[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl](2-hydroxypropyl)dimethylammonium chloride

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015-12-11 to 2016-09-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

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:

2015-12-11 to 2016-09-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 March 1996

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco, Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks old
- Weight at study initiation: 220 to 235 g for males, 184 to 205 g for females
- Housing: from arrival to pairing animals were housed up to 5 of one sex to a cage in polysulfone solid bottomed cages. During mating, animals were housed one male to one female in clear polysulfone cages 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 for the gestation period, birth and lactation.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 19 days (main goups), 26 days (recovery groups)

DETAILS OF FOOD AND WATER QUALITY: There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet.

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

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The formulation was prepared daily at concentrations of 4, 10 and 25 mg/mL. Formulations were maintained under magnetic stirring for approximately 1 hour at room temperature prior to use and up until the time of dosing of the last animal. Concentrations were calculated and expressed in terms of test item as supplied.

ADMINISTRATION OF TEST ITEM:
The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation analysis
Analysis was performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory (RTC Study No. A0762). The proposed formulation procedure for the test item was checked in the range from 2 to 25 mg/mL by chemical analysis (concentration and homogeneity) during the pre-treatment period, to confirm that the method was suitable. Stability after 28 hours at room temperature and at +5 °C for 8 days (range from 2 to 25 mg/mL) 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. Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. A0762) using spectrophotometric analysis. The software used for this activity was SkanIt® version 2.4.2.55 (Thermo Scientific).

Duration of treatment / exposure:

Main groups:
Males: once a day, 7 days a week for 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy. Males were treated for a total of 36 or 37 days.
Females: 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 (for at least 41 days).
Recovery groups:
Animals were dosed once a day, 7 days a week, for a minimum of 4 consecutive weeks. No treatment was given during the recovery period.

Frequency of treatment:

once daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1, Control

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10 (main groups 1-4); 5 (recovery groups)

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 high dose group) and the post-exposure period was two weeks

Positive control:

not necessary

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 once a week 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. 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 36 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 11).
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 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 28 of the study (during treatment) and once during Week 2 of recovery (Day 11)

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. 69 (Group 4) which did not give birth after 25 days of post coitum period was sacrificed shortly on Day 26. This animal was 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 males were killed after the mating of all females, after a total of 37 or 38 days of dosing. 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. 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. At the discretion of the Study Director, pups with abnormalities were retained in an appropriate fixative.

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) all abnormalities in all groups.
Since changes were observed between control and high dose animals, the histopathological examination was extended to the liver on a) the remaining 5 males and 5 females (animals not evaluated for clinical pathology) of Groups 1 and 4 (main groups), b) the animals (males and females) of Groups 2 and 3 (main groups) and c) the animals (males and females) killed after 2 weeks of recovery period (recovery groups, 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 the Williams test. The criterion for statistical significance was p<0.05.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

RESULTS:
Mortality and fate of females:
No mortality occurred throughout the study. All females proved to be pregnant, with the exception of one female of Group 4 (250 mg/kg body weight /day). The number of females with live pups on Day 4 post partum was 10 in the control, 10 in the low dose group (40 mg/kg bw/day), 10 in the mid-dose group (100 mg/kg bw/day) and 9 in the high dose group (250 mg/kg bw/day).

Clinical signs:
No relevant clinical signs were observed throughout the study in all treated main animals of both sexes.

Neurotoxicity assessment:
The assessment did not reveal changes attributable to the test item.

Body weight :
Body weight of treated males was comparable to the control group throughout the study. Statistically significant decrease in body weight gain was recorded in males receiving 250 mg/kg body weight/day and in those receiving 100 mg/kg body nweight/day, on Day 8 before pairing and on Day 15, during mating phase, respectively. Some variations occurred in body weight during gestation and post partum periods in treated females receiving 250 mg/kg body weight/day. However these changes, even if associated in some occasions at statistical significance, were of minimal severity and therefore considered irrelevant. Body weight gain of treated females was unaffected by treatment in all phases of the study. 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:
Food consumption of females dosed at 250 mg/kg body weight/day was lower (with statistical significance) than the control group on Day 7 post coitum ( -11%). No changes were noted in treated males and in females before pairing and during post partum period. No differences were noted in animals of the recovery groups, both during the treatment and recovery periods.

