1,3,6-Naphthalenetrisulfonic acid, 7-[2-[2-[(aminocarbonyl)amino]-4-[[4-chloro-6-[[4-[[2-(sulfooxy)ethyl]sulfonyl]phenyl]amino]-1,3,5-triazin-2-yl]amino]phenyl]diazenyl]-, sodium salt

Endpoint summary

Description of key information

The test substance did not cause toxicologically relevant adverse effects after suacute or subchronic continuous oral administration. The no observed adverse effect level (NOAEL) for the substance is 1000 mg/kg bw/day after 28 or 90 days of continuous oral treatment.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

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

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)

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.

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

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.

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.

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

Key result

Critical effects observed:

no

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.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 July 2007 to 29 August 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: Crl:(WI) BR rats
Source: TOXI COOP Ltd. 1103 Budapest, Cserkesz u. 90.
Justification of strain: The Wistar rat as a rodent is one of the preferred species of toxicity studies.
Hygienic level at arrival: SPF
Hygienic level during the study: Good conventional
Number of animals: 30 male and 30 female (nulliparous, non-pregnant) rats
Age of animals: Young adult rats, 6 - 8 weeks old
Body weight range at randomisation: Male: 190 - 209 g
Female: 170 - 190 g
Acclimatisation period: 7 days
Animal health: Only healthy animals were used for the study. The veterinarian ascertained healthy status.
Room: 242/2
Housing: Five animals /cage
Cage type: Type III polypropylene/polycarbonate
Bedding: Laboratory bedding, changed together with the cages twice a week
Illumination: Artificial light, from 6 a.m. to 6 p.m.
Temperature: 22 ± 3 °C
Relative humidity: 30 - 70 %
Ventilation: 8-12 air exchanges/hour by central air-condition system.
Food and Water Supply: The animals received ssniff® SM R/M-Z+H autoclavable complete feed for rats and mice breeding and maintenance produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany and tap water from municipal supply from 500 ml bottle, ad libitum.
The diet and drinking water are routinely analysed and are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.
Animal Identification: The individual identification was performed by tattoo numbers on the tail.
The boxes were marked by identity cards, with information about study code, sex, dose group, cage number and individual animal numbers.

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

The test item was formulated in concentrations of 6.25 mg/ml, 25 mg/ml and 100 mg/ml prepared with distilled water.
Animals were dosed with the single dose of the test item by gavage daily seven days each week for a period of 28 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of concentration of dosing formulations was conducted on days 5 and 22. All the measured concentrations varied in the range of 100 % and 107 % of the nominal concentrations.

Duration of treatment / exposure:

Duration of treatment: 28 days

Frequency of treatment:

Single dose of the test item daily seven days each week for a period of 28 days.

Dose / conc.:

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

Dose / conc.:

250 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:

GROUP Dose mg/kg bw/day Concentration mg/ml No. of Animals
Male Female
Group 1 Control 0 5+5* 5+5*
Group 2 62.5 6.25 5 5
Group 3 250 25 5 5
Group 5 1000 100 5+5* 5+5*
* animals for recovery groups.

Control animals:

yes

Details on study design:

The doses were chosen on the basis of the results of repeated dose toxicity studies of compounds with similar chemical structure.

Animals were dosed with the single dose of the test item by gavage daily seven days each week for a period of 28 days. Animals in the control group were treated with the vehicle and handled in an identical manner to those in the test groups. Treatment was carried out at similar times (± 2 hours) in the morning each day. The first treatment day was considered as day 0. Animals were not treated on the day of necropsy.

Duration of treatment: 28 days

Animals of the recovery groups were not treated from day 28. Daily and detailed weekly clinical observation, weekly body weight and food consumption measurements were conducted for a 14 days period. All recovery animals were processed on the same way as animals at termination of the treatment.

