4-[[2-methoxy-4-[(4-nitrophenyl)azo]phenyl]azo]phenol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

Group 8 animals were allowed a 4-day wash-out period and not 3 days. Pituitary was weighed even if not stated in the study protocol. These deviations were considered to have not affected the integrity of the study.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-[[2-methoxy-4-[(4-nitrophenyl)azo]phenyl]azo]phenol

EC Number:

243-325-5

EC Name:

4-[[2-methoxy-4-[(4-nitrophenyl)azo]phenyl]azo]phenol

Cas Number:

19800-42-1

Molecular formula:

C19H15N5O4

IUPAC Name:

4-[[2-methoxy-4-[(4-nitrophenyl)azo]phenyl]azo]phenol

Test material form:

solid: particulate/powder

Remarks:

Dark brown

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- A total of 130 Hsd: Sprague Dawley SD rats (65 males and 65 virgin females), 6 to 7 weeks old and weighing 176 to 200 g for males and 151 to 175 g for females, were ordered from Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy. 5 animals per cage.
- Acclimatisation period of approximately 3 to 9 weeks, depending on the type of treatment.
- Temperature and relative humidity: 22+/- 2°C and 55 +/- 15%, respectively.
- Artificial light for 12 hours.
- Feed (commercially available laboratory rodent diet (4 RF 21) and water: ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

The test item was administered orall by gavage to animals at 5 mL/kg bw.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical method validation:
An analytical method was validated in the range
from 10 to 120 mg/mL. Linearity, accuracy and precision were within the
limits stated in RTC SOPs for suspensions (r > 0.98; accuracy 90-110%;
precision CV < 5%).

Validation of the analytical method:
The analytical method was validated in RTC Study no. A0769 in the range
from 10 to 120 mg/mL. Linearity, accuracy and precision were within the
limits stated in RTC SOPs for suspensions (r > 0.98; accuracy 90-110%;
precision CV < 5%).

Formulation sampling:
Formulations of the test item were prepared as suspensions in PEG 400
- water (1:1). Concentration and homogeneity of the low and high dose
level were assessed by taking six analytical aliquots in different positions.
For the intermediate levels, only concentration was assessed by taking two
different analytical aliquots. Each analytical aliquot was analysed separately.
Concentration was evaluated as the mean of the single determinations and
homogeneity as the coefficient of variation of the sextuplicate set.

Stability:
In RTC Study no. A0769, a 28 hour stability at room temperature and a 8
day at 5°C ± 3°C was verified in the range from 10 to 120 mg/mL. According to RTC SOPs, solutions are considered to be stable if concentration and
homogeneity, after the defined period of storage, are still acceptable (85%-
115% for concentration and CV < 10% for homogeneity).

Duration of treatment / exposure:

28 days, followed by a 2 week treatment-free recovery period

Frequency of treatment:

Once daily for 28 days

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

controls

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Remarks:

This level was stopped after 3 weeks of treatment and the animals were sacrificed

Doses / concentrations 4

Dose / conc.:

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

Remarks:

This level was stopped after 6 days of treatement and the animals were sacrificed

Doses / concentrations 5

Dose / conc.:

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

Remarks:

This level was stopped after 3 days of treatment and the animals were allowed a 4-day washout period

Doses / concentrations 6

Dose / conc.:

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

Remarks:

This level was given after a 4-day washout period

No. of animals per sex per dose:

10 for treated groups with the test substance
5 for recovery and positive control groups

Control animals:

yes

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily throughout the study

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and at least once daily during the study

BODY WEIGHT: Yes
- Time schedule for examinations:
Main groups
Males were weighed weekly from allocation to termination.
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.

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
Animalswereweighed on the day of allocation, on the day of dosing and just prior to necropsy. These data are not tabulated in this report but archived with all raw data.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
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 2 of the recovery period, blood samples were withdrawn from all surviving
animals under identical conditions in order to re-evaluate the haematology and clinical
chemistry parameters, which showed possible treatment-related changes at measurements
performed during the treatment period.
Blood samples were not taken from animals of Groups 3 and 4 sacrificed after 21 and 6 days
of treatment, respectively.
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

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes

Clinical Observations (Functional Observation Battery Tests) (Main and Recovery
groups):
Once before commencement of treatment and at least once aweek from the start of treatment
until termination, 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). Changes in fur, skin,
eyes, mucous membranes, occurrences of secretions and excretions were also recorded.

Grip strength and sensory reactivity to stimuli (Main and Recovery groups):
Once during the study, towards the end of treatment, 5 males and 5 females were randomly
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).
Once duringWeek 2 of recovery, these evaluations were also performed in all animals.