Haematology:
Slight reduction of erythrocytes, haemoglobin and haematocrit was recorded in males dosed at 250 mg/kg body weight/day. Haematocrit was also reduced in males dosed at 100mg/kg body weight/day. Changes, significant at statistical analysis, were of minimal severity (up to -11%), therefore considered not adverse. In addition, female no. 79 (250 mg/kg body weight/day) showed leucocytosis (100% above mean control data), mainly due to an increase of neutrophils and lymphocytes. Due to the low incidence, this change cannot be conclusively attributed to treatment. For the coagulation parameters it can be stated: Activated partial thromboplastin time was reduced in animals of both sexes receiving 250 mg/kg body weight/day and in treated females dosed at 100 mg/kg/body weight/day,with no dose-relation. Changes were approximately -21%. Due to the direction, this finding was not considered adverse. The reduction of erythrocytes, haemoglobin and haematocrit was still present in treated males (-7% to -12%) in the recovery groups, even though a partial reversibility was recorded.

Clinical Chemistry:
Statistically significant fluctuations of some biochemical parameters were recorded, such as: decrease of creatinine in males dosed at 250 mg/kg body weight/day (-10%), decrease of albumin/globulin ratio in those (males) receiving 40 and 250mg/kg body weight/day (approximately -11%), increase of phosphorus in males dosed at 100 and 250 mg/kg body weight/day (approximately 20%), decrease of potassium in those treated at 40 and 100 mg/kg body weight/day (approximately -14%) and increase of glucose in females dosed at 250 mg/kg body weight/day (+26%). The severity of the findings observed was not considered to be suggestive of tissue/organ injury. No relevant changes were recorded in the recovery groups, confirming complete reversibility.

Terminal body weight and organ weights:
Terminal body weight was unaffected by treatment in both sexes. An increase in absolute (+19% in males and +13% in females) and relative (+24% in males
and +20% in females) liver weights was the principal alteration noted in males and females receiving 250 mg/kg body weight/day, although these changes were not significant at statistical analysis. In addition some other changes were also noted, such as:
– statistically significant decrease in absolute adrenals weight in females receiving the dose levels 100 mg/kg body weight/day;
– statistically significant increase in relative brain and heart weights in males and females respectively, receiving the dose level of 100 mg/kg body weight/day;
– statistically significant increase in relative kidneys weight in males receiving the dose level of 250 mg/kg body weight/day.

In general, all the above mentioned changes were not accompanied by histopathological findings and therefore were considered unrelated to treatment. In particular, the alterations noted in the liver weights were associated with a histopathological finding such as: minimal centrilobular hepatocellular hypertrophy. However it was considered an hepatic adaptive effect and not an injurious change. In addition, similar finding was not observed at the end of the recovery period. In the recovery groups after 2 weeks of recovery period, terminal body weight of animals previously dosed at 250 mg/kg body weight/day was comparable to the control group. Lower absolute testes (-10%) weight was noted in males previously dosed at 250 mg/kg body weight/day.

Gross pathology:
No remarkable differences were noted at post mortem examination in treated animals, either sacrificed at the end of treatment or after 2 weeks of recovery period, when compared with controls.