Positive control:

No data

Observations and examinations performed and frequency:

Clinical Observations: Animals were inspected for signs of morbidity and mortality twice daily (at the beginning and end of each day).
General clinical observations were made once a day, after treatment at approximately the same time.
Detailed clinical observations were made on all animals outside the home cage in a standard arena once, prior to the first exposure and once a week thereafter.
Observations were performed on the skin, fur, eyes and mucous membranes, autonomic activity (lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions), circulatory and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted on all animals in the fourth exposure week (day 27). General physical condition and behaviour of animals were tested. A modified Irwin test was performed. (Irwin, S.: Comprehensive Observational Assessment: Ia. A systematic, Quantitative procedure for Assessing the Behavioral and Physiologic State of the Mouse, Psychopharmacologia (Berl) 13 222-257 1968).

Body Weight Measurement: Body weight was measured on day 0 (beginning of treatment), then weekly with a precision of 1 g.

Food Consumption Measurement: The food consumption was determined weekly by re-weighing the non-consumed diet with a precision of 1 g.

LABORATORY EXAMINATIONS
Blood sampling for laboratory examinations including haematology and clinical chemistry were conducted one day after the last treatment and at the end of the recovery period. After an overnight food deprivation (approximately 16 hours), animals were anaesthetised with Euthanyl (Batch No.: 42WG; Expiry date: July 2009; Bimeda-MTC, Animal Health Inc., Cambridge, ON N3C 2W4) and blood samples were collected by heart puncture. Three samples were taken from each animal: one for haematology (tubes contained K3-EDTA as anticoagulant), one for determination of blood clotting times (APTT and PT; tubes contained sodium citrate as anticoagulant) and the third one to obtain serum samples (tubes did not contain any anticoagulant) for clinical chemistry. Clinical chemistry parameters were measured after one day storage of serum at 2-8 ⁰C because of a transient failure of Vitros 250 equipment.

Haematology: parameters listed in table format submitted under any other information.

Clinical Chemistry: parameters listed in table format submitted under any other information.

Sacrifice and pathology:

Necropsy: A gross necropsy was performed on each animal just after the blood harvesting for clinical pathology examinations. One day after the last treatment, animals were sacrificed by exsanguination under pentobarbital anaesthesia (Euthanyl; Batch No.: 42WG; Expiry date: July 2009; Bimeda-MTC, Animal Health Inc., Cambridge, ON N3C 2W4). After examination of the external appearance the cranial, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed. Any abnormality was recorded with details of the location, colour, shape and size.

The following organs and tissues were preserved in 10 % formaldehyde solution for histological evaluation:
Gross lesions, lymph nodes (submandibular, mesenteric) sternum, skin and female mammary gland, salivary glands (submandibular), femur + bone marrow, spinal cord (cervical, lumbar, thoracic level), pituitary, thymus, trachea, lungs (with main stem bronchi), heart, thyroid + parathyroid, oesophagus, stomach, caecum, duodenum, ileum, jejunum, colon, rectum, urinary bladder, liver, pancreas, spleen, kidneys, adrenals, prostate, testes with epididymides, ovaries, uterus with vagina, brain (including cerebrum, cerebellum, pons and medulla oblongata), eyes with optic nerve, lachrymal gland with Harderian gland, seminal vesicle, muscle (quadriceps), sciatic nerve and aorta.

Organ Weight Measurement
The following organs:
The following organ weights were weighed and recorded (paired organs were weighed together).
With precision of 0.01g: Liver, kidneys, testes, epididymides, thymus, spleen, brain and heart.
With precision of 0.001g: Adrenals

Histopathology: Histological examinations were performed on the preserved organs or tissues of the animals of the control and high dose groups including animals of recovery period. In groups 2 and 3, liver and kidneys were also processed histologically. The listed organs or their specimens were embedded into paraffin after dehydration. Slides were stained with haematoxylin eosin and examined by a light microscope.

Statistics:

Statistical analysis was done with SPSS PC+ software package for the following data:
- body weight
- haematology
- clinical chemistry
- organ weight
The heterogeneity of variance between the groups was checked by Bartlett's homogeneity of variance test. Where no significant heterogeneity was detected, a oneway analysis of variance was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of the inter-group differences. Where significant heterogeneity was found, the normal distribution of data by Kolmogorov-Smirnov test was used. In case of not-normal distribution, the nonparametric method of Kruskal-Wallis One-Way analysis of variance was applied. If positive results were obtained, the inter-group comparisons were performed using Mann-Whitney U-test.
Frequency of clinical symptoms, necropsy and histopathological findings and daily mean food consumption were calculated. At the end of recovery period, the homogeneity of variance between groups was determined by F-test. Depending on the result Poled or Separate variance estimate of the Two-Sample t-test was performed. For data, which was not normally distributed, the data were compared between groups according to Mann-Whitney U-test.

