2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium methyl sulphate

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:

2016-12-06 to 2018-02-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

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:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium chloride

EC Number:

248-053-0

EC Name:

2-[[4-[ethyl(2-hydroxyethyl)amino]phenyl]azo]-6-methoxy-3-methylbenzothiazolium chloride

Cas Number:

26850-47-5

Molecular formula:

C19H23ClN4O2S

IUPAC Name:

2-[(E)-{4-[Ethyl(2-hydroxyethyl)amino]phenyl}diazenyl]-6-methoxy-3-methyl-1,3-benzothiazol-3-ium chloride

Test material form:

solid

Details on test material:

see below

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was the species and strain of choice since it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy; supplied by Charles River Germany, Sandhofer Weg 7, 97633 Sulzfeld, Germany.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10-11 weeks
- Weight at study initiation: females: 181-217 g; males: 206-223 g
- Fasting period before study: no
- Housing: From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm. During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor. After mating, the males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages measuring 42.5×26.6×18.5 cm for the gestation period, birth and lactation. Nesting material was provided inside suitable bedding bags. In addition, suitable nesting material (Scobis 0 Mucedola) was provided as necessary and changed at least 2 times a week.
- Diet (e.g. ad libitum): RF 21, Mucedola S.r.l.; ad libitum, except when bleeding was performed
- Water (e.g. ad libitum): Drinking water was supplied ad libitum to each cage via water bottles
- Acclimation period (From arrival): An acclimatisation period of 18 days (main groups) and 40 days (recovery groups) was allowed before the start of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 2 °C
- Humidity (%): 55% ± 15%
- Air changes (per hr): approximately 15 to 20 air changes per hour
- Photoperiod (hrs dark / hrs light): the rooms were lit by artificial light for 12 hours each day

IN-LIFE DATES: From: 13 December 2016 (allocation to the study) To: 22 February 2017 (Last day of necropsy)

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was suspended in the vehicle. The formulation was prepared daily or up to weekly at concentrations of 4, 8, 16 or 12 mg/mL from day 17 on. Formulations were maintained under magnetic stirring for ca. 1 hour at room temperature prior to use and up until the dosing of the last animal.

- VEHICLE
- Justification for use and choice of vehicle (if other than water): no toxic effects
- Amount of vehicle (if gavage): 10 mL/kg bw

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 of the formulation in the range from 0.05 to 25 mg/mL was satisfactory. Stability after 28 hours at room temperature and 5 +/- 3 °C for 8 days was also verified. In the present study, samples of the formulations prepared during the study were also analysed to check the concentration and homogeneity (two occasions during the study). Results of all analyses were within the acceptability limits (85-115% for concentration and CV <10% for homogeneity). Chemical analysis was carried out using spectrophotometric analysis. The software used for this activity was SkanIt version 2.4.2.55 (Thermo Scientific).

Duration of treatment / exposure:

Main groups (Groups 1 to 4):
Males: Animals were dosed for 2 consecutive weeks prior to pairing, through the mating period and thereafter for a total of 28 days.

Females: Animals were dosed for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 3 post partum (for at least 41 days).

Recovery groups (Groups 5 and 6):
Animals were dosed for a minimum of 4 consecutive weeks. No treatment was given during the recovery period.

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1, control (main study)

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Group 2, low dose (main study)

Doses / concentrations 3

Dose / conc.:

80 mg/kg bw/day (nominal)

Remarks:

Group 3, medium dose (main study)

Doses / concentrations 4

Dose / conc.:

160 mg/kg bw/day (nominal)

Remarks:

Group 4, high dose (main study) - until day 16

Doses / concentrations 5

Dose / conc.:

120 mg/kg bw/day (nominal)

Remarks:

Group 4, high dose (main study) - from day 17

Doses / concentrations 6

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 5, control (recovery)

Doses / concentrations 7

Dose / conc.:

80 mg/kg bw/day (nominal)

Remarks:

Group 6, medium dose (recovery)

No. of animals per sex per dose:

Main groups (groups from 1 to 4): 10
Recovery groups (Groups 5 and 6): 5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a previous dose range finding study
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: to assess recovery from any delayed toxicity or peristence of adverse effects observed during the dosing phase
- Post-exposure recovery period in satellite groups: Two recovery groups were assigned (control and medium dose group) and the post-exposure period was two weeks.

Positive control:

not necessary

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: General clinical observations were made at least once a day for all animals. Twice daily all animals were checked for mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least daily during the study each animal. 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. All observations were recorded for individual animals.