Histopathology:
Treatment-related changes were noted in the liver of most treated males and females dosed at 250 mg/kg/day. The pathological lesions consisted of minimal, multifocal centrilobular hepatocytic hypertrophy [1], the most common histological change associated to drug-metabolizing enzyme (DME) induction. Inflammatory reactions, mainly represented by mononuclear cells (plasma cells, lymphocytes and/or histiocytes) were in some instances associated to single cell necrosis (hepatocyte) and were mostly reported in control and treated males. In the recovery groups no treatment-related changes were seen in the liver of treated males and females. In conclusion, a minimal centrilobular hepatocellular hypertrophy was seen in high dose males and females; however in the absence of other histologic findings and being this finding not evident after 2 weeks of recovery period, the hepatic drug metabolizing enzyme (DME) induction was considered an hepatic adaptive and not injurious change.

Spermatogenic cycle:
A detailed qualitative examination of the testes was performed in five randomly selected control and high dose group males. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952 and referred to the comprehensive reviews on the subject Russell, 1990; Creasy, 1997; Creasy, 2002. The PAS-H stained sections were used to identify the spermatogenic stages. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

[1] References Liver Hypertrophy: A Review of Adaptive (Adverse and Non-adverse) Changes - Conclusions from the 3rd International ESTP Expert Workshop - Toxicologic Pathology, 40: 971-994, 2012.

Key result

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects were observed related to the test substance

Critical effects observed:

not specified

Conclusions:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test no adverse effects were found after oral administration of the test item in male and female rats. Based on the results, the NOAEL is considered to be 250 mg/kg bw/day.

Executive summary:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) the test item (87.9% purity) was administered orally to 10 male and female Sprague Dawley rats/dose in water by gavage at dose levels of 0, 40, 100 and 250 mg/kg bw/day. The animals were treated with the test item formulation on 7 days per week once per day. Males were treated for a total of 36 or 37 days, females until day 3 post partum (for at least 41 days).

No adverse effects of the test item were found up to the dose level of 250 mg/kg body weight/day.

No mortality occurred in the control or in any of the dose groups during the treatment period of this study. There were no clinical signs of toxicological relevance in the dose groups when compared to the control group. The neurotoxicity assessment (motor activity, grip strength and sensory activity) did not reveal any changes attributable to the test item. There were changes of minimal severity in body weight and body weight gain in both male and female animals which were considered irrelevant. There were no adverse effects on food consumption of males and females of the dosing groups compared to control animals during the study period. Furthermore, no adverse changes were measured for haematology and clinical chemistry parameters. Additionally, no adverse effects were seen in gross and histopathology when compared with controls.

The NOAEL in this study is considered to be 250 mg/kg bw/day. This study is classified as acceptable and satisfies the guideline requirement for an oral repeated dose toxicity study in rat. 

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

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:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl](2-hydroxypropyl)dimethylammonium chloride

EC Number:

259-033-6

EC Name:

[2-[[4-[(2-chloro-4-nitrophenyl)azo]phenyl]ethylamino]ethyl](2-hydroxypropyl)dimethylammonium chloride

Cas Number:

54229-13-9

Molecular formula:

C21H29ClN5O3.Cl

IUPAC Name:

[2-({4-[(E)-2-(2-chloro-4-nitrophenyl)diazen-1-yl]phenyl}(ethyl)amino)ethyl](2-hydroxypropyl)dimethylazanium chloride

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:

TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco, Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 7-8 weeks old
- Weight at study initiation: 220 to 235 g for males, 184 to 205 g for females
- Housing: from arrival to pairing animals were housed up to 5 of one sex to a cage in polysulfone solid bottomed cages. During mating, animals were housed one male to one female in clear polysulfone cages 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 for the gestation period, birth and lactation.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 19 days (main goups), 26 days (recovery groups)

DETAILS OF FOOD AND WATER QUALITY: There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet.

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

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The formulation was prepared daily at concentrations of 4, 10 and 25 mg/mL. Formulations were maintained under magnetic stirring for approximately 1 hour at room temperature prior to use and up until the time of dosing of the last animal. Concentrations were calculated and expressed in terms of test item as supplied.

ADMINISTRATION OF TEST ITEM:
The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The dose was administered to each animal on the basis of the most recently recorded body weight and the volume administered was recorded for each animal.

