL
- Amount of vehicle (if gavage): 10 mL/kg
- Lot/batch no. (if required): NA
- Purity: NA
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the preparations made in Weeks 1, 7 and 13 were analysed to check the concentration.
Results of the analyses were within the acceptability limits 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 / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
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 in consultation with the Sponsor based on information from a previous sub-acute toxicity study in rats (Study No. 07/425-100P, supplied by the Sponsor)
- Rationale for animal assignment (if not random): Random
- Rationale for selecting satellite groups: to show reversibility of apparent effects
- Post-exposure recovery period in satellite groups: 4 weeks
- Section schedule rationale (if not random): Random

Examinations

Observations and examinations performed and frequency:
Mortality
Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holiday a similar procedure was
followed except that the final check was carried out at approximately mid-day. Two animals died during the study and a complete necropsy was performed.

Clinical signs and neurotoxicity assessment
All clinical signs were recorded for individual animals. Once before commencement of treatment and once daily during the study, each animal was observed and any clinical signs recorded. Observations were performed at the same time interval each day (2 - 2.5 hours post-dose).
Once before commencement of treatment and at least once per week during the study from the start of treatment, each animal was given a detailed clinical examination. Each animal was observed in an open arena. The test included observation of 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 were also recorded.
Once during Week 13 of treatment and once during Week 4 of recovery an evaluation of
sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive
stimuli) and an assessment of grip strength was also performed.

Motor activity assessment
The motor activity (MA) of all animals was measured once during Week 13 of treatment
and once during Week 4 of recovery by an automated activity recording. Measurements
were performed using a computer generated random order.

Body weight
Each animal was weighed on the day of allocation to treatment group, on the day that
treatment commenced, weekly thereafter and just prior to necropsy.

Food consumption
The weight of food consumed by each cage of rats was recorded at weekly intervals starting
from treatment. The group mean daily intake per rat was calculated.

Ophthalmoscopy
Both eyes of all animals were examined prior to the commencement of treatment by means
of an ophthalmoscope, and by a slit-lamp microscope, after the instillation of 0.5% Tropicamide
(Visumidriatic®, Visufarma, Rome, Italy). One male initially allocated to Group 2
(no. 46) and another one allocated to Group 4 (no. 88), showing ocular lesions were replaced
with spare animals showing no ocular abnormality, from the batch initially ordered for the
study. Observations carried out on the spare animals were not tabulated in the report and
will be archived with the raw data of the study.
The eyes of all animals from high dose and control groups were re-examined during Week 13 of treatment.

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

Clinical pathology investigations
Before necropsy, blood samples were collected, from all male and female animals from each
main phase group, under isoflurane anaesthesia from the retro-orbital sinus for thyroid
hormones determination, haematology and clinical chemistry, under conditions of food
deprivation.
During the necropsy procedure, blood samples were collected under isoflurane anaesthesia
from the abdominal vena cava for coagulation determination.
Further blood samples were taken under identical conditions at the end of the recovery
period.
Blood samples were collected and analysed in the same order. The blood samples collected
were divided into tubes as follows:
– EDTA anticoagulant for haematological investigations
– Heparin anticoagulant for biochemical tests
– Citrate anticoagulant for coagulation tests
– No anticoagulant for hormone determination

Immunoanalysis -Thyroid hormone determination (T3, T4 and TSH) (delegated phase)
Immunoanalysis was carried out by the Test Site, BioVetim (Study Phase number: BIOVA4043),
according to immunoanalytical methods validated at the Test Site:
– RV-T3/R/S-BKM/RIA-002 for T3
– RV-T4/R/S-BKM/RIA-002 for T4
– RV-TSH/R/S-IZO/RIA-002 for TSH
Samples were transferred into tubes containing no anticoagulant and centrifuged at room
temperature. The serum obtained (at least 250 μL) was divided in two aliquots, the first
with 250 μL of serum and the second with the remaining amount. Samples were stored
at -80°C pending analysis. Samples were assayed to determine the serum levels of Total
triiodothyronine (total T3), Total thyroxine (total T4) and Thyroid stimulating hormone
(TSH) by RadioImmuno Assay (RIA). In the first instance, the determination was performed
in the main phase animals only.
Theoretical number of samples: 80 × 2 aliquots.
Actual number of samples: 79 × 2 aliquots (1 sample was not collected due to premature
death).
Samples collected at the end of recovery period were not analysed and will be destroyed
following finalisation of the report.
Sacrifice and pathology:
Euthanasia
Animals in extremis and those 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.