Motor activity assessment (MA) (Main and Recovery groups):
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). Once, duringWeek 2 of recovery, the MA was
also performed in all animals.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
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 (excluding animals sacrificed for humane reasons or
found dead) and the required tissue samples preserved in fixative and processed for histopathological examination.

Females (Main groups)
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 apparently non-pregnant females or uteri of females with no visible implantations
were immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation.

Pups
All pups found dead in the cage or sacrificed for humane reasons were examined for external and internal abnormalities. All live pups sacrificed at termination were killed and examined
for external abnormalities and sex confirmation by gonadal inspection.

ORGAN WEIGHTS: Yes
Organ weights (Main and Recovery groups)
From all animals completing the scheduled test period, the organs indicated in section 4.5.6
were dissected free of fat and weighed. The ratios of organ weight to body weight were
calculated for each animal.
Organ weights were not taken from animals of Groups 3 and 4 sacrificed after 21 and 6 days of treatment, respectively.

HISTOPATHOLOGY: Yes
The tissues required for histopathological examination are listed in section 4.5.6. 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.
In the first instance, the examination was restricted as detailed below:
i Tissues specified in section 4.5.6 from 5 males and 5 females randomly selected (animals
evaluated for clinical pathology) in the control and high dose group (Group 2)
killed at term.
ii Tissues specified in section 4.5.6 from all animals killed or dying during the treatment
period, with the exception of Group 4 animals.
iii All abnormalities in all groups
The examination was extended to include the remaining females (animals not evaluated for
clinical pathology) of the control, the low and the high dose groups and from the females
killed after 2weeks of recovery, the thymus, in which a suspicion of treatment-related changes was observed in the high dose group.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

- Signs of toxicity such as decreased activity, piloerection, kyphosis, staining of the perianal region/muzzle and pallor were seen in almost all animals receiving 250 mg/kg bw/day, during the first week of mating period and in almost all animals receiving 600 mg/kg bw/day, during the first week of treatment.
- Piloerection, kyphosis and/or decreased activity and staining of the perianal region/muzzle were seen in 2 females when receiving 350-50 mg/kg bw/day; no clinical signs were seen in males and females when receiving 50 mg/kg bw/day. Males receiving 100 mg/kg bw/day did not show any clinical signs throughout the study. Kyphosis, piloerection, dyspnoea and/or pallor, swollen ventral region of the neck and/or staining of the perianal region, decreased activity, cold to touch were the major signs of toxicity seen in 3 females which were sacrificed for humane reasons, on Day 1 post partum.
- Recovery groups: No clinical signs were seen in treated animals throughout the study, during treatment and recovery periods.

Mortality:

mortality observed, treatment-related

Description (incidence):

- Two males receiving 600 mg/kg bw/day were found dead on Day 5 and 7 of treatment. Kyphosis, piloerection and/or staining on the perianal region were seen the day before death. The remaining animals of this group were killed after 6 days of treatment.
- Five males and 6 females receiving 250 mg/kg bw/day showed kyphosis, piloerection, pallor and/or decreased activity, staining of perianal region, swollen neck and cold to touch and were killed for humane reasons. The remaining animals in this group were killed after 21 days of treatment.
- Four unscheduled deaths occurred in females receiving 100 mg/kg/day. One female was found dead and 3 females were killed for humane reasons on Day 1 post partum. The clinical signs observed in these last three females were similar to those observed in humanely killed animals of the 250 and 600 mg/kg bw/day groups, suspended due to high toxicity.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

During the study:
- No relevant differences in body weight and body weight gain were noted between control and treated groups, of both sexes, during the study, with the exception of statistically significant decreases in body weight and body weight gain, seen in the 100 mg/kg bw/day group of females when compared to controls, during the post partum period (-13% and -4 fold on Day 4, respectively).
- Recovery groups: during treatment, slight decrease in body weight gain was seen on Day 8 of treatment, in the 100 mg/kg bw/day group of males (statistically significant) and females, when compared to controls. During recovery, no relevant differences in body weight and body weight gain were noted between control and treated group, of both sexes.
Terminal body weight:
- Terminal body weight was unaffected by treatment in males, while a slight statistically significant decrease (13%) in terminal body weight was seen in females (100 mg/kg bw/day), when compared to controls. Terminal body weight was also unaffected by treatment in both sexes of recovery groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Slight statistically significant decrease in food consumption was recorded in females dosed at 100 mg/kg bw/day on Day 7 post coitum (-6%) and on Day 4 post partum (-47%), compared to controls. No changes were noted in treated males during treatment.
- Recovery groups: no relevant differences in food consumption were noted in recovery group animals (both sexes), during the study.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- When compared with controls, reticulocytosis was recorded in treated males (47%) with no relevant changes of erythrocytes/haemoglobin. In addition, treated females showed a slight decrease of haemoglobin and haematocrit (approximately 9%) and increase of platelets (38%). Due to the slight magnitude, these changes were not considered to be adverse. The differences of leucocytes between control and treated females were mainly due to the high values of one control animal.
- Recovery groups: changes recorded during the dosing phase were no longer observed, confirming complete reversibility.
- Coagulation: no changes were recorded.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

