2,2'-[[3-acetamido-4-[(2-chloro-4-nitrophenyl)azo]phenyl]imino]diethyl diacetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 December 2014 to 05 March 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OECD Guideline no. 474 adopted on September 2014.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

methyl 2-({4-[2-(2-bromo-4,6-dinitrophenyl)diazen-1-yl]-5-acetamido-2-methoxyphenyl}(2-methoxy-2-oxoethyl)amino)acetate

EC Number:

808-279-8

Cas Number:

88938-51-6

Molecular formula:

C21H21BrN6O10

IUPAC Name:

methyl 2-({4-[2-(2-bromo-4,6-dinitrophenyl)diazen-1-yl]-5-acetamido-2-methoxyphenyl}(2-methoxy-2-oxoethyl)amino)acetate

Test material form:

solid: particulate/powder

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

A total of 125 Sprague Dawley [Crl:CD(SD)BR] rats (65 males and 60 virgin females), 7 to 8 weeks old and weighing 206-237 g for males and 180-187 g for females, were received from Charles River Italia S.p.A., Calco (Lecco), Italy. After arrival the weight range for each sex was determined and the
animals were temporarily identified within the cage by means of a coloured mark on the tail. A health check was then performed by a veterinarian.
An acclimatisation period of 14 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.
The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C +/-2°C and 55% +/- 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
In-life phase: 31 december 2015 - 05 March 2015 (last necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

other: The vehicle for the test item was Polyethylene glycol (PEG) 400 in softened water (by reverse osmosis) - 1:1 v/v.

Details on oral exposure:

The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The required amount of the substance was suspended in the vehicle, Polyethylene glycol (PEG) 400 in softened water (by reverse osmosis) - 1:1 v/v. The formulations were prepared daily (concentrations of 12.5, 50 and 200 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed to confirm that the proposed formulation procedure was acceptable and that the stability (28 hours at room temperature in the range of 5 - 250 mg/mL) of the formulation was satisfactory (RTC Study No. A0671).
In the present study, samples of the formulations prepared during the study were also analysed to check the concentration and homogeneity (the first and
the last week of treatment, where possible). Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated
method (RTC Study No. A0671) by a spectrophotometer analysis.
The software used for this activity was Skanlt® version 2.4.2.55 (Thermo Scientific).

Duration of treatment / exposure:

Main groups (Groups 1 to 4)

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 through the day before necropsy (Days 37 and 38 of study). Males were treated for a total of 36 or 37 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 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum. Dose volumes were adjusted once per week for each animal according to the last recorded body weight. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.

Recovery groups (Groups 5 and 6)
Animals were dosed once a day, 7 days a week, for 4 consecutive weeks. No treatment was given during the recovery period.

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
62.5, 250, 1000 mg/kg bw/day
Basis:
actual ingested

Doses / concentrations 2

Remarks:

Doses / Concentrations:
12.5, 50 and 200 mg/mL
Basis:
other: concentration in vehicle

No. of animals per sex per dose:

Each main group comprised 10 male and 10 female rats (Groups 1 to 4).
Two groups (control and high dose levels) of 5 animals per sex were sacrificed after 3 weeks of recovery (Groups 5 and 6).

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Parental animals: Observations and examinations

Mortality
Throughout the study, all animals were checked early in each working day and again 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

Main and Recovery groups
Once before commencement of treatment and at least once daily during the study, each animal 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.

Observations of the cage tray
Observations of the cage tray during the pre-mating period were performed for all groups and were recorded three times weekly.
During mating period these observations were performed for all main groups and were recorded daily.
After mating (only males in main groups) and during gestation (only females in main groups), the observations were performed for all groups and were recorded three times weekly.

Neurotoxicity assessment (removal of animals from the home cage and open arena)
Once before commencement of treatment and at least once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests 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, pupil size, unusual respiratory pattern). All observations were recorded for individual animals.
Animals were examined in an open arena for a minimum of three minutes.