Details on mating procedure:

Mating was monogamous (one male to one female). A vaginal smear was taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray). The female was paired with the same male until positive identification occurred or 14 days had elapsed. The pairing combination of one animal which did not have positive identification of mating after 14 days of pairing was changed within the treatment group (female no. 21). The subsequent pairing was monitored for mating as described above.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Formulation analysis
Analysis was performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory (RTC Study No. A0762). The proposed formulation procedure for the test item was checked in the range from 2 to 25 mg/mL by chemical analysis (concentration and homogeneity) during the pre-treatment period, to confirm that the method was suitable. Stability after 28 hours at room temperature and at +5 °C for 8 days (range from 2 to 25 mg/mL) 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. Chemical analysis was carried out by the Analytical Chemistry Department at RTC according to a validated method (RTC Study No. A0762) 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 once a day, 7 days a week, for 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 36 and 37 of study). Males were treated for a total of 37 or 38 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 (for at least 41 days). 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.

Frequency of treatment:

daily

Details on study schedule:

according to guideline

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1, control

Doses / concentrations 2

Dose / conc.:

40 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10 (main groups 1-4); 5 (recovery groups)

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 high dose group) and the post-exposure period was two weeks

Positive control:

not necessary

Examinations

Parental animals: Observations and examinations:

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 once a week 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. 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 36 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 11).
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 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 28 of the study (during treatment) and once during Week 2 of recovery (Day 11).

Oestrous cyclicity (parental animals):

The vaginal smear data were examined to determine the following:
- anomalies of the oestrous cycle,
- pre-coital interval (i.e., the number of nights paired prior to the detection of mating).

Sperm parameters (parental animals):

Parameters examined male parental generation: testis weight, epididymis weight, for the testes, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). Afterwards, the morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.

Litter observations:

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.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: The males were killed after the mating of all females, after a total of 37 or 38 days of dosing (main groups). For the recovery groups the males were killed after 2 weeks of recovery
- Maternal animals: 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.

GROSS NECROPSY
- The clinical history of the animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination. All females were examined also for the following: 1. external and internal abnormalities; 2. number of visible implantation sites (pregnant animals); 3. 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.

HISTOPATHOLOGY
The tissues indicated in Table 1 (see box "any other information on materials and methods incl. tables") were prepared for microscopic examination. The examination was restricted, in the first instance, as detailed in the following: a) tissues specified iin the Table 1 from 5 males and 5 females randomly selected in the control and high dose group killed at term, b) all abnormalities in all groups. Since changes were observed between control and high dose animals, the histopathological
examination was extended to the liver on a) the remaining 5 males and 5 females (animals not evaluated for clinical pathology) of Groups 1 and 4 (main groups), b) the animals (males and females) of Groups 2 and 3 (main groups) and c) the animals (males and females) killed after 2 weeks of recovery period (recovery groups, Groups 5 and 6).

ORGAN WEIGHTS
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.

Postmortem examinations (offspring):

SACRIFICE
- Pups were euthanised by intraperitoneal injection of Sodium Thiopenthal (on Day 4 post
partum)

GROSS NECROPSY
- 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.

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 the Williams test. The criterion for statistical significance was
p< 0.05.

Reproductive indices:

Males:
- Copulation Index (%) = (no. of animals mated / no. of animals paired) x 100
- Fertility Index (%) = (no. of males which induced pregnancy /no. of animals paired) x 100
Females:
- Copulatory Index (%) = (no. of animals mated / no. of animals paired) x 100
- Fertility Index (%) = (no. of pregnant females / no. of females paired) x 100
- Pre-implantation loss = ((no. of corpora lutea - no. of implantations) / no. of corpora lutea) x 100
- Pre-natal loss: ((no. of visible implantations - live litter size at birth) / no. of visible implantations) x 100

Offspring viability indices:

- Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter.
- Pup loss at birth = ((Total litter size - live litter size) / Total litter size) x 100
- Post-natal loss on day 4 post partum = ((live litter size at birth - live litter size at day 4 post partum) / live litter size at birth) x 100

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

- Mortality:
No mortality occurred throughout the study.

