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Administrative data

Description of key information

No treatment-related adverse effects were observed in a subacute toxicity study in male and female rats. The NOAEL (No Observed Adverse Effect Level) was considered to be 1000 mg/kg body weight/day for males and females.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2014 to July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
One genetox endpoint was included
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy
- Age at study initiation: 7 to 8 weeks (day of allocation)
- Weight at study initiation: (P) Males: 251 g; Females: 213 g
- Fasting period before study: no
- Housing: 5 animals/sex/cage
- Diet (e.g. ad libitum): ad libitum except for clinical pathology
- Water (e.g. ad libitum): ad libitum except for clinical pathology
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/- 2°C
- Humidity (%): 55%+/-15%
- Air changes (per hr): approximately 15 to 20 air change per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: from 03 October (day of allocation) to 09 December 2014 (last day of necropsy)
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: up to 7 days interval

DIET PREPARATION
- Rate of preparation of diet (frequency): n/a
- Mixing appropriate amounts with (Type of food): n/a
- Storage temperature of food: n/a

VEHICLE
- Justification for use and choice of vehicle (if other than water): n/a
- Concentration in vehicle: 6.25, 25, 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw
- Lot/batch no. (if required): n/a
- Purity: n/a
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Before treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable. Samples of the formulations prepared during the study, on the first and last week of treatment (when all females were present) were also analysed to check the concentration. In addition, in the present study a 8 day stability at +4°C was verified at 6.25 mg/mL. The overall results of the analyses were within the limits of acceptance stated
in RTC SOPs for concentration (90-110%).

Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. A0001) by a spectrophotometer analysis. The software used for this activity was Skanlt® version 2.4.2.55 (Thermo Scientific).
Duration of treatment / exposure:
Males
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter until the day before necropsy for a total of 48 days.

Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until at least up to, and including, Day 3 post partum or the day before sacrifice. Dose volumes were adjusted once per week for each animal according to the last recorded body weight. One female, no. X0010033 (Group 2), was erroneously dosed on Day 4 post partum.
During the gestation period, dose volumes were calculated according to individual body weights on Days 0, 7, 14 and 20 post coitum and on
Day 1 post partum. Thereafter individual dose volumes remained constant.

Recovery groups
Animals were dosed once a day, 7 days a week, for a minimum of 4 consecutive weeks. No treatment was given during the recovery period.

Positive control group
Animals (males only) received a single dose approximately 24 hours before sacrifice.
Frequency of treatment:
once a day
Remarks:
Doses / Concentrations:
62.5, 250, 1000 mg/kg/day
Basis:
nominal in water
No. of animals per sex per dose:
Main groups
3 groups of 10 males and 10 females each dosed at 62.5, 250 and 1000 mg/kg/day bw. One control group (group 1) of 10 males and 10 females
received the vehicle alone.

Recovery groups
1 group of 5 males and 5 females dosed at 1000 mg/kg/day bw. One control group (group 5) of 5 males and 5 females received the vehicle alone.

Positive control group
One positive control of 5 males only was administered with Mitomycin-C (2 mg/kg) once by intraperitoneal injection at the dose volume of 10 mL/kg
body weight.
Control animals:
yes, concurrent vehicle
Details on study design:
The Sprague Dawley SD rat was the species and strain of choice because it is accepted by many regulatory authorities for reproductive toxicology studies and there is ample experience and background data on this species and strain. The test item was administered orally by gavage to parental animals at 10 mL/kg body weight. Dose levels of 62.5, 250 and 1000 mg/kg/day were selected by the Sponsor based on information from previous studies. The oral route was selected as it is a possible route of exposure of the test item in man.

The study design was in agreement on the following guidelines:
OECD Guideline no. 422 adopted on 22 March 1996.
OECD Guideline no. 474 adopted on July 1997.
The test item was administered orally by gavage at 10 mL/kg body weight. Dose levels of 62.5, 250 and 1000 mg/kg/day were selected by the
Sponsor based on information from previous studies. The oral route was selected as it is a possible route of exposure of the test item in man.
Positive control:
One positive control group of 5 males only was administered once with Mitomycin-C by intraperitoneal injection at the dose volume
of 10 mL/kg bw. This group served as positive control for genotoxicity assessment.
Observations and examinations performed and frequency:
MORTALITY
Throughout the study, all animals were checked early in each working day in the morning and in the afternoon. At weekends and Public Holidays
a similar procedure was followed except that the final check was carried out at approximately mid-day.