Grip strength and sensory reaction to stimuli
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reaction to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli); an assessment of grip strength was also performed.
Measurements were performed using a computer generated random order (for the main groups).
For males (main groups) the tests were performed on Day 35 of the study and for females on Day 3 post partum (main groups). For animals of the recovery groups the tests were performed on Day 27 of the study (during treatment) and once during Week 2 of recovery (Day 13).

Motor activity assessment (MA)
Once during the study, 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 (main groups) the tests were performed on Day 35 of the study and for females on Day 3 post partum (main groups). For animals of the
recovery groups the tests were performed on Day 27 of the study (during treatment) and once during Week 2 of recovery (Day 13).

Body weight
Main groups
Males were weighed weekly from allocation to termination.
Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also
weighed on Days 1 and 4 post partum.

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.

Food consumption
Main groups
The weight of food consumed by each cage of males and females were recorded weekly (whenever possible) during the pre-mating period starting
from allocation. Individual food consumption for the females were measured on Days 7, 14 and 20 post coitum 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 were recorded at weekly intervals following allocation.

Clinical pathology investigations
During the necropsy 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 conditions of food deprivation.
Since some changes were detected at the clinical chemistry investigations performed during treatment, blood samples were taken under identical
conditions during Week 3 of the recovery period (see below).

Main
Group Animal Number
Males Females
1 10 12 14 16 20 7 13 15 17 19
2 22 26 30 34 40 21 25 31 35 39
3 42 44 48 56 60 43 47 49 51 57
4 62 68 74 78 80 61 65 69 77 79

Recovery Animal Number
Groups Males Females
5 82 84 86 88 90 81 83 85 87 89
6 92 94 96 98 100 91 93 95 97 99

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
The measurements performed on blood samples are listed below:

Haematology

– 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
Coagulation tests in males were repeated during Week 3 of the recovery period.

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

Glucose, triglycerides and total protein in males and inorganic phosphorus in females were repeated during Week 3 of the recovery period.

Sacrifice and pathology:

Postmortem examinations (Parental animals)

Main and Recovery groups
Animals were killed under carbon dioxide asphyxiation. Animals that had completed the scheduled test period and selected for blood collection,
were
killed by exsanguination under isofluorane anaesthesia. Animals that had completed the scheduled test period and not selected for blood
collection,
were killed under carbon dioxide asphyxiation.

Positive Control group
Positive Control group animals were killed under carbon dioxide asphyxiation.

Parental males (Main groups)

The males were killed after the mating of all females after 36 and 37 day of treatment.

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 (Days 26 and 27 post coitum).

Males and females (Recovery groups)
Animals were killed after 3 weeks of recovery.

Necropsy (Main and recovery groups)

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 (excluding one animal found dead: no. X0100094) and the required tissue
samples preserved in fixative and processed for histopathological examination

Females
All females were examined also for the following:
– external and internal abnormalities;
– number of visible implantation sites (pregnant animals);
– 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 period, selected organs according to guideline 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 (all parental animals) collected according to guideline were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol).

Histopathological examination (All groups)
The tissues required for histopathological examination were selected according to guideline. 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 also performed.

The examination was restricted as detailed below:
1. Tissues specified in Annex 1 (section 4.5.6) from 5 males and 5 females (main groups) randomly selected (animals evaluated for clinical
pathology) in the control and high dose group killed at term
2. Tissues specified in Annex 1 (section 4.5.6) from all animals killed or dying during the treatment period
3. All abnormalities in all main groups

On the basis of the histopathological differences observed between control and high dose groups (main groups), the examination was extended to
the stomach of the remaining males and females of the control and high dose groups (main groups), all animals of Groups 2 and 3 (main groups)
and all animals killed after 3 weeks of the recovery period.