- Fate of females:
One female receiving 250 mg/kg body weight/day was found not pregnant at necropsy (no. 69). The number of females with live pups on Day 4 post partum was: 10 in the control, 10 in the low dose (40 mg/kg body weight/day), 10 in the mid-dose (100 mg/kg body weight/day) and 9 in the high dose (250 mg/kg body weight/day) groups.

- Clinical signs:
No relevant clinical signs were observed throughout the study in all treated main animals of both sexes

- Neurotoxicity assessment:
The assessment did not reveal changes attributable to the test item.

- Body weight:
Body weight of treated males was comparable to the control group throughout the study. Statistically significant decrease in body weight gain was recorded in males receiving 250 mg/kg body weight/day and in those receiving 100 mg/kg body nweight/day, on Day 8 before pairing and on Day 15, during mating phase, respectively. Some variations occurred in body weight during gestation and post partum periods in treated females receiving 250 mg/kg body weight/day. However these changes, even if associated in some occasions at statistical significance, were of minimal severity and therefore considered irrelevant. Body weight gain of treated females was unaffected by treatment in all phases of the study. 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. The terminal body weight was unaffected by treatment in both sexes.

- Food consumption:
Food consumption of females dosed at 250 mg/kg body weight/day was lower (with statistical significance) than the control group on Day 7 post coitum ( -11%). No changes were noted in treated males and in females before pairing and during post partum period. No differences were noted in animals of the recovery groups, both during the treatment and recovery periods.

- Haematology
Slight reduction of erythrocytes, haemoglobin and haematocrit was recorded in males dosed at 250 mg/kg body weight/day. Haematocrit was also reduced in males dosed at 100mg/kg body weight/day. Changes, significant at statistical analysis, were of minimal severity (up to -11%), therefore considered not adverse. In addition, female no. 79 (250 mg/kg body weight/day) showed leucocytosis (100% above mean control data), mainly due to an increase of neutrophils and lymphocytes. Due to the low incidence, this change cannot be conclusively attributed to treatment. For the coagulation parameters it can be stated: Activated partial thromboplastin time was reduced in animals of both sexes receiving 250 mg/kg body weight/day and in treated females dosed at 100 mg/kg/body weight/day,with no dose-relation. Changes were approximately -21%. Due to the direction, this finding was not considered adverse. The reduction of erythrocytes, haemoglobin and haematocrit was still present in treated males (-7% to -12%) in the recovery groups, even though a partial reversibility was recorded.

- Clinical Chemistry:
Statistically significant fluctuations of some biochemical parameters were recorded, such as: decrease of creatinine in males dosed at 250 mg/kg body weight/day (-10%), decrease of albumin/globulin ratio in those (males) receiving 40 and 250 mg/kg body weight/day (approximately -11%), increase of phosphorus in males dosed at 100 and 250 mg/kg body weight/day (approximately 20%), decrease of potassium in those treated at 40 and 100 mg/kg body weight/day (approximately -14%) and increase of glucose in females dosed at 250 mg/kg body weight/day (+26%). The severity of the findings observed was not considered to be suggestive of tissue/organ injury. No relevant changes were recorded in the recovery groups, confirming complete reversibility.

- Oestrous cycle, reproductive parameters, pairing combination and mating performance:
Oestrous cycle and reproductive parameters (pre-coital intervals, copulatory and fertility indices) were similar in treated and control groups.

- Implantation, pre-implantation loss data, pre-natal loss data and gestation length of females:
Gestation periods were similar between treated and control group females. All pregnant dams gave birth on Day 22 post coitum (mean value).
Corpora lutea, implantations, total litter size and pre-natal loss (percentage) were similar in control and treated groups.