- Fluctuations of liver/metabolic markers were recorded in some treated animals. Compared with mean control data, 1 male showed increase of alanine aminotransferase (64%), aspartate aminotransferase (3.5 fold), bilirubin (6.3 fold) and bile acids (2.5 fold) and another one showed increase of cholesterol (78%) and triglycerides (93%). An increase of aspartate aminotransferase (2.1fold), urea (59%) and a decrease of triglycerides (74%) was observed in one female, and an increase of phosphorus (34%) was detected in another female.
- Recovery groups: full recovery was observed for all changes recorded during the dosing phase.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

- During the last week of treatment for in the 250 mg/kg bw/day group (Week 3), females showed slow handling reactivity at the removal from the cages; in the open arena hunched gait and/or mobility impairment was seen in males and females.
- In the 0, 50, and 100 mg/kg bw/day groups: neurotoxicity assessment (removal of animals from the home cage and in an open arena) did not reveal changes attributable to the test substance, with the exception of one female of the 100 mg/kg bw/day group which showed slow handling reactivity at the removal from the cage and moderately hunched gait in the open arena.
- No relevant differences in motor activity, grip strength and sensory reactivity to stimuli were noted between control and treated groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Statistically significant changes seen in organ weights of the 350-50 mg/kg bw/day group (increases in epididymides and prostate weights, decrease in heart and pituitary weights) were considered of no toxicological significance since they were minimal and/or related to the low dose only and not accompanied by histopathological findings. The most relevant changes observed were the decreases (statistically significant) in absolute and/or relative thymus weight, seen in males and females of the group 350-50 mg/kg bw/day, and thyroid weights, seen in males of the group 350-50 mg/kg bw/day. Males of the 100 mg/kg bw/day showed statistically significant increases in absolute and/or relative spleen weight. In addition, a statistically significant decrease in thymus and increase in adrenal weights were seen in females dosed at 100 mg/kg bw/day. These changes were accompanied by histopathological findings, such as atrophy of thymus. The other changes (kidneys, brain, heart) were minimal and seen only in one sex. In addition, these changes were not accompanied by histopathological findings and therefore they were considered unrelated to treatment.
- No relevant changes in organ weights were noted in the recovery groups, with the exception of increase of absolute and relative pituitary weights and in relative seminal vescicles weight in high dose males.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

effects observed, treatment-related
Unscheduled death:
- Seventeen unscheduled deaths occurred during the study, in 100, 250, and 600 mg/kg bw/day groups.
- Group 600 mg/kg bw/day: 2 males were found dead within the first week of the premating phase. The macroscopic findings observed were: reduced size of the spleen and/or thymus and dark fluid content mainly in intestinal tract, and only for one male in the urinary bladder. Black staining of the skin of urogenital region was also observed. No histopathological evaluations were performed.
- Group 250 mg/kg bw/day: 5 males and 6 females were humanely sacrificed during the first week of the mating phase, with the exception of one female which was sacrificed on Day 14 of the premating phase. The findings seen at macroscopic observations were: dark and/or red colour and fluid or mucoid content of gastro-intestinal tract, reduced size of thymus, presence of dark/clear fluid in the thoracic cavity and staining in the urogenital region and/or head and, in few instances,oedematous consistency. Major findings at microscopic observation included: lymphocytolysis and/or atrophy in the spleen and/or thymus in both sexes. In addition, mucosal congestion of ileum was observed in few instances. The factors contributory to the illness status of the animals were attributed to the immuno depression seen in thymus and spleen.
- Group 100mg/kg bw/day: 3 females of were sacrificed for humane reasons and one was found dead. The findings observed at macroscopic examination were: oedematous consistency in the cervical lymphnodes, salivary glands and skin (ventral neck orurogenital region) in most animals. In addition, dark and/or red colour and/or fluid or mucoid content of gastro intestinal tract in few animals were also observed. Major findings at microscopic observation included: lymphocytolysis and/or atrophy in the spleen and/or thymus in both sexes and diffuse oedema in the ventral neck or urogenital region. The factors contributory to the illness status were attributed again to immunodepression (spleen and thymus) and haemodynamic imbalance. The immunodepression could be considered as stress-related.
Final sacrifice:
- Groups 250 and 600 mg/kg bw/day (sacrificed before term).The major findings observed at macroscopic examination were: dark content of intestinal tract and brown staining in the urogenital or perianal region (at 600 mg/kg bw/day only).
- Groups 100 and 350-50 mg/kg bw/day (sacrificed at term). The only change noted was reduced size of thymus in females.
Recovery sacrifice:
- No remarkable treatment-related changes were observed when compared to the controls.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