CLINICAL SIGNS
Once before commencement of treatment and at least once daily during the study, each animal of the main and recovery groups, was observed and
any clinical signs were recorded.
Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose
reactions.
Once before commencement of treatment and at least once daily during the study each animal of the positive control group was observed and any
clinical signs were recorded. These data are not not presented in this report but retained and archived as study raw data.

CLINICAL OBSERVATIONS (FUNCTIONAL OBSERVATION BATTERY TESTS)
Once before commencement of treatment and at least once per week 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.
All observations were recorded for individual animals.

MOTOR ACTIVITY
Once towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order (for the main groups). For males the tests were performed 7 days before necropsy and for females on Day 3 post partum. Once during Week 4 of recovery, the
motor activity was also performed for all animals.

GRIP STRENGTH AND SENSORY REACTIVITY TO STIMULI
Once towards the end of treatment, 5 males and 5 females were selected from each group for evaluation of sensory reactivity to stimuli of different
modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength. Measurements were performed using a computer
generated random order (for the main groups). For males the tests were performed 6 days before necropsy and for females on Day 3 post partum.
Once during Week 4 of recovery, these evaluations were also performed in all animals.

BODY WEIGHT
Main groups
Males were weighed weekly from allocation to termination. Body weights measured at term (days 34 and 35) are not presented in this report, but
retained and archived as study raw data. Females were weighed weekly from allocation to positive identification of mating and on gestation Days 0,
7, 14 and 20. Dams were also weighed on Days 1 and 4 post partum.
For females, body weights measured weekly during the mating phase are not presented in this report but retained and archived as study raw data.

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

Positive control group
Animals were weighed on the day of allocation, on the day of dosing and just prior to necropsy. These data are not presented in this report but
retained and archived as study raw data.

FOOD CONSUMPTION
Main groups
The weight of food consumed by each cage of males and females was recorded weekly (whenever possible) during the pre-mating period starting
from allocation. Individual food consumption for the females was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and
on Day 4 post partum starting from Day 1 post partum.

Recovery groups
The weight of food consumed by each cage of rats was recorded at weekly intervals following allocation.

BLOOD CLOTTING TIME
During the last week of treatment, a measure of blood clotting time was performed once on the same 5 males and 5 females
randomly selected from each group as specified in Clinical-pathology section (only main groups).

CLINICAL PATHOLOGY
As a part of the sacrificial procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males
and 5 females (females with viable litters) randomly selected from each group, under condition of food deprivation. At the end of
Week 4 of the recovery period, blood samples were also taken from all surviving animals under identical conditions in order to re-evaluate
haematology parameters, gamma-glutamyltransferase and bilirubin which showed treatment-related changes at measurements performed
during the treatment period.

The blood samples collected were divided into tubes as follows:
EDTA anticoagulant fro haematological investigations
Heparin anticoagulant for biochemical tests
citrate anticoagulant for coagulation tests

The measurments performed on blood samples are listed below:
Haematocrit
Haemoglobin
Red blood cell count
Reticulocyte count
Mean red blood cell volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
White blood cell count
Differential leucocyte count
– Neutrophils
– Lymphocites
– Eosinophils
– Basophils
– Monocytes
– Large unstained cells

Platelets

Coagulation
- Prothrombin time
- Activated partial thromboplastin time
- Blood clotting time (in vivo test)

Clinical chemistry
Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Bile acids
Inorganic phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride

Urinalysis (only males) (main groups)
At the same time interval of the clinical pathology investigations, individual overnight urine samples were also collected from the same animals
under the same conditions. Before starting urine collection, water bottles were removed from each cage and each animal received approximately
10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis.
The measurements performed on the urine samples are listed below:
Appearance
Volume
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Blood

The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, was examined microscopically for:

Epithelial cells
Leucocytes
Erythrocytes
Crystals
Spermatozoa and precursors
Other abnormal components
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Parental males (Main groups)
The males were killed after the mating of all females. The total treatment period was up to 48 days.