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:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

MORTALITY: There was no mortality during the study.

CLINICAL OBSERVATIONS
Daily and Detailed Weekly Clinical Observations: Decreased activity, piloerection, hunched back, wryneck, tremor and stereotype movement were noted for one female rat (No.: 6764) at 62.5 mg/kg bw/day. The first signs were observed on day 3 (decreased activity, piloerection and hunched back) followed wryneck, tremor and stereotype movement from day 9 up to the end of the treatment. These symptoms were noted at the weekly observations, as well.
There were no clinical symptoms related to the test item in the other animals in the control, 62.5 mg/kg bw/day, 250 mg/kg bw/day or 1000 mg/kg bw/day groups in the course of the observation period. The physiological state and behaviour of animals were considered to be normal at the daily and at the detailed weekly observation.
In summary: No test item related clinical signs appeared. Neuro-behavioral symptoms of female animal No.: 6764 were considered to be individual disorder and not related to the test item.

Functional Observation Battery
Group 1 - Control: In the male animals, reduced (6/10) or increased (1/10) transfer arousal, lack of finger approach (4/10) or finger withdrawal (1/10), increased touch escape response (2/10), positional struggle to a marked degree (5/10) and vocalisation (5/10) were found. Negative tail pinch reaction was found in one rat (1/10). In the female animals, increased (2/10) or reduced transfer arousal (2/10), reduced finger approach (2/10), lack of finger withdrawal (1/10), increased touch escape response (2/10) and vocalisation (3/10) were noted.

Group 2 - 62.5 mg/kg bw/day: In the male animals, reduced transfer arousal (2/5) lack of finger approach (2/5) or elongated finger approach (1/5) and positional struggle to a marked degree (1/5), no tail pinch reaction (1/5) and vocalisation (1/5) were noted. In the female animals, head searching (1/5), circling (1/5), tremor (1/5), decreased transfer arousal (2/5) and finger approach (2/5) or lack of finger withdrawal (1/5), marked touch escape response (2/5) or positional struggle (2/5) and vocalisation (1/5) were observed.

Group 3 - 250 mg/kg bw/day: In the male animals, reduced (1/5) or increased (1/5) transfer arousal, lack of finger approach (1/5), marked touch escape response (1/5), increased positional struggle (2/5) and vocalisation (1/5) were found. In the female animals, reduced (1/5) or increased (1/5) transfer arousal, reduced finger approach (2/5) or marked finger withdrawal (2/5) positional struggle to a marked degree (2/5) and vocalisation (2/5) were observed.

Group 4 - 1000 mg/kg bw/day: In the male animals, reduced (3/10) or marked (3/10) transfer arousal, lack of finger withdrawal (4/10) marked finger withdrawal (3/10), marked touch escape response (3/10), positional struggle to a marked degree (3/10) and vocalisation (3/10) were observed. In the female animals, reduced (3/10) or increased (2/10) transfer arousal, lack of finger withdrawal (3/10), or marked finger withdrawal (4/10), increased touch escape response (5/10), increased positional struggle (1/10) and vocalisation (1/10) were noted.

In summary: There were no test item effects identified in the functional observation battery. Variations in transfer arousal, finger approach and finger withdrawal, in touch escape response, positional struggle, tail pinch reaction and vocalisation were observed with similar incidences in each group representing inter individual differences in animals behaviour and reactions to different type of stimuli or manipulations. Neuro-behavioural symptoms of female animal at 62.5 mg/kg bw/day were considered to be individual changes because of the singular occurrence and in the lack of any signs in the higher dose groups.

BODY WEIGHT AND BODY WEIGHT GAIN: There were no significant differences in the mean body weight or mean body weight gain between the control and 62.5, 250 or 1000 mg/kg bw/day of Gelb Sulfato groups during the entire treatment period. The mean body weight and mean body weight gain remained similar to the control value in the male and female animals during the recovery period. A significant body weight loss was noted in female animal No.: 6764, (62.5 mg/kg bw/day) during the first week as individual alteration corresponding with clinical signs.