- Final sacrifice
Groups 250 and 600 mg/kg bw/day (sacrificed before term): Few males and females showed minimal to moderate congestion in the gastro intestinal tract and lymphocytolysis of spleen and thymus in a single female.
Groups 100 and 350-50 mg/kg bw/day (sacrificed at term). Treatment-related changes in females, consisting in minimal to marked atrophy of thymus, were observed. No treatment-related histopathological changes were observed in males.
- Recovery sacrifice
No histopathological changes were noted in the thymus of control and treated females sacrificed after 2 weeks of recovery period.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: General toxicity

2

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Remarks:

94.8% test substance purity

Sex:

male/female

Basis for effect level:

other: General toxicity

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: Thyroid

Organ:

other: Thyroid

Treatment related:

yes

Dose response relationship:

not specified

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

Based on these results, the systemic NOAEL for the study was considered to be 100 mg/kg/day (i.e. 94.8 mg a.i./kg bw/day). 

Executive summary:

A study was conducted to determine the toxicity to reproduction of the test substance (in the form of a dark brown powder of 94.8% purity) according to OECD Guideline 422. Male and female Sprague Dawley rats (10/sex/dose for the main study and 5/sex/dose for the recovery groups) were exposed to the following concentrations (dissolved in polyethylene glycol, at a constant volume of 10 mL/kg bw):

 

Group	Dose (mg/kg bw/day)	Treatment period	Comment	Recovery?
1	0	Males: 2 weeks prior to pairing and during pairing with females until the day before necropsy: 29 days Females: 2 weeks prior to pairing, during pairing and throughout gestation and lactation until Day3 post-partum: 41-51 days	Controls	Yes
2	100	Males: 2 weeks prior to pairing and during pairing with females until the day before necropsy: 29 days Females: 2 weeks prior to pairing, during pairing and throughout gestation and lactation until Day3 post-partum: 41-51 days	-	Yes
3	250	-	This level was stopped after 3 weeks of treatment and animals were sacrificed	No
4	600	-	This level was stopped after 6 days of treatment and animals were sacrificed	No
8	350	Males: 2 weeks prior to pairing and during pairing with females until the day before necropsy: 38 days Females: 2 weeks prior to pairing, during pairing and throughout gestation and lactationuntil Day3 post-partum: 49-64 days	This level was stopped after 3 days of treatment and animals were allowed a 4-day wash-out period	No
8	50		This level was given after a 4-day wash-out period	No

 

Animals in the controls and 100 mg/kg bw/day groups were assigned to a 2 week recovery period after end of treatment.

 

In the main groups, mortality and clinical signs were recorded daily. Neurotoxicity, motor activity, grip strength and sensory reactivity to stimuli were assessed once during the study. Body weight and food consumption were measured weekly (and dams were also weighed on Days 1 and 4 post-partum). At necropsy, blood samples were collected for haematology and clinical chemistry investigations. Also, a gross macroscopical examination was conducted, and organs were weighed. Routine histopathological examination was performed on control and high dose (100 mg/kg bw/day) animals (5/sex/group randomly selected) and on 250 mg/kg bw/day animals. In addition, thymus was examined in all females at 0, 100 and 350-50 mg/kg bw/day. Animals of the recovery groups were treated for a total of 4 consecutive weeks and sacrificed after 2 weeks of recovery. The following parameters were evaluated in these animals: clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction tostimuli), body weight, food consumption, clinical pathology investigations (haematology and clinical chemistry), macroscopic observations, organ weights and histopathological evaluation of the thymus in all females, only.

 

Mortality occurred at 100, 250 and 600 mg/kg bw/day. Signs of toxicity such as decreased activity, piloerection, kyphosis, staining of the perianal region/muzzle and pallor, cold to touch were seen at most does levels, apart from rats at 50 mg/kg bw/day and in the recovery groups.