Parental females (Main groups)
The females with live pups were killed on Day 4 post partum. The females which did not give birth 25 days after positive identification of mating were killed
shortly after.

Males and females (Recovery groups)
Animals were killed after 4 weeks of recovery.

The clinical history of adult 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 and processed for histopathological
examination.

Females (Main groups)
All females were examined also for the following:
1. number of visible implantation sites (pregnant animals);
2. number of corpora lutea (pregnant animals).
Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

Organ weights – Main and recovery groups
From all animals completing the scheduled test periods, the organs indicated in Annex 1 of Study Protocol were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.

Tissues fixed and preserved – Main and recovery groups
Samples of all the tissues listed in Annex 1 of Study Protocol were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and
Harderian glands, testes and epididymides which were fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol).

HISTOPATHOLOGY: Yes
Histopathological examination – Main and recovery groups
The tissues required for histopathological examination are listed in Annex 1 of Study Protocol. After dehydration and embedding in paraffin wax, sections
of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3
micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic
cycle) was performed.
The examination was restricted as detailed below:

1.Tissues specified in Annex 1 of Study Protocol from 5 males and 5 females randomly selected (animals evaluated for clinical pathology)
in the control and high dose group killed at term.
2. All abnormalities in all main groups.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means were assessed by
Dunnett’s test or a modified t test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the other
parameters. The criterion for statistical significance was p < 0.05.
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
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
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
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred throughout the study.
No signs of toxicological significance were found. The majority of high dose animals showed slight to moderate staining of the tail throughout the
study. This finding was also recorded in the mid-dose group. No other clinical signs were recorded.
No signs were recorded in the low dose group throughout the study.
At observation of the cage tray, violet staining and/or dark faeces were recorded in the mid- and high dose groups during treatment.
These findings disappeared few days after the end of treatment during the recovery phase.

BODY WEIGHT AND BODY WEIGHT GAIN
No effects on body weight were found. A transient reduction in body weight gain was recorded in the high dose males of the main group after 2 weeksof treatment and at the end of the dosing period.

FOOD CONSUMPTION
Food consumption was unaffected by treatment. No differences were noted during the recovery period.

BLOOD CLOTTING TIME
No differences were noted in the in vivo coagulation performed at the end of the treatment phase in the main groups.

HAEMATOLOGY
No clear toxicological relevance was given to the neutrophilia and lymphopenia observed in some high dose males of the main group. Neutropenia
was instead observed in the high dose females. Comparable value of neutrophil was recorded in recovery animals. No change was recorded in the
coagulation test.

CLINICAL CHEMISTRY
Increased values of γ-glutamyltransferase and bilirubin were recorded in the mid- and high dose groups. At the end of recovery phase, almost
complete reversibility was noted.

URINALYSIS (main groups only)
No treatment-related changes were recorded.

ORGAN WEIGHTS
Some statistically significant differences were noted in the absolute or relative organ weights, such as:
Decrease in absolute thymus weight in males of the main groups receiving 1000 mg/kg bw/day (16.6%);
Increase in absolute thyroid weight in males of the recovery group receiving 1000 mg/kg bw/day (21.4%);
Increase in relative testes weight in males of the main groups receiving 1000 mg/kg bw/day (6%).
The above differences were not accompanied by histopathological findings. Therefore, they were considered not
treatment-related.

GROSS PATHOLOGY
Dark mucoid contents in the caecum and dark colour in the mesenteric lymph nodes, testes and kidneys were mainly reported in high dose males.
This renal change was also noted in most high dose females. In addition, dark staining was also reported in the tail.

HISTOPATHOLOGY
No treatment-related changes were reported.

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no test substance-related adverse effects were noted
Critical effects observed:
not specified
Conclusions:
On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for both general toxicity and reproduction/developmental toxicity could be considered 1000 mg/kg/day for males and females.
Executive summary:

Study design

A reproductive/developmental toxicity study, including evaluation of systemic toxicity after repeated dose of Reactive Brown DYHY 0331/0334, was conducted in Sprague Dawley SD rats up to Day 4 post partum. The animals received the test item, dissolved in softened water, at the dosages of 62.5, 250 and 1000 mg/kg bw/day as indicated in the scheme below. Three groups of 10 males and 10 females each were administered orally by gavage with the test item at constant volume of 10 mL/kg body weight. A control group of the same number of animals/sex was administered with softened water only. In addition, two groups (Groups 5 and 6) of five animals/sex were included and administered for four consecutive weeks at the same treatment conditions. A 4-week treatment-free period was allowed for these two additional groups, in order to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase.