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 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 was recorded weekly (whenever possible) during the pre-mating period starting from allocation. Individual food consumption for the females was 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 was recorded at weekly intervals following allocation.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: as part of the sacrificial procedure
- Anaesthetic used for blood collection: Yes, isoflurane
- Animals fasted: Yes
- How many animals: 5 males and 5 females randomly selected
- Parameters checked were: 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; to assess blood coagulation: prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: as part of the sacrificial procedure
- Animals fasted: Yes
- How many animals: 5 males and 5 females randomly selected
- Parameters checked were: 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

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during the study, towards the end of treatment
- Dose groups that were examined: all
- Battery of functions tested:
Sensory activity / grip strength:
5 males and 5 females were randomly selected from each main 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 23 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 25 of the study (during treatment) and once during Week 2 of recovery (Day 9).
Motor activity: 5 males and 5 females were randomly selected from each main 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 24 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 24 of the study (during treatment) and once during Week 2 of recovery (Day 9)

OTHER:

VAGINAL SMEARS:
Vaginal smears were taken daily in the morning starting two weeks before pairing throughout the mating period until a positive identification of copulation was made. The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle;
2. pre-coital interval (i.e., the number of nights paired prior to the detection of mating).

PARTURITION CHECK and GESTATION LENGHTH (Main groups):
A parturition check was performed from Day 20 to Day 25 post coitum. Female no. 11 (Group 1) and 43 (Group 3) which did not give birth after 25 days of post coitum period was sacrificed on Day 27. This animals were found not pregnant at necropsy.

PUPS IDENTIFICATION, WEIGHT and OBSERVATION (Main groups):
As soon as possible, after parturition was considered complete (Day 0 or 1 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1 and 4 post partum. Pups dying during the lactation period were weighed before the despatch to necropsy. Observation was performed once daily for all litters.

Sacrifice and pathology:

EUTHANASIA (All groups):
Animals selected for blood collection were killed by exsanguination under isofluorane anaesthesia. Animals not selected for blood collection were killed under carbon dioxide asphyxiation. Pups were euthanised by intraperitoneal injection of Sodium Thiopenthal (on Day 4 post partum). The parental males were killed after the mating of all females, after a total 28 days of dosing. The parental 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. Animals from the recovery groups were killed after 2 weeks of recovery.

GROSS PATHOLOGY: Yes
The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination. All females were examined also for the following a) external and internal abnormalities b) number of visible implantation sites (pregnant animals) and c) number of corpora lutea (pregnant animals). Uteri of apparently non-pregnant female was immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation. All pups found dead in the cage were examined for external and internal abnormalities. All live pups at termination were killed and examined for external abnormalities and sex confirmation by gonadal inspection.

HISTOPATHOLOGY: Yes
The examination was restricted, in the first instance, as detailed in the following: a) tissues specified in table 1 from 5 males and 5 females randomly selected in the control and high dose group killed at term, b) tissues from all animals killed or dying during the treatment period and c) all abnormalities in all groups. Since changes were observed in the heart and spleen between control and high dose animals, the histopathological examination was extended to: 1) the remaining 5 males and 5 females of Main Groups 1 and 4, 2) the animals of Main Groups 2 and 3, 3) the animals killed after 2 weeks of recovery period (Recovery groups 5 and 6).

ORGAN WEIGHTS: Yes
From all animals completing the scheduled test period, the organs indicated in table 1 were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal.

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 if n was more than 5. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of theWilliams test. The criterion for statistical significance was
p< 0.05.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Main groups:
Clinical signs mainly observed in the treated animals were blue staining in different regions of the body surface (related to the nature and intended use of the test item) and salivation. Other clinical signs such as piloerection, damaged ear and cyphosis were generally observed in a limited number of animals during the study.
Recovery groups:
Clinical signs observed during the treatment period were blue staining on the dorsum/tail in all treated animals and salivation in one treated male (no. 96) and three treated females (nos. 91, 93 and 99). Hairloss on the head was observed in one control female (no. 87). During the recovery phase, blue staining of the tail was still observed in all treated animals, in the first week of the recovery period. Hairloss on the head was observed in two males (nos. 84 and 86) and two females (nos. 81 and 87) of the control group. In addition, control females showed brown staining on the head (animal no. 81) and scab on the head ( animal no. 87).
Recovery groups:
Clinical signs observed during the treatment period were blue staining on the dorsum/tail in all treated animals and salivation in one treated male (no. 96) and three treated females (nos. 91, 93 and 99). Hairloss on the head was observed in one control female (no. 87). During the recovery phase, blue staining of the tail was still observed in all treated animals, in the first week of the recovery period. Hairloss on the head was observed in two males (nos.
84 and 86) and two females (nos. 81 and 87) of the control group. In addition, control females showed brown staining on the head (animal no. 81) and scab on the head ( animal no. 87).