- Litter data at birth, on Day 1 and on Day 4 post partum of females and sex ratio of pups:
An increased incidence of pups loss value (percentage) on Day 1 post partum and post natal loss value on Day 4 post partum (percentage) was observed in females receiving 250 mg/kg body weight/day. These changes were not statistically significant and no dose-relationship was observed. Sex ratios at birth and on Day 4 post partum did not show differences between groups, when calculated as the percentage of males.

- Clinical signs of pups:
Cold to touch, apparently no food intake (milk), small and pale appearance were the main clinical signs noted in control and treated pups. In addition, found dead or missing pups were also observed both in control and treated groups. One pup at the high dose level (250 mg/kg body weight/day) showed right hindlimb damaged. This finding was considered incidental.

- Necropsy findings in deceased pups and in pups sacrificed on Day 4 post partum:
Autolysed abdominal organs were observed in one pup of Group 4 (250 mg/kg body weight/day) which died during the lactation period. An increase incidence of pups without milk in stomach was noted in Group 4 (250 mg/kg body weight/day) compared to the control group.

- Terminal body weight and organ weights:
Terminal body weight was unaffected by treatment in both sexes. An increase in absolute (+19% in males and +13% in females) and relative (+24% in males and +20% in females) liver weights was the principal alteration noted in males and females receiving 250 mg/kg body weight/day, although these changes were not significant at statistical analysis. In addition some other changes were also noted, such as:
– statistically significant decrease in absolute adrenals weight in females receiving the dose levels 100 mg/kg body weight/day;
– statistically significant increase in relative brain and heart weights in males and females respectively, receiving the dose level of 100 mg/kg body weight/day;
– statistically significant increase in relative kidneys weight in males receiving the dose level of 250 mg/kg body weight/day.

In general, all the above mentioned changes were not accompanied by histopathological findings and therefore were considered unrelated to treatment. In particular, the alterations noted in the liver weights were associated with a histopathological finding such as: minimal centrilobular hepatocellular hypertrophy. However it was considered an hepatic adaptive effect and not an injurious change. In addition, similar finding was not observed at the end of the recovery period. In the recovery groups after 2 weeks of recovery period, terminal body weight of animals previously dosed at 250 mg/kg body weight/day was comparable to the control group. Lower absolute testes (-10%) weight was noted in males previously dosed at 250 mg/kg body weight/day.

- Gross pathology:
No remarkable differences were noted at post mortem examination in treated animals, either sacrificed at the end of treatment or after 2 weeks of recovery period, when compared with controls.

- Histopathology:
Treatment-related changes were noted in the liver of most treated males and females dosed at 250 mg/kg/day. The pathological lesions consisted of minimal, multifocal centrilobular hepatocytic hypertrophy [1], the most common histological change associated to drug-metabolizing enzyme (DME) induction. Inflammatory reactions, mainly represented by mononuclear cells (plasma cells, lymphocytes and/or histiocytes) were in some instances associated to single cell necrosis (hepatocyte) and were mostly reported in control and treated males. In the recovery groups no treatment-related changes were seen in the liver of treated males and females. In conclusion, a minimal centrilobular hepatocellular hypertrophy was seen in high dose males and females; however in the absence of other histologic findings and being this finding not evident after 2 weeks of recovery period, the hepatic drug metabolizing enzyme (DME) induction was considered an hepatic adaptive and not injurious change.