According to the study design, male animals were dosed 2 weeks prior to pairing, continuously thereafter during pairing and up to the day before necropsy (Week 7). Females were dosed throughout the study including 2 weeks before pairing, thereafter during pairing, gestation and lactation periods until Day 3 post partum.

The following parameters were evaluated in parental animals: body weight, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology, clinical chemistry and urinalysis), litter data, macroscopic observations and organ weights. Histopathological examination was performed only on control and high dose groups (five animals/sex/group randomly selected). It included identification of the stages of the spermatogenic cycle in the selected five males. Measurement of body weight, clinical signs and macroscopic observations of pups were also performed.

Recovery group animals were administered daily for four consecutive weeks followed by a 4-week recovery period. The following parameters were evaluated in these animals: body weight, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), food consumption, clinical pathology investigations (haematology and clinical chemistry), macroscopic observations and organ weights. As no adverse effects were noted in main group animals, histopathology was not performed in these animals.

Mortality and fate of females

No mortality occurred throughout the study.

Clinical signs, neurotoxicity assessment and observation of cage tray

Brown staining of the tail was recorded in the mid- and high dose groups. This finding was related to the colour of the test item (dark brown textile dye). No signs were recorded in the low dose group throughout the study.

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item. In addition, measurements of motor activity, grip strength and sensory reaction to stimuli were comparable between control and treated groups.

At observation of the cage tray, violet staining and/or dark faeces were recorded in the mid- and high dose groups during treatment. These findings disappeared few days after the end of treatment during the recovery phase.

Body weight and body weight gain

No difference in body weights was recorded in animals of both sexes compared to the relative control groups (Groups 1 and 5), throughout the study. However, transient reduction in body weight gain was recorded in the high dose males of the main group after 2 weeks of treatment and at the end of the dosing period.

Food consumption

No effects on food consumption were observed in either males or females of all groups when compared to control groups.

Haematology

At the end of the treatment period, neutrophilia and lymphopenia were observed in some high dose males of the main group. On the contrary, neutropenia was observed in some high dose females. Due to the inconsistency between sexes, these changes were not conclusively attributed to treatment and therefore not considered of toxicological significance. At the end of recovery period, neutrophilia showed almost complete recovery in males, whereas lymphocytes and neutrophils were still lower in treated males and females respectively, when compared to controls. No changes of toxicological relevance were recorded in the coagulation tests (PT and APTT and blood clotting time).

Clinical chemistry

A dose-related increase of bilirubin was recorded in males and females of the main groups. Increased values of gamma-glutamyltransferase were also recorded, especially in the high dose group. These changes, especially for bilirubin, were expected and likely due to the staining of the plasma by the test item (a dark brown textile dye), therefore interfering with the photometric determination of the parameters.

At the end of recovery phase, almost complete reversibility was noted.

Urinalysis

No treatment-related changes were recorded.

Terminal body weight and organ weights

Terminal body weight was unaffected by treatment in both sexes. No toxicological differences were seen in the absolute and/or relative organ weights between groups.

Macroscopic observations

The most relevant changes at macroscopic observations were dark mucoid contents in the caecum and/or dark colour in the mesenteric lymph nodes, testes and kidneys in the high dose group.

This colouration was attributed to the chemical properties of the test item, which is a dark brown textile dye.

Microscopic observations

No treatment-related changes were reported. In addition, no abnormalities were found at the evaluation of the spermatogenic cycle, regular layering in the germinal epithelium of seminiferous tubules was recorded.