Necropsy
The clinical history of the animals was studied and a detailed post mortem examination
was conducted (including examination of the external surface and orifices). Changes were
noted, the requisite organs weighed and the required tissue samples preserved in fixative.

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

Tissues fixed and preserved
Samples of all the tissues listed below were fixed and preserved in 10% neutral buffered formalin (except eyes, testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol).
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)
Kidneys
Liver
Lungs (including mainstem bronchi)
Lymph nodes – cervical
Lymph nodes – mesenteric
Mammary area (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
Thyroid gland
Trachea
Urinary bladder
Uterus – cervix
Vagina

Histopathological examination
The tissues required for histopathological examination are listed below:
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 area (Males and Females)
Oesophagus
Ovaries
Oviducts
Pancreas
Parathyroid glands
Pituitary gland
Prostate gland
Rectum
Salivary glands
Sciatic nerve
Seminal vesicles
Skeletal muscle
Skin
Spinal cord (cervical, thoracic, lumbar)
Spleen
Stomach
Testes
Thymus
Thyroid gland
Trachea
Urinary bladder
Uterus – cervix
Vagina

After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 µm thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides of main group animals were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed initially in all animals in the control and high dose groups dying during the treatment period or killed at the end of the 13 weeks of treatment. Sections from the vagina, uterus and ovaries from the control and high dose females were examined for evaluation of oestrus cycle.
Since treatment-related findings were found in the kidneys of high dose animals of both sexes, as requested by the Sponsor the histopathological evaluation of the kidneys was initially extended to animals of both sexes of the intermediate dose group and to the animals sacrificed after recovery.
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, non-treatment-related
Description (incidence and severity):
No clinical signs were noted in control and treated males during the dosing period, except
for tooth missing recorded in one animal of control group (no. 28) from Day 19 up to Day
26 of the dosing period. This animal apperead emaciated, showing hunched posture and it
was isolated in cage.
A palpable mass on the left side of the thoracic region was noted in one female receiving
300mg/kg body weight/day (no. 61) from Day 36 up to Day 92. A second palpable mass
was recorded on the ventral left side in the same animal on Day 93. Granulomatous inflammation,
characterized by the presence of a central core of necrosis with cell debris and
degenerated macrophages and lymphocites, were observed at histophology.

Weekly detailed clinical signs
No changes of toxicological significance were observed at the weekly clinical examination,
which included an evaluation of neurotoxicity.
During the dosing period, slight fluctuations (decreases or increases) in the number of
rearing, statistically significant, were occasionally observed in males and females dosed at
100, 300 and 1000 mg/kg body weight/day, when compared to controls. Due to the absence
of a clear dose-relation and to the inconsistency of the variations (increases or decreases),
these changes were not considered to be toxicologically significant (incidental).
A statistically significant increase in urination of approximately 3-folds than controls, was
observed on Day 90 of dosing in males treated at 1000 mg/kg body weight/day. Since this
change was observed only once, it was considered incidental.
No changes were noted during the recovery period.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Animal no. 82, treated at 1000mg/kg body weight/day, was found dead on Day 25 of dosing.
No clinical signs were observed in this animal during the study.
At macroscopic and microscopic examinations no findings were observed except this
animal having been partially cannibalized. The cause of death of this animal could not be
determined.
Animal no. 28 (control male) was found dead on Day 29 of recovery. No clinical signs were
observed before the death.
At macroscopic observations there was a single dark red gelatinous mass involving the right
kidney and right adrenal gland, associated with tubular dilation and presence of gelatinous
material of the right ureter. At microscopic observations the most relevant finding was
unilateral nephropathy characterised by tubular cell basophilia and dilation associated
with presence of hyaline casts in cortex and medulla mainly of proximal and distal tubules.
Renal findingswere considered the factors contributory to the death of the above mentioned
animal.
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
Description (incidence and severity):
Before the start of treatment, animals showing no ocular abnormality at the ophthalmoscopic
examination were selected for the study. The eyes of all animals were examined
during week 13 of treatment (Study Day 87). No ocular findings were detected.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Dosing phase
Some statistically significant differences between control and treated males were recorded,
such as: increase of haematocrit in males dosed at 300 and 1000mg/kg bw/day (6% and 5%,
respectively), increase of mean corpuscular volume in those receiving
1000mg/kg bw/day (4%), decreases of mean corpuscular haemoglobin concentration
in animals dosed at 300 and 1000 mg/kg bw/day (2%, both) and decrease of lymphocytes
in those dosed at 300mg/kg bw/day (33%). Changes were not dose-related, not consistent
with sexes and/or of minimal severity, therefore they were considered to be unrelated to
treatment.
Recovery Phase
No changes were seen in treated animals.