FOOD CONSUMPTION: No test item related differences were observed in the daily food consumption between the control and Gelb Sulfato treated groups at 62.5 mg/kg bw/day, 250 mg/kg bw/day or 1000 mg/kg bw/day. The mean daily food intake was also similar in the control and high dose animals during the recovery period.

HAEMATOLOGY
Group 2 - 62.5 mg/kg bw/day: In the male animals, the mean platelet (thrombocyte) count (PLT) was slightly less and the percent of large unstained cells (LUC) was higher than the control value. In the female animals, the red blood cell count (RBC) was slightly less than the control.

Group 3 - 250 mg/kg bw/day: In the male animals, the percent of large unstained cells (LUC) was higher than the control value. In the female animals, the red blood cell count was slightly less than the control.

Group 4 - 1000 mg/kg bw/day: There were no statistically significant differences in the examined parameters when compared to the control in the male or in the female animals. At the end of the recovery period, the red blood cell distribution width (RDW) and percent of basophile cells (BA) were less, the percent of lymphocytes (LY) was higher than the control value in the male group of 1000 mg/kg bw/day. The haemoglobin content of plasma (HGB), the haematocrite (HTC), the mean corpuscular haemoglobin content of erythrocytes (MCHC) and the percent of monocytes (MO) were slightly less than the control value in the female animals.

In summary: There was no test item related adverse effect on the examined haematological parameters. Slight statistically significant differences from the control were noted for some parameters (PLT, LUC, RBC) only at 62.5 and 250 mg/kg bw/day, which were of low magnitude and within the normal physiological range (historical control) and were independent from dose. The differences of some parameters at the end of the recovery period represent also the biological variations of these parameters (RDW, BA, LY, HGB, HTC, MCHC, MO). So these were not considered to be toxicologically significant.

CLINICAL CHEMISTRY
Group 2 - 62.5 mg/kg bw/day: All examined clinical chemistry parameters were similar to the control in the male the female animals.

Group 3 - 250 mg/kg bw/day: In the male animals, the activity of alkaline phosphatase (ALKP) was slightly higher than the control value. In the female animals, there were no differences from the control in the examined parameters.

Group 4 - 1000 mg/kg bw/day: In the male animals, the ALKP activity was less than the control. In the female animals, the concentration of chloride (Cl-) was less than the control. In the recovery period, no significant differences were found in the examined parameters between the control and high dose animals.

In summary: There were no test item related changes in the examined clinical chemistry parameters. The slight differences in ALKP activity and chloride concentrations terminally represented physiologic variation. These were considered to be normal values independent from the treatment.

NECROPSY
Group 1 – Control: In the male animals, in the lungs pinprick-sized haemorrhages (2/5) and in the kidney one side pyelectasis (1/5) were observed. In the female animals, pinprick-sized (1/5) or point like (1/5) haemorrhages were found in the lungs and the liver was pale (1/5). At termination of the recovery period, pinprick-sized haemorrhages in the lungs (1/5, male) and hydrometra (1/5, female) were detected.

Group 2 - 62.5 mg/kg bw/day: In the male animals, pale raised areas in the lungs (1/5) and one side pyelectasis in the kidney (2/5) occurred. In the female animals, reddish mottled lungs (1/5) and hydrometra (1/5) were detected.

Group 3 - 250 mg/kg bw/day: In the male animals, in the lungs pinprick-sized haemorrhages (1/5) and pale kidneys (both sides, 2/5) and one side pyelectasis (1/5) were observed. In the female group, the liver (1/5) and kidneys (1/5) were pale in one rat.

Group 4 - 1000 mg/kg bw/day: In the male animals, pinprick-sized (1/5) or point like (1/5) haemorrhages were noted. In one female animal hydrometra (1/5) were observed. In the male recovery group, in the lungs pinprick-sized haemorrhages (1/5) were observed. Pinprick sized haemorrhages (2/5) and hydrometra (1/5) were noted for female recovery group.