Statistics:

Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test. The non-parametric Kruskal-
Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups
were assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p < 0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off. Further tests were used as considered appropriate.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Slight decrease in body weight was noted in males receiving the dose levels >= 250 mg/kg body weight/day. Body weight gain was also decreased in males receiving 1000 mg/kg body weight/day on Days 8 and 15 of the study.

Food efficiency:

no effects observed

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Details on results:

One male of the recovery group receiving 1000 mg/kg body weight/day was found dead on Day 22 of the dosing phase. Clinical signs, such as staining on body surface, rales and salivation were recorded the days before or the day of death of this animal. On the basis of the macroscopic and microscopic findings the factor contributory to the death of this animal could be attributed to a misdosing.
During the in vivo phase, the principal clinical sign recorded was staining on the body surface, particularly evident in males and females receiving
the dose levels 250 mg/kg body weight/day. This finding was also confirmed by green or orange staining noted inside the cage which was likely due to the compound eliminated by the animals. No findings of toxicological relevance were recorded at daily clinical observations including neurotoxicity
assessment.
Decreases in body weight and body weight gain were recorded in males receiving the dose levels more of 250 mg/kg body weight/day during the
treatment period. This change was more evident in those receiving 1000 mg/kg body weight/day. No relevant differences in body weights or in body weight gain were recorded in females before pairing or during post coitum and post partum periods.
No differences in food consumption were observed in treated animals compared to the control group.
No changes of toxicological significance were recorded at haematological investigation or at coagulation in animals sacrificed at the end of the
study.
At clinical chemistry evaluation, changes noted at the end of dosing period in animals receiving the dose levels more of 250 mg/kg body weight/day
(increases in the glucose and triglycerides levels in few males, decrease in protein also in males and increase of phosphorus level in females),
showed a trend of reversibility when evaluated at the end of the recovery period.
Motor activity and sensory reaction to stimuli did not show any differences between control and treated groups.

No differences in terminal body weight or organ weights were noted between control and treated animals of the main groups.
At macroscopic observations single dark/red depressed area in the glandular region of the stomach was noted in two males receiving the test item
at 62.5 or 250 mg/kg body weight/day, respectively. Multiple pale raised areas with central depression in non-glandular region of the stomach associated, in one instance, with thickness of the same gastric region were also observed in two females receiving 1000 mg/kg body weight/day. Dark staining of the tail was observed in all animals dosed at 250 or 1000 mg/kg body weight/day and in one female dosed at 62.5 mg/kg body weight/day. Dark staining in the dorsum of the skin was also seen in some treated males and females.
At microscopic observation, treatment related changes in the stomach (squamous epithelial hyperplasia in non-glandular region) were observed in
animals receiving the dose levels more of 250 mg/kg body weight/day. In addition to the above changes, gastric ulceration was detected in some males (mild) and females (minimal to moderate) dosed at 1000 mg/kg body weight/day and in one male sacrificed after the recovery period (mild).
Furthermore, no changes were recorded in the spermatogenic cycle.

Effect levels

1

Dose descriptor:

NOAEL

Remarks:

local effects

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on the findings in the non-glandular stomach, the NOAEL (No Observed Adverse Effect Level) in rats for local effects was considered to be 250 mg/kg body weight/day.

2

Dose descriptor:

NOAEL

Remarks:

systemic

Effect level:

1 000 mg/kg bw (total dose)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the findings in the non-glandular stomach in the combined repeat-dose/developmental screening study, the NOAEL (No Observed Adverse Effect Level) in rats for local effects was considered to be 250 mg/kg body weight/day. Due to the fact that the histopathological findings observed in the non- glandular region of the stomach are a local reaction due to the test material overload, the NOAEL for systemic effects was considered to be 1000 mg/kg body weight/day for male and female rats. However, as humans do not possess a non-glandular stomach, this finding is not relevant for humans and consequently for the risk assessment.