Observations of the cage tray
– Blue staining on the cage tray, slight to marked, was observed during the pre-mating
and mating periods in animals of all main groups of both sexes.
– Blue staining on the cage tray, slight to marked, was observed after mating phase in all
treated males (main groups) up to the end of treatment.
– Blue staining on the cage tray, slight to moderate, was seen during the gestation and
post partum periods in females receiving dose levels of 80 and 120 mg/kg body weight/day.
– During the dosing phase, recovery group (males and females) showed blue staining
on the cage tray, slight to moderate. This sign disappeared from Day 2 of the recovery
phase.
The above mentioned findings were considered related to the colour of the test item which was eliminated by the animals.

Mortality:

mortality observed, treatment-related

Description (incidence):

Thirteen high dose group animals died or were sacrificed for humane reasons during the treatment period. Four males (nos. 70, 74, 76 and 80) and 7 females (nos. 61, 65, 69, 73, 75, 77 and 79) of Group 4 died during the treatment phases (premating and mating phase for males and premating, gestation and post partum period for females). In addition, two females were sacrificed for humane reasons, one (no. 63) on Day 22 of the gestation period, due to a prolapse of the uterus occurred and the second one (and its litter) on Day 1 of the post partum period, due to a poor health condition (no. 71). In the high dose group, starting from Day 17 of treatment, the dose level was reduced from 160 mg/kg body weight/day to 120 mg/kg body weight/day. However, the animals continued to die although the dose level was reduced. Five mid-dose group animals died during the treatment period. One male (no. 46) which died on Day 4 mating phase and 4 females died on Days 21 and 22 of the gestation period (nos. 57 and 59, respectively) and on Day 1 of the post partum period (nos. 47 and 51). Generally, clinical signs observed were blue staining in different regions of the body surface (related to the nature and intended use of the test item) and salivation. In addition, clinical signs such as: hairloss, piloerection, cannibalisation by cage mates were generally observed in a limited number of animals. The changes observed at post mortem examination were represented by single instances of multiple/single red or dark depressed areas in the stomach (glandular region) and reduced size of the thymus. Blue staining of head, salivary gland, tail or blue content of the stomach were seen and considered related to the test item. Moreover, animal no. 74 (Group 4) showed red fluid in the thoracic cavity related to a possible misdosing, while animal no. 75 (Group 4) showed a subcutaneous mass in the skin, microscopically recognised as adenocarcinoma in the mammary glands. This latter finding was considered an incidental pathology rather than treatment-related.
The histopathological evaluation identified the factors contributory to the death of these animals in the cardiomyopathy, represented by mild to marked multifocal mononuclear cell infiltrations, fibrosis, cardiomyocytes necrosis, associated or not with multifocal mild necrosis in the liver and/or adrenals and/or kidneys. Immunodepression was also reported in some instances in the thymus.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Main groups
No differences of toxicological significance were noted throughout the study in the body
weight of treated males, when compared to the control group. On Day 8 of the study, during the mating phase, body weight and body weight gain were significantly decreased (at statistical analysis) in males of the low dose group (-7 and -82%, respectively). Changes in body weight and body weight gain observed at the end of the mating period were not significant, at statistical analysis, in any treated groups, not dose related and therefore, not considered treatment related. Before pairing, body weight of treated females was comparable to the control group. At the end of the post coitum period (Day 20), body weight of females of the high dose group was slightly lower than controls (-6%) but statistically significant. At post partum period (Day 4), body weight of females of the mid- and high dose levels was lower than controls (-12% and -11%, respectively), statistically significant in the mid-dose group. Significant reduction in body weight gain was observed in all groups of females on Day 7 of the post coitum period (ranging from -27% to -33%). In addition, a statistically significant reduction in body weight gain was noted in the mid-dose group at the end of the post partum period. Reduced body weight gain at the end of the post partum period was also observed in the unique female of the high dose group, which survived treatment and reached the end of the study.