In summary: No test item related macroscopic findings were detected at the necropsy. Kidney alterations occurred at the control and lower doses only (one side pyelectasis; male control, 62.5 and 250 mg/kg bw/day, pale kidneys at 250 mg/kg bw/day male and female). Pale liver occurred in single female animals of the control and 250 mg/kg bw/day groups but not in high dose group. Thus both kidneys and liver findings were considered to be independent from the test item. The haemorrhages, pale raised areas and reddish mottled colour in the lungs of animals were caused by the euthanasia procedures. Hydrometra indicative of the sexual cycle of animals was present in some female animals of the control and treated groups.

ORGAN WEIGHT
62.5 mg/kg bw/day, 250 mg/kg bw/day, 1000 mg/kg bw/day
The only statistically significant difference from the control was noted for male animals at 1000 mg/kg bw/day in the spleen weight relative to the body weight at the termination of the treatment. There were no differences in other examined organ weights (absolute and relative to the body and brain weights) between the control and any test item treated group.
At the end of the recovery phase, the heart and epididymides weights both relative to the body weights were slightly higher than the control value in male animals. There were no differences in the examined organs between the control and treated female animals.

In summary: There was no test item effect on the examined organ weights. The difference in spleen weight at 1000 mg/kg bw/day was low and not considered to be significant because of singular occurrence (there were no related haematological or histopathological findings). Heart and epididymides weights were not influenced at the termination of the treatment, so the small differences at the end of the recovery period were representative of biological variations and were not considered to have toxicological significance.

HISTOPATHOLOGY
Histopathological examinations revealed focal alveolar emphysema or haemorrhages in the lungs as follows:

Dose mg/kg bw/day Focal alveolar emphysema Focal haemorrhages
Male Female Male Female
Control 1/5 2/5 2/5 3/5
Control Recovery 1/5 1/5 1/5 0/5
1000 1/5 0/5 2/5 2/5
1000 Recovery 0/5 0/5 1/5 1/5

For male animals, in the kidneys one side pyelectasia was noted in the control (1/5), 62.5 mg/kg bw/day (2/5) and 250 mg/kg bw/day (1/5) groups.
In female groups, uterus dilatation was observed in some animal (1/5 in control recovery group; 1/5 in 1000 mg/kg bw/day terminal group, 1/5 in 1000 mg/kg bw/day recovery group).

No morphological evidence of acute or subacute injury of the alimentary tract, the pancreas, the cardiovascular system, the haematopoietic system, the immune system, the skeleton, the muscular system, the male and female reproductive system, or the central or peripheral nervous system was detected. The structure and the cell morphology of the endocrine glands was the same at the control and treated animals.

In summary: Histopathological examination did not reveal any toxic or other test item related findings. The focal alveolar emphysema and haemorrhages in the lungs were caused by hypoxia, dyspnoea and circulatory disturbance developed during exsanguination. The incidence and severity of these lesions were similar in the control and high dose treated groups. The pyelectasia (unilateral) in the kidney was slight individual disorders without toxicological significance. The dilatation of uterine horns in one female animal was a slight neuro-hormonal phenomenon in connection with the sexual function of the inner genital organs.

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Test substance caused no toxic effects at 62.5 mg/kg bw/day, 250 mg/kg bw/day or 1000 mg/kg bw/day doses in CRL:(W) BR rats after 28-day continuous oral (by gavage) administration.

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Conclusions:

Gelb Sulfato caused no toxic effects at 62.5 mg/kg bw/day, 250 mg/kg bw/day or 1000 mg/kg bw/day doses in CRL:(W) BR rats after 28-day continuous oral (by gavage) administration.
The no observed effect level (NOEL) of Gelb Sulfato was 1000 mg/kg bw/day.

Executive summary:

The study was performed in accordance with the study plan agreed upon by Sponsor, the OECD Guidelines for Testing of Chemicals No. 407, Directive 96/54/EC B.7 and the Principles of Good Laboratory Practice (GLP) and reported with a GLP certificate.