Executive summary:

The toxic effects on rats after repeated dosing with the test item as well as any effects of the test item on male and female reproductive performance, such as gonadal function, conception, parturition and early lactation of the offspring, were investigated. A 3-week treatment-free period was allowed in order to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase.

The animals received the test item, dissolved in Polyethylene glycol (PEG) 400 in softened water (by reverse osmosis) - 1:1 v/v, at a constant volume of 5 mL/kg body weight as indicated in the scheme below.

 

Main groups

Group	Treatment (mg/kg body weight/day)	Number of animals
1	0	10M+10F
2	62.5	10M+10F
3	250	10M+10F
4	1000	10M+10F

 

 Recovery groups

Group	Treatment (mg/kg body weight/day)	Number of animals
5	0	5M+5F
6	1000	5M+5F

 

Main groups

Males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 37/38 days (Groups 1, 2, 3, 4). Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 3 post partum or approximately 41 days. (Groups 1, 2, 3, 4). The following investigations were performed in all groups: 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 and clinical chemistry), litter data, macroscopic observations, organ weights and histopathological examination. Routine histopathological examination was performed only on control and high dose groups (five animals/sex/group randomly selected). Since histopathological changes of the stomach were observed in high dose animals, the examination was extended to the stomach of the remaining animals of the control and high dose groups and in all animals of Groups 2 and 3. The identification of the stages of the spermatogenic cycle was performed in all males of the control and high dose groups.

Body weight, clinical signs and macroscopic observations of pups were also performed.

Recovery groups

Animals of the recovery groups were treated for 4 consecutive weeks and sacrificed after 3 weeks of recovery (Groups 5, 6).

The following parameters were evaluated in these animals: mortality, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), body weight, food consumption, clinical pathology investigations (haematology and clinical chemistry), macroscopic observations, organ weights and histopathological examination.The histopathological examination was performed on the stomach only.

 

Results

One male of the recovery group receiving 1000 mg/kg bodyweight/day was found dead on Day 22 of the dosing phase. Clinical signs, such as staining on body surface (Days 21 and 22) rales and salivation (Day 21) were recorded the days before or the day of death of this animal. At macroscopic observations, the most remarkable changes noted were abnormal dark soft contents in the thoracic cavity, red staining on the muzzle, as well as red colour and/or dark soft contents in most of the gastro-intestinal tract.

At microscopic observations, moderate atrophy of the thymus with oedema in the surrounding connective tissue and squamous hyperplasia in the non glandular region of the stomach were noted. The factor contributory to the death of this animal could be attributed to a misdosing.

 

The major clinical sign recorded during the treatment period was staining (green/blue) of the body surface, particularly evident in males and females receiving the dose levels >=250 mg/kg body weight/day. This discolouration was due to the colour of the test item and its properties as textile dye.

Also during the recovery period, staining of the body surface was noted in animals previously treated with 1000 mg/kg body weight/day.

Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item. No relevant differences in motor activity, grip strength and sensory reaction to stimuli were noted between control and treated groups.

As a consequence of the colour of the test item, green or orange staining was observed inside the cage of all treated animals (main and recovery groups during the treatment period) of both sexes receiving the dose levels >=62.5 mg/kg body weight/day, before pairing, during pairing (cage tray) until termination (only main males). Treated females did not show any discoloration of the cage during the gestation period. After cessation of treatment in the recovery group, no abnormal colour of the cage tray was observed in these animals.

The above mentioned finding was considered related to the green/dark colour

of the test item which was eliminated by the animals.

In main group animals, slightly lower mean body weights compared to control weights were noted in males receiving the dose levels >= 250 mg/kg body weight/day starting from Day 8 until termination reaching statistically significance on Days 8 and 15 in the high dose groups. This was caused by lower body weight gains in males receiving 1000 mg/kg body weight/day on Days 8 and 15 of the study, reaching statistically significance on Day 8.

No relevant differences in body weights or in body weight gain were recorded in females before pairing or during post coitum and post partum periods.