Coagulation
Dosing Phase
No changes were seen in treated animals.
Recovery Phase
No changes were seen in treated animals.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Dosing Phase
No treatment-related changes were recorded.
Statistically significant differences between control and treated animals were observed, such as: decreases of chloride in males dosed at 300 and 1000 mg/kg bw/day (2% and 3%, respectively), decrease of phosphorus in males receiving 300 mg/kg bw/day (11%), increase of creatinine in all treated females (10% to 17%) and increase of phosphorus in females dosed at 1000 mg/kg bw/day (25%). The changes in phosphorus were not dose-related and not consistent between sexes, the decrease of chloride and creatinine were of minimal severity, therefore, the recorded findings were considered to be incidental.
Recovery Phase
No treatment-related changes were recorded. The increase of alanine and aspartate aminotransferases recorded in the male no. 100 (1000 mg/kg bw/day) was not observed at the
end of the Dosing Phase, therefore it was considered to be incidental.
Endocrine findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were observed.
A statistically significant increase of T4 was recorded in males dosed at 1000 mg/kg bw/day (22%). This change was of low severity and no other related findings were observed (e.g. decrease of TSH, thyroid and/or liver histopathological changes), therefore the increase of T4 was considered to be incidental.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related differences were observed at functional tests (sensory reactivity,
landing foot splay, grip strength) performed at the end of treatment and recovery periods.
At the end of dosing period, statistically significant increase in motor activity measurements
was recorded in males treated at 300 and 1000 mg/kg bodyweight/day, while no remarkable
differences were noted in females, when compared to controls. Due to the absence of a
dose-relation and to the inconsistency between sexes, these variations were not considered
to be treatment-related (incidental).
No remarkable differences in motor activity measurements were recorded at the end of
recovery period.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Final sacrifice
No changes were observed in terminal body weight of treated animals, when compared to
the controls.
A slight statistically significant increase in relative mean kidneyweights (+12% mean relative
kidney) in high dose treated males was observed. The change observed in kidney weights
correlated microscopically with accumulation of yellow pigment (test item like) in the
renal (mainly proximal) tubular epithelial cells of the cortex. Any organ weight changes
other than those listed above were within the range of occasionally observed and expected
spontaneous changes in rats of the same age and considered unrelated to treatment.
Recovery sacrifice
There were no relevant treatment-related changes in terminal body weight and organ
weights at the end of the recovery period. Any organ weight variations were in the range
of expected spontaneous changes in rats of the same age and considered unrelated to
treatment.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Macroscopic observations (Table 16; Appendix 19; Addendum 8)
Final sacrifice
At the end of the treatment period, treatment-related macroscopic observation consisted
of yellow discolouration of the following organs/tissues:
Sex Males Females
Dose (mg/kg/day) 1000 1000
Group 4 4
Stomach 1/9 1/10
Salivary glands - 1/10
Prostate glands 2/9 -
Epididymides 2/9 -
Testes 3/9 -
Cervical lymph nodes - 1/10
Uterus - 2/10
- = No abnormalities detected
The yellow discolouration was considered to be related to the colour of the test item. The
discolouration noted macroscopically had no microscopic correlate. Any macroscopic
observations other than that listed above had a comparable incidence in control and
treated groups and/or are characteristically seen in untreated rats of the same age and were
considered incidental and unrelated to treatment.