 

A 28-day repeated dose oral (by gavage) toxicity study was performed in CRL:(WI) BR rats (n= 5 animals/sex/group and 5 animals/sex in the control and Group 4 for recovery) on Gelb Sulfato with dose levels of 0 (vehicle only), 62.5, 250 and 1000 mg/kg bw/day for 28 days. Animals were treated by daily oral gavage with test item or vehicle (distilled water) at a constant dosing volume of 10 ml/kg body weight. Recovery animals were retained for further 14-day observation without treatment.

The stability of test item in this vehicle was found to be adequate (at least 72 hours at room temperature). The concentration of dosing solutions varied in the range of 100 % to 107 % of the nominal concentrations.

General clinical observations were made daily and detailed clinical observations were performed weekly. A functional observation battery was conducted in week 4. Body weight and food consumption were measured weekly. Clinical pathology and gross pathology were conducted one day after the last treatment. Selected organs were weighed. A full histological examination was performed on the preserved organs and tissues of the animals of the Control group and Group 4 (1000 mg/kg bw/day). The liver and kidneys were evaluated histologically in Group 2 and Group 3. Animals of recovery groups were processed on the same way as animals at termination of the treatment.

 

Results

Mortality:There was no mortality.

Clinical observations:No test item related clinical symptoms were noted. There were no test item effect on the behaviour and reaction of animals to different type of stimuli (Functional Observation Battery).

Body weight:No test item influence on the body weight development was found.

Food consumption:There were no test item related differences in the mean food consumption between any dose levels.

Clinical pathology:Haematological and clinical chemistry investigations did not reveal any toxic changes related to test item in the examined parameters.

Organ pathology:There were no pathological findings related to the test item at gross necropsy, organ weight or histopathological examinations.

 

Conclusions

Gelb Sulfato caused no toxic effects at 62.5 mg/kg bw/day, 250 mg/kg bw/day or 1000 mg/kg bw/day doses in CRL:(W) BR rats after 28-day continuous oral (by gavage) administration.

 

The no observed effect level (NOEL) for Gelb Sulfato was 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A 28-day repeated dose oral (by gavage) toxicity study was performed in CRL:(WI) BR rats (n= 5 animals/sex/group and 5 animals/sex in the control and Group 4 for recovery) on Reactive Yellow 176 with dose levels of 0 (vehicle only), 62.5, 250 and 1000 mg/kg bw/day for 28 days. Animals were treated by daily oral gavage with test item or vehicle (distilled water) at a constant dosing volume of 10 ml/kg body weight. Recovery animals were retained for further 14-day observation without treatment.

The stability of test item in this vehicle was found to be adequate (at least 72 hours at room temperature). The concentration of dosing solutions varied in the range of 100 % to 107 % of the nominal concentrations.

General clinical observations were made daily and detailed clinical observations were performed weekly. A functional observation battery was conducted in week 4. Body weight and food consumption were measured weekly. Clinical pathology and gross pathology were conducted one day after the last treatment. Selected organs were weighed. A full histological examination was performed on the preserved organs and tissues of the animals of the Control group and Group 4 (1000 mg/kg bw/day). The liver and kidneys were evaluated histologically in Group 2 and Group 3. Animals of recovery groups were processed on the same way as animals at termination of the treatment.

There was no mortality throughout the study. No test item related clinical symptoms were noted. There were no test item-related effects on the behaviour and reaction of animals to different type of stimuli (Functional Observation Battery). No test item influence on the body weight development was found. There were no test item related differences in the mean food consumption between any dose levels. Haematological and clinical chemistry investigations did not reveal any toxic changes related to test item in the examined parameters. There were no pathological findings related to the test item at gross necropsy, organ weight or histopathological examinations.

In conclusion, Reactive Yellow 176 caused no toxic effects at 62.5 mg/kg bw/day, 250 mg/kg bw/day or 1000 mg/kg bw/day doses in CRL:(W) BR rats after 28-day continuous oral (by gavage) administration.

The no observed effect level (NOEL) for Reactive Yellow 176 was 1000 mg/kg bw/day.

 

 

The toxicity of Reactive Yellow176 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 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.

Justification for classification or non-classification

The above results triggered no classification under the Dangerous Substance Directive (67/548/EEC) and the CLP Regulation (EC No 1272/2008). No classification for prolonged effects is therefore required.