In recovery groups, between Days 8 and 29 of the study, treated males receiving 1000 mg/kg bodyweight/day showed lower body weight, not reaching statistically significance, compared to the control group resulting from a lower body weight gain of treated males on Days 8, 15 and 22, reaching statistically significance on Day 8. Statistically significant decrease in body weight gain was also seen in females on Day 8 of the study. No difference in body weight or body weight gain was recorded in animals of both sexes during the recovery phase.

Food consumption was lower than in the control group (without statistically significance) on Days 8 and 15 in high dose males. No effects on food consumption were observed in treated females throughout the study.

No haematological changes of toxicological significance were noted.

In main group animals, a slight but statistically significant decrease of activated thromboplastin time was recorded in males dosed with 1000 mg/kg bodyweight/day. Due to the slight severity and the direction of change, this finding was not considered adverse.

At the end of Week 3 of the recovery period, coagulation analyses were performed on males. No differences between control and treated animals were observed, confirming complete reversibility.

In main group animals, some changes were observed in animals receiving the dose levels of 250 mg/kg body weight/day and above, such as: increases in the glucose and triglycerides levels in few males without reaching statistically significance at a group mean level and a statistically significant decrease in protein in males and increase of phosphorus level in females at the highest dose level.

At the end of Week 3 of the recovery period, glucose, triglycerides and total protein were analysed in males and inorganic phosphorus in females. Triglycerides in males and phosphorus in females showed reversibility, even if they were still slightly higher than controls. Concerning glucose in males, data were comparable with controls, although values were similar to those observed during the dosing phase. Protein showed a complete recovery.

 

No statistically significant differences in terminal body weight were noted between controls and treated animals, both in main and recovery groups. There were no treatment related changes in absolute and relative organ weights.

In main groups at macroscopic observations, single dark/red depressed area in the glandular region of the stomach was noted in each one male receiving the test item at 62.5 or 250 mg/kg body weight/day. Multiple pale raised areas with central depression in non-glandular region of the stomach associated, in one instance, with thickness of the same gastric region were also observed in two females receiving 1000 mg/kg body weight/day. Dark staining of the tail was observed in all animals dosed at >=250 mg/kg body weight/day and in one female dosed at 62.5 mg/kg body weight/day. Dark staining of the dorsum of the skin was also seen in some treated males and females. The only relevant changes noted at post mortem examinations in treated animals of the recovery group, when compared with controls, were dark staining of the tail. The dark colour reported in the skin and tail was considered related to the colour of the test item and its properties as textile dye.

Microscopically, treatment-related changes in the main groups consisted of minimal to marked squamous epithelial hyperplasia in the non-glandular region of the stomach reported in males and females dosed at 250 and 1000 mg/kg body weight/day sacrificed at the end of study. This gastric change with a reduced severity degree was reported in all males (minimal to moderate) and in 3 out of 5 females (minimal to mild), sacrificed after 3weeks of recovery period. No pathological changes were noted in the non-glandular region of the stomach in animals dosed at 62.5 mg/kg body weight/day (low dose group). In addition to the above changes, gastric ulceration in the non glandular region of the stomach was detected in some males and females dosed at 1000 mg/kg body weight/day and in one male sacrificed after the recovery period. The epithelial hyperplasia in the forestomach could be considered not relevant since humans do not have a forestomach equivalent. The glandular region of the stomach did not show pathological changes. In addition, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.

Based on the findings in the non-glandular stomach, the NOAEL (No Observed Adverse Effect Level) in rats for local effects was considered to be 250 mg/kg body weight/day. Due to the fact that the histopathological findings observed in the non- glandular region of the stomach are a local reaction due to the test material overload, the NOAEL for systemic effects was considered to be 1000 mg/kg body weight/day for male and female rats. However, as humans do not possess a non-glandular stomach, this finding is not relevant for humans and consequently for the risk assessment.