Recovery groups
Means of body weight and body weight gain were comparable between controls and the treated group both in males and females throughout the treatment phase. During the recovery period, no differences in body weight and body weight gain were recorded in animals of both sexes, when compared to the control group.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Main groups:
Food consumption measured in treated males and females, before pairing, was comparable to the control group. On Day 7 post coitum, food consumption was significantly lower (at statistical analysis) than the control group in females of the mid- and high dose groups (-11% and -12%, respectively).
Recovery groups:
During treatment and recovery periods, no differences from controls were noted in food consumption in the treated groups in both sexes receiving 80 mg/kg body weight/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Main groups:
Leucocytes were lower than controls in a number of males treated at 40 and 80 mg/kg body weight/day. Mean group values were 40% and 32% below controls, respectively. Eosinophils were also decreased in males receiving 160/120 mg/kg body weight/day (77%). In addition, some females from all treated groups (nos. 21, 41, 55 and 67) showed lymphocytes higher than controls: the increments were approximately 2.1 fold, with no dose-relation. Moreover, eosinophils were decreased in all treated females (83% to 100%). Due to the absence of dose-relation, the above white blood cell findings were not conclusively attributed to treatment. Concerning the red blood cell parameters, erythrocytes, haemoglobin, haematocrit and
mean corpuscular volume were lower than controls and mean corpuscular haemoglobin concentration was higher than controls and mean corpuscular haemoglobin concentration was higher than controls in some males from all treated groups, without dose-dependency. Changes were approximately 20% for haematocrit and approximately 10% for the other parameters. Changes were insufficient in severity to represent an adverse anaemia. The statistically significant decrease of reticulocytes recorded in males receiving 40 mg/kg body weight/day was not dose-related, therefore it was considered incidental.

Coagulation
Prolonged prothrombin time was recorded in males dosed at 80 and 160/120 mg/kg bodyweight/day. Changes were 16% and 18% above mean control data, respectively.

Recovery phase - Recovery groups
Changes recorded during the dosing phase were no longer observed. The statistically significant differences between control and treated animals (slight increases of lymphocytes in males and erythrocytes in females) were not observed during the dosing phase, therefore they were considered unrelated to treatment.
Coagulation
No changes were recorded, confirming complete reversibility.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups
Most of the statistically significant differences between control and treated males (alanine aminotransferase, aspartate aminotransferase, cholesterol, triglycerides, phosphorus and potassium) were mainly due to the changes recorded in some control males (nos. 4, 10 and 14), which showed increases of many biochemical parameters. By the contrary, the decreases of urea, creatinine, protein, albumin, globulin and calcium were not associated with the control animal changes. However, due to the absence of dose relation, these findings cannot be definitively attributed to treatment. One female receiving 80 mg/kg/body weight/day (no. 49) showed moderate increases of both transaminases enzymes, total bilirubin and urea. Female no. 67 (160/120 mg/kg body weight/day) showed reduced triglycerides and increased glucose. Due to the limited incidence and the reduced number of animals dosed at 160/120 mg/kg body weight/day, no conclusions could be made for such changes.

Recovery groups
Most of the findings recorded during the dosing phase were completely recovered with few exceptions, which showed partial reversibility. In particular, protein, albumin and calcium were still slightly lower than controls in males (-6%, -9% and -4%, respectively), and globulin was slightly higher in females (7%). In addition, statistically significant differences of sodium (-1%) in treated females and phosphorus (13%) in treated males were recorded. Due to the minimal severity, the above changes
were considered of no toxicological relevance.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Main groups
Observations of treated animals at removal from the cage and in an open arena did not reveal changes attributable to the test item. Sensory reaction to stimuli and motor activity did not show relevant differences between control and treated groups in both sexes. Significant reduction, at statistical analysis, in grip
strength (second trial) and the relative mean data was noted in all treated male groups, while no relevant differences were observed in female animals.

Recovery groups
Observations of treated animals at removal from the cage and in an open arena did not reveal changes attributable to the test item. No significant alterations in sensory reaction to stimuli were observed in the treated group at the examination performed at the end of treatment. A statistically significant decrease in grip strength (first, second trial and mean) was noted in treated males. In addition, a statistically significant increase in motor activity measurement was noted in treated females. For males, due to the low control values measured (when compared to all other control values in the study), no relevance was attributed at the statistically significant increase measured. At the end of the recovery period, no variations in motor activity, grip strength or sensory reaction to stimuli were observed.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant decrease was observed in the terminal body weight of mid-dose females (-11%), when compared to the controls. Slight reduction, not statistically significant, was noted in terminal body weight of low and high dose males (-6% and -4%, respectively). The reduction observed in males and females was not considered toxicologically relevant, due to the very slight severity (ranging from -4% to -11%), even if statistically significant for females. A statistically significant increase in absolute and relative heart weight was noted in females receiving 80 mg/kg body weight/day. No relevant changes were observed in the remaining absolute and relative organ weights of treatment groups of both sexes, when compared to control data, with the exception of the decreased absolute and relative liver weight of low and high dose male groups, absolute and relative thymus weight in low dose males. However, since histomorphologies of these organs were comparable to control animals, no clear toxicological significance could be attributed to these findings.