Spermatogenic cycle:
A detailed qualitative examination of the testes was performed in five randomly selected control and high dose group males. The evaluation, taking into account the tubular stages of the spermatogenic cycle, was conducted in order to identify treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952 and referred to the comprehensive reviews on the subject Russell, 1990; Creasy, 1997; Creasy, 2002. The PAS-H stained sections were used to identify the spermatogenic stages. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

[1] References Liver Hypertrophy: A Review of Adaptive (Adverse and Non-adverse) Changes - Conclusions from the 3rd International ESTP Expert Workshop - Toxicologic Pathology, 40: 971-994, 2012.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Remarks:

maternal toxicity

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse effects observed

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

see below

Body weight and weight changes:

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

see below

Gross pathological findings:

no effects observed

Description (incidence and severity):

see below

Histopathological findings:

effects observed, treatment-related

Description (incidence and severity):

see below

Details on results (F1)

- Litter data at birth, on Day 1 and on Day 4 post partum of females and sex ratio of pups:
An increased incidence of pups loss value (percentage) on Day 1 post partum and post natal loss value on Day 4 post partum (percentage) was observed in females receiving 250 mg/kg body weight/day. These changes were not statistically significant and no dose-relationsship was observed. Sex ratios at birth and on Day 4 post partum did not show differences between groups, when calculated as the percentage of males. No effects on mean litter weight were noted.

- Clinical signs of pups:
Cold to touch, apparently no food intake (milk), small and pale appearance were the main clinical signs noted in control and treated pups. In addition, found dead or missing pups were also observed both in control and treated groups. One pup at the high dose level (250 mg/kg body weight/day) showed right hindlimb damaged. This finding was considered incidental.

- Necropsy findings in deceased pups and in pups sacrificed on Day 4 post partum:
Autolysed abdominal organs were observed in one pup of Group 4 (250 mg/kg body weight/day) which died during the lactation period. An increase incidence of pups without milk in stomach was noted in Group 4 (250 mg/kg body weight/day) compared to the control group.

- Terminal body weight and organ weights (Dosing Phase -Main groups)
Terminal body weight was unaffected by treatment in both sexes. Some changes (increase in absolute and relative liver weight in males and females, decrease in absolute adrenals weight in females, increase in relative brain and heart weights in males and females, increase in relative kidneys weight in males) were observed in animals receiving the dose levels 100 mg/kg body weight/day. With the exception of the liver, the alterations were not associated with histopathological findings. Liver lesion was considered hepatic adaptive effect rather than an injurious change.

Effect levels (F1)

Key result

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no toxicological relevant effects observed

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results obtained from a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422), the NOAEL for general toxicity as well as for reproductive and developmental toxicity was considered to be 250 mg/kg bw/day.

Executive summary:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) the test item (87.9% purity) was administered orally to 10 male and female Sprague-Dawley rats/dose in water by gavage at dose levels of 0, 40, 100, or 250 mg/kg bw/day. The animals were treated daily with the test item formulation on 7 days per week. Females were treated two 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). Males were treated for a total of 36 or 37 days. Litter was not exposed. There were no treatment-related effects found regarding mortality, clinical signs, neurotoxicity assessment, body weights, food consumption, histopathology, organ weights, haematology and clinical chemistry. Concerning the reproductive parameters, no relevant differences were found in terms of mating performance including the pre-coital interval (number of days paired to sperm positive day), copulatory evidence (the positive identification of mating i.e. the presence of sperm and/or copulation plug in situ or in the cage) or fertility index. All pregnant females had a comparable length of gestation period and gave birth on day 22 post coitum (mean value). An increased incidence of pups loss value (percentage) on day 1 post partum and post natal loss value on day 4 post partum (percentage) was observed in females receiving 250 mg/kg bw/day without any dose relationsship. Sex ratios at birth and on day 4 post partum did not show differences between groups, when calculated as the percentage of males. Similar clinical signs were recorded in control and treated pups during the lactation period. At necropsy, pups of females receiving 250 mg/kg bw/day and sacrificed at termination showed an increased incidence of no milk in stomach. Deceased pups did not show relevant findings both in control and treated groups.

Based on the results of the present study, the NOAEL for maternal and reproductive/developmental toxicity was considered to be 250 mg/kg bw/day. This study is classified as acceptable and satisfies the guideline requirements for an oral repeated dose toxicity study in rat.