Recovery sacrifice
At the end of the recovery period, there were no relevant treatment-related changes following
gross pathology examination. Any observations had a comparable incidence in control
and high dose treated groups of both sexes and /or are characteristically seen in untreated
Sprague Dawley SD rats of the same age and were considered incidental and unrelated to
treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Final sacrifice
Changes considered to be associated with the oral administration of Reactive Yellow 176
were present only in the kidneys of animals of both sexes dosed with
1000 mg/kg body weight/day (high dose). There were no microscopic observations in
the kidney in animals treated at 300 mg/kg body weight/day (mid-dose).
Kidneys - Presence of pigment in the cortex, characterised by yellow brown material (test
item like) in the renal (mainly proximal) tubular cells was observed in all high dose males
(9/9) and females (10/10). The severity of the renal finding in the high dose animals of both
sexes was minimal to mild. This storage of dye material did not result in microscopical
visible organ damage. This change correlated with increased kidney mean weights in high
dose males and was considered therefore treatment-related.
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 teated
males of the high dose.
Normal physiology of the estrous cycle (estrous, metestrous, diestrous and proestrous) was
noted in control and treated high dose females.
Any microscopic observation other than those listed above, were within the range of occasionally
observed and expected spontaneous changes in rats of the same age and therefore
considered incidental and unrelated to treatment.

Recovery sacrifice
After 4 weeks of recovery, histopathological change consisting in the presence of pigment in
the cortex of minimal to mild degree, was observed in the kidneys of all high dose females
(5/5) and two high dose males (2/5).
Any microscopic observation other than those listed above were within the range of occasionally
observed and expected spontaneous changes in rats of the same age and therefore
considered incidental and unrelated to treatment.
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 no relevant changes in clinical chemistry parameters were observed , the
treatment-related change in kidneys of animals treated at 1000 mg/kg body weight/day
was not considered adverse.
Other effects:
no effects observed
Details on results:
One control male and one high dose male (1000 mg/kg body weight/day) were found
dead on Days 29 of recovery and 25 of dosing period, respectively. The cause of death could
not be determined for the animal of the high dose group, while the death of the animal of
the control group was attributed to the renal findings.
No relevant clinical signs were recorded during the study. No signs indicating neurotoxic
effects were seen at neurobehavioural test performed 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 clinical chemistry parameters. No treatment-related
changes were recorded for thyroid hormones.
The most relevant macroscopic change observed at post-mortem observations was yellow
discolouration, considered to be related to the colour of the test item, of most tissues
and organs in most high dose animals of both sexes, including stomach, salivary glands,
prostate glands, epididymes, testes, cervical lymph nodes and uterus, when compared to
controls. This change was no longer apparent at the end of recovery period, indicating the
reversibility of this effect.
Accumulation of dye particles, characterized by the presence of yellow brown material (test
item like) in the renal (mainly proximal) tubular cells was seen in the kidneys of the high
dose group animals. This change was correlated to a slight statistically significant increase
in relative mean kidney weights in high dose treated males.
At the end of recovery period, the presence of pigment in the cortex was observed in all of
high dose females but only in few males. 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 no relevant changes in clinical
chemistry parameters were observed, the treatment-related change in kidneys of animals
treated at 1000 mg/kg body weight/day was not considered 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 high dose females during histopathological evaluation.