Conclusion

On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) was considered to be 1000 mg/kg body weight/day for males and females.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
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 reproductive/developmental toxicity study, including evaluation of systemic toxicity after repeated dose of Reactive Brown DYHY 0331/0334, was conducted in Sprague Dawley SD rats up to Day 4 post partum. The animals received the test item, dissolved in softened water, at the dosages of 62.5, 250 and 1000 mg/kg bw/day as indicated in the scheme below. Three groups of 10 males and 10 females each were administered orally by gavage with the test item at constant volume of 10 mL/kg body weight. A control group of the same number of animals/sex was administered with softened water only. In addition, two groups (Groups 5 and 6) of five animals/sex were included and administered for four consecutive weeks at the same treatment conditions. A 4-week treatment-free period was allowed for these two additional groups, in order to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase.

According to the study design, male animals were dosed 2 weeks prior to pairing, continuously thereafter during pairing and up to the day before necropsy (Week 7). Females were dosed throughout the study including 2 weeks before pairing, thereafter during pairing, gestation and lactation periods until Day 3 post partum.

The following parameters were evaluated in parental animals: body weight, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology, clinical chemistry and urinalysis), litter data, macroscopic observations and organ weights. Histopathological examination was performed only on control and high dose groups (five animals/sex/group randomly selected). It included identification of the stages of the spermatogenic cycle in the selected five males. Measurement of body weight, clinical signs and macroscopic observations of pups were also performed.

Recovery group animals were administered daily for four consecutive weeks followed by a 4-week recovery period. The following parameters were evaluated in these animals: body weight, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), food consumption, clinical pathology investigations (haematology and clinical chemistry), macroscopic observations and organ weights. As no adverse effects were noted in main group animals, histopathology was not performed in these animals.

Mortality and fate of females

No mortality occurred throughout the study.

Clinical signs, neurotoxicity assessment and observation of cage tray

Brown staining of the tail was recorded in the mid- and high dose groups. This finding was related to the colour of the test item (dark brown textile dye). No signs were recorded in the low dose group throughout the study.

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item. In addition, measurements of motor activity, grip strength and sensory reaction to stimuli were comparable between control and treated groups.

At observation of the cage tray, violet staining and/or dark faeces were recorded in the mid- and high dose groups during treatment. These findings disappeared few days after the end of treatment during the recovery phase.

Body weight and body weight gain

No difference in body weights was recorded in animals of both sexes compared to the relative control groups (Groups 1 and 5), throughout the study. However, transient reduction in body weight gain was recorded in the high dose males of the main group after 2 weeks of treatment and at the end of the dosing period.

Food consumption

No effects on food consumption were observed in either males or females of all groups when compared to control groups.

Haematology

At the end of the treatment period, neutrophilia and lymphopenia were observed in some high dose males of the main group. On the contrary, neutropenia was observed in some high dose females. Due to the inconsistency between sexes, these changes were not conclusively attributed to treatment and therefore not considered of toxicological significance. At the end of recovery period, neutrophilia showed almost complete recovery in males, whereas lymphocytes and neutrophils were still lower in treated males and females respectively, when compared to controls. No changes of toxicological relevance were recorded in the coagulation tests (PT and APTT and blood clotting time).

Clinical chemistry

A dose-related increase of bilirubin was recorded in males and females of the main groups. Increased values of gamma-glutamyltransferase were also recorded, especially in the high dose group. These changes, especially for bilirubin, were expected and likely due to the staining of the plasma by the test item (a dark brown textile dye), therefore interfering with the photometric determination of the parameters.

At the end of recovery phase, almost complete reversibility was noted.

Urinalysis

No treatment-related changes were recorded.

Terminal body weight and organ weights

Terminal body weight was unaffected by treatment in both sexes. No toxicological differences were seen in the absolute and/or relative organ weights between groups.

Macroscopic observations

The most relevant changes at macroscopic observations were dark mucoid contents in the caecum and/or dark colour in the mesenteric lymph nodes, testes and kidneys in the high dose group.

This colouration was attributed to the chemical properties of the test item, which is a dark brown textile dye.

Microscopic observations

No treatment-related changes were reported. In addition, no abnormalities were found at the evaluation of the spermatogenic cycle, regular layering in the germinal epithelium of seminiferous tubules was recorded.

Conclusion

On the basis of the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) was considered to be 1000 mg/kg body weight/day for males and females.

 

Justification for classification or non-classification