Recovery sacrifice
After 2 weeks of recovery period, no relevant changes were observed on terminal body weight, absolute and relative organ weights in the recovery phase animals previously dosed
at 80 mg/kg body weight/day.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The changes observed at post mortem as blue staining of tail, head, clitoral or salivary glands were attributed to the colour of the test item. Reduced size of the thymus seen in control and treated females was considered related to physiological changes, often seen in this kind of study in some treated Sprague Dawley rats of the same age.

Recovery sacrifice
No remarkable differences were noted at post mortem examination in treated animals, when compared with controls.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Treatment-related changes were seen in the heart of all high dose males and the remaining surviving high dose female, consisting in moderate to marked cardiomyopathy, represented by multiple mononuclear cell infiltrations, fibrosis and cardiomyocyte necrosis. Cardiomyopathy was also observed, with minimal to moderate degree in males and mild to marked degree in females of the mid-dose group. Increased incidence or presence of minimal inflammatory cell foci was seen in the heart of low dose males and females, respectively, when compared with controls. This finding consisted mainly in mononuclear cells such as macrophages and lymphocytes in some cases accompanied by necrosis of single cardiomyocyte and fibrosis. The remaining lesions reported in control and/or treated animals such as nephropathy of kidneys were considered to be an expression of spontaneous and/or incidental pathology commonly seen in this species and age under our experimental conditions. Following the extension of the histopathological evaluation of the spleen to all treated males and females, this organ was considered with normal morphological aspect, when compared with all control animals.

Recovery sacrifice
At the histopathology investigations performed on the heart, cardiomyopathy was still observed in most treated rats of both sexes with minimal to moderate degree in males and minimal to mild degree in females, after a wash-out period of two weeks.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

LOAEL

Effect level:

40 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

40 mg/kg bw/day (nominal)

System:

cardiovascular

Organ:

heart

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Applicant's summary and conclusion

Conclusions:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422), adverse effects were found after oral administration of the test item in male and female rats. Based on the occurrence of the histopathological findings observed in the heart of all dose levels, the NOAEL (No Observed Adverse Effect Level) can not be determined and thus the LOAEL for systemic toxicity is considered to be 40 mg/kg bw/day.

Executive summary:

The test item (90.6% purity) was tested in a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) at dose levels of 0, 40, 80 and 160 mg/kg bw/day in 10 animals per sex/dose. Due to the toxicity observed in the high dose group, the high dose level was reduced to 120 mg/kg bw/day from day 17 on. Males were treated for a total of 28 days. Females were treated for 2 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 3 post-partum, for at least 41 days. During the study, a total of 13 high dose animals died or were sacrificed for humane reasons during the treatment period. Five mod-dose group animals died during the treatment period. Generally, clinical signs observed were blue staining in different regions of the body surface (related to the nature and intended use of the test item) and salivation. In addition, clinical signs such as hairloss, piloerection, cannibalisation by cage mates were generally observed in a limited number of animals. No toxicological significant effects on body weight were noted throughout the study in males. Before pairing, body weight of treated females was comparable to the control. At the end of the post coitum period, the body weight of females of the high dose group was only slightly lower than controls, but statistically significant. At post-partum period, body weight of females in the mid and high dose levels were lower than the control group with statistical significance in the mid-dose group. In the recovery groups no effect on body weight were seen. Food consumption in treated males and females, before pairing, was comparable to the control group. On day 7 post coitum, food consumption was slightly but statistically significant lower than the control in females of the mid-and high dose groups. In the recovery groups, no differences from controls were noted in food consumption. A prolonged prothrombin time was recorded in mid and high dose male animals. In the recovery groups this effect was complete reversible. Decreases of urea, creatinine, protein, albumin, globulin and calcium were observed in treated males. However, due to the absence of a dose-relationship, these findings cannot be definitively attributed to the treatment.

A statistically significant decrease was observed in terminal body weight of mid-dose females when compared to controls. A significant increase in absolute and relative heart weight was noted in females of the mid-dose group. Treatment related changes were seen in the heart of all high dose males and one surviving female, consisting in moderate to marked cardiomyopathy, which was also observed with minimal to moderate degree in males and mild to marked degree in females of the mid-dose group, while mononuclear cell infiltration associated or not with single necrosis of cardiomyocytes was also reported in low dose males and females.

Based on the occurrence of the histopathological findings observed in the heart of all dose levels, the NOAEL for general toxicity cannot be determined. Thus, based on the findings from the study, the LOAEL is considered to be 40 mg/kg bw/day for systemic toxicity.