In conclusion, the daily oral administration of Reactive Yellow 176 for 13 consecutive weeks
at dose levels of 100, 300 and 1000 mg/kg body weight/day induced treatment-related effects
in animals of the high dose group, consisting in increases in kidney weights associated
with histopathological changes in the kidneys (accumulation of yellow brown material in
tubular cells), indicating the kidney as target organ. In the absence of remarkable changes
in clinical chemistry and organ damage, the treatment-related changes in kidneys were not
considered to be adverse.
No treatment-related changes were observed at the low and mid-dose of 100 and
300 mg/kg body weight/day.

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
gross pathology
histopathology: non-neoplastic
organ weights and organ / body weight ratios
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:
The daily oral administration of Reactive Yellow 176 for 13 consecutive weeks
at dose levels of 100, 300 and 1000 mg/kg body weight/day induced treatment-related effects
in animals of the high dose group, consisting in increases in kidney weights associated
with histopathological changes in the kidneys (accumulation of yellow brown material in
tubular cells) du to re-resorption of excreted test material. In the absence of related changes
in clinical chemistry and organ damage, the treatment-related changes in kidneys were not
considered to be adverse.
No treatment-related changes were observed at the low and mid-dose of 100 and
300 mg/kg body weight/day.
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.
Executive summary:

The toxicity of Reactive Yellow176 in rats after oral administration for 13weeks 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 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 and motor activity, bodyweight, food consumption, ophthalmoscopy, clinical pathology investigations (including thyroid hormones determination), terminal body weight, organ weights, macroscopic observations, histopathological examination.


One male of the high dose group (1000 mg/kg bodyweight/day) and one male of the control group were found dead on Day 25 of dosing and Day 29 of recovery period, respectively. No clinical signs were observed prior to death. The cause of death could not be determined for the animal of the high dose group, while the death of the animal of the control group was attributed to the renal findings.


No clinical signs of toxicological relevance were observed during the study. Weekly detailed clinical signs (removal from cage and open field measurements) Observation of animals at removal from the cage and in an open arena did not reveal treatment-related changes.


No treatment-related changes were observed in treated and control animals, at functional tests (sensory reactivity, landing foot splay, grip strength) and 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 ophthalmoscopic examination.


No treatment-related anomalies were noted in the estrous cycle of treated females, when compared to controls.


No treatment-related changes were recorded in haematological, coagulation, clinical chemistry parameters or thyroid hormone determination (T3, T4 and TSH) at the end of dosing and recovery periods.


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 statistically significant increase in relative mean kidney weights was observed in the high dose treated males at the end of the treatment phase. The change observed in kidney weights correlated microscopically with the accumulation of yellow pigment (test item like) in the renal (mainly proximal) tubular epithelial cells of the cortex. There were no relevant treatment-related changes in terminal body weight and organ weights at the end of the recovery period.


The most relevant change observed at post-mortem observations at the end of the treatment phase was yellow discolouration of most tissues and organs in most animals of both sexes treated at 1000 mg/kg body weight/day, including stomach, salivary glands, prostate glands, epididymides, testes, cervical lymph nodes and uterus, when compared to controls. At the end of the recovery period, there were no relevant treatment-related changes following gross pathology examination.


At microscopic observations, presence of pigment in the cortex, characterised by yellow brown material in the renal tubular cells, was observed only in male and female animals treated at 1000 mg/kg body weight/day. The severity of the renal finding in high dose animals of both sexes was minimal to mild. After 4 weeks of recovery period, the presence of pigment in the cortex was still observed in the kidneys of all of high dose females and two high dose males, however to a lesser degree. As no organ damage was caused by this pigment storage effect, this treatment-related renal change was not considered to be adverse.


In conclusion, the daily oral administration of Reactive Yellow 176 for 13 consecutive weeks at dose levels of 100, 300 and 1000 mg/kg body weight/day induced treatment-related effects in animals of the high dose group, consisting of an increase in kidney weights associated with histopathological changes in the kidneys (accumulation of yellow brown material in tubular cells). Due to the absence of related changes in clinical chemistry or organ damage, the treatment-related changes in kidneys were not considered to be adverse. No treatment-related changes were observed at the low and mid-doses of 100 and 300 mg/kg body weight/day.


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.