 

During the last week of treatment (Week 3), 250 mg/kg bw/day females showed slow handling reactivity at the removal from the cages; in the open arena hunched gait and/or mobility impairment was seen in males and females. Neurotoxicity assessment (removal of animals from the home cage and in an open arena) did not reveal changes attributable to the test item, with the exception of one female at 100 mg/kg bw/day which showed slow handling reactivity at the removal from the cage and moderately hunched gait in the open arena. No relevant differences in motor activity, grip strength and sensory reactivity tostimuliwere noted between control and the other treated groups.

 

No relevant differences in body weight and body weight gain were noted between control and treated groups during the study, with the exception of statistically significant decreases in body weight and body weight gain, in females at 100 mg/kg bw/day when compared to controls during thepost-partumperiod. During recovery, no relevant differences in body weight and body weight gain were noted between control and treated group.

 

Slight statistically significant decrease in food consumption was recorded in 100 mg/kg bw/day females on Day 7post coitumand on Day 4post-partum, compared to controls. No changes were noted in treated males during treatment. No relevant differences in food consumption were noted in recovery group animals.

 

When compared with controls, reticulocytosis was recorded in treated males of the main groups, with no relevant changes of erythrocytes/haemoglobin. In addition, treated females showed a slight decrease of haemoglobin and haematocrit and increase of platelets. Due to the slight magnitude, these changes were not considered to be adverse. The differences of leucocytes between control and treated females were mainly due to the high values of one control animal. In the recovery phase, changes recorded during the dosing phase were no longer observed, confirming complete reversibility.

 

No changes were seen on coagulation parameters of main group animals.

 

Fluctuations of liver/metabolic markers were recorded in some main groups treated animals. Full recovery was observed for all changes recorded during the dosing phase.

 

Terminal body weight was unaffected by treatment in main group males, while a slight statistically significant decrease in terminal body weight was seen 100 mg/kg bw/day females when compared to controls. Statistically significant changes seen in organ weights of 350-50 mg/kg bw/day males (increases in epididymides and prostate weights, decrease in heart and pituitary weights) were considered of no toxicological significance since they were minimal and/or related to the low dose only and not accompanied by histopathological findings. The most relevant changes observed were the decreases (statistically significant) in absolute and/or relative thymus weight, seen in 350-50 mg/kg bw/day males and females, and thyroid weights, seen in males of this dose group. Males at 100 mg/kg bw/day showed statistically significant increases in absolute and/or relative spleen weight. In addition, a statistically significant decrease in thymus and increase in adrenal weights were seen in 100 mg/kg bw/day females. These changes were accompanied by histopathological findings such as atrophy of thymus. The other changes (kidneys, brain and heart) were minimal and seen only in one sex. In addition, these changes were not accompanied by histopathological findings and therefore they were considered unrelated to treatment. Terminal body weight was unaffected by treatment in both sexes of recovery groups. No relevant changes in organ weights were noted in the recovery groups, with the exception of increase of absolute and relative pituitary weights and in relative seminal vesicles weight in 100 mg/kg bw/day males.

 

Seventeen unscheduled deaths occurred during the study at 100 mg/kg bw/day and above. Gross pathology findings in these animals included reduced size of the spleen and/or thymus, dark and/or red fluid content mainly in the thoracic cavity, the intestinal tract (and mucoid content) and the urinary bladder, black staining of the skin of urogenital region and head, oedematous. Major findings at microscopic observation included lymphocytolysis and/or atrophy in the spleen and/or thymus in both sexes, as well as mucosal congestion of the ileum. At sacrifice (at 100 mg/kg bw/day and above), gross pathology findings included dark content of intestinal tract and brown staining in the urogenital or perianal region, and reduced size of thymus in females. No findings were seen in the recovery group. In the final sacrifice groups at 250 and 600 mg/kg bw/day (sacrificed before term), minimal to moderate congestion was seen in the gastro-intestinal tract of a few males and females and lymphocytolysis of the spleen and thymus were noted in a single female. At 100 and 350-50 mg/kg bw/day (sacrificed at term), treatment-related histopathological changes in females consisted in minimal to marked atrophy of thymus. No treatment-related changes were observed in males. In the recovery group (final sacrifice), no histopathological changes were noted in the thymus after 2 weeks of recovery.

 

Few males and females receiving 250 mg/kg/day showed minimal to moderate congestion in the gastrointestinal tract and lymphocytolysis of spleen and thymus in a single female.Treatment-related changes in females receiving 50 and 100 mg/kg/day consisted in minimal to marked atrophy of thymus. No treatment-related histopathological changes were observed in males. No histopathological changes were noted in the thymus of control and treated females sacrificed after 2 weeks of recovery period.

 

Based on these results, the systemic NOAEL for the study was considered to be 100 mg/kg/day (i.e. 94.8 mg a.i./kg bw/day) (Rossiello, 2016).