Violanthrene-5,10-dione

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 August 2016 - Date of Study Director’s signature on this report

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

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)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

16,17-dimethoxyviolanthrene-5,10-dione

EC Number:

204-896-6

EC Name:

16,17-dimethoxyviolanthrene-5,10-dione

Cas Number:

128-58-5

Molecular formula:

C36H20O4

IUPAC Name:

16,17-dimethoxyanthra[9,1,2-cde]benzo[rst]pentaphene-5,10-dione

Test material form:

solid: particulate/powder

Details on test material:

Name: Vat Green 1

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The Sprague Dawley rat was the species and strain of choice because it is accepted by many regulatory authorities and there is 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: Envigo RMS srl., San Pietro al Natisone (UD), Italy
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 187.8-206.4 g for males and 150.2-171.7 g for females
- Weight ordered: 176 to 200 g for males and 151 to 175 g for females
- Assigned to test groups randomly: yes, by computerised stratified randomisation to give approximately equal initial group mean body weights
- Fasting period before study:
- Housing: 5 of one sex to a cage - main groups from arrival to pairing; main group males after pairing; recovery groups throughout the entire study period
one male to one female- main groups during pairing
single - main group females after pairing
- Diet (ad libitum): 4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019, Settimo Milanese (MI), Italy
- Water (ad libitum): tap water
- Acclimation period: 2 weeks

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

IN-LIFE DATES: From: To:

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route by gavage was selected as it is a possible route of exposure of the test item in man and provides the best internal exposure possible for this kind of dye.

Vehicle:

other: Sesame oil

Details on oral exposure:

The selected vehicle was sesame oil, as the test substance is neither soluble in water nor in organic solvents. A formulation in sesame oil provides a homgenous suspension.
The required amount of Vat Green 1 was suspended in sesame oil. The formulations were prepared daily (concentrations of 12.5, 50 and 200 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.
The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight.
Control animals received the vehicle alone at the same dose volume. The 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:

no

Remarks:

the test item is neither extractable in hydrophilic nor in lipophilic solvents, hence analytical verification of test item concentrations is not possible

Details on analytical verification of doses or concentrations:

The correct preparation of the respective dose levels was monitored in the weighing record of the test item in each formulation process

Duration of treatment / exposure:

MAIN GROUPS (Groups 1 to 4)
- Males:
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter at least untilthe minimum total dosing period of 28 days had been completed including the day before necropsy.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
- Females:
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post
partum periods until at least up to, and including, Day 3 post partum or the day before sacrifice.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and then on Day 1 post
partum. Thereafter individual dose volumes remained constant.

RECOVERY GROUPS (Groups 5 and 6)
Animals were dosed once a day, 7 days a week, for 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 and 5

Doses / concentrations 2

Dose / conc.:

62.5 mg/kg bw/day (nominal)

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4 and 6

No. of animals per sex per dose:

Each main group comprised 10 male and 10 female rats (Groups 1 to 4).
Two groups (control and high dose levels) included 5 animals per sex to be sacrificed after 2 weeks of recovery (Groups 5 and 6) following a 4-week treatment period in unmated animals.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the test substance did not cause any adverse effects in acute oral toxicity studies up to dose levels of 10000 mg/kg bw

Examinations

Observations and examinations performed and frequency:

Clinical signs:
Once before commencement of treatment (data not presented but retained and archived together with all raw data) and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day.

Clinical observations ((Functional Observation Battery Tests):
Once before commencement of treatment and once a week thereafter, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respirator pattern).

Grip strength and sensory reactivity to stimuli:
Once during the study, towards the end of treatment, 5 males and 5 females (*) were randomly selected from each group for evaluation of sensory reactivity to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli) and for assessment of grip strength.
Measurements were performed using a computer generated random order (for the main groups).
Once towards the end of treatment and once duringWeek 2 of recovery these evaluations were also performed in all recovery animals.
(*) The same 5 males and 5 females, randomly selected from each main group, were also assessedfor motor activity, clinical pathology investigation, histopathology evaluation and micronucleus test.
Male nos.:
Group 1: 2, 4, 14, 16, 18
Group 2: 26, 28, 36, 38, 40
Group 3: 44, 46, 48, 52, 58
Group 4: 64, 66, 70, 72, 74

Female nos.:
Group 1: 3, 9, 11, 13, 19
Group 2: 21, 29, 35, 37, 39
Group 3: 43, 45, 49, 55, 59
Group 4: 65, 69, 71, 77, 79

Motor activity:
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order (for the main groups).

Body weight:
- Main groups: Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum.
Dams were also weighed on Days 1 and 4 post partum.
- Recovery groups: Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.
Once towards the end of treatment and once during Week 2 of recovery, this evaluation was also performed in all recovery animals.

Food consumption:
- Main groups: The weight of food consumed by each cage ofmales and females was recorded weekly 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.

Clinical pathology:
As a part of the sacrificial procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females, randomly selected from each main group, under condition of food deprivation (females only, with viable litters) and under condition of food and water deprivation (males only). At the same time interval, individual overnight urine samples were also collected from the same males under the same conditions. Before starting urine collection, water bottles were removed from each cage and each animal received approximately 10 mL/kg body weight of drinking water by gavage, in order to obtain urine samples suitable for analysis (proof of absorption). At the end of Week 2 of the recovery period, blood samples were also taken from all surviving animals under condition of food deprivation in order to re-evaluate haematology (no coagulation) and clinical chemistry parameters which showed possibly treatment-related changes at measurements performed during the treatment period.

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

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

– Platelets

Coagulation
– Prothrombin time
– Activated partial thromboplastin time

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

Sacrifice and pathology:

EUTHANASIA
Main and Recovery groups:
Animals selected for blood collection were killed by exsanguination under isofluorane anaesthesia.
Animals not selected for blood collection were killed by carbon dioxide asphyxiation.
Parental males - Main groups:
The males were killed after the mating of all females, after at least 28 days of treatment period.
Parental females - Main groups:
The females with live pups were killed on Day 4 post partum. The females showing no evidence of copulation were killed 25 days after the last day of the mating session. The females which did not give birth 25 days after positive identification of mating were killed shortly after.
Males and females - Recovery groups
Animals were killed after 2 weeks of recovery following a 28-day treatment period.

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.
Females
All females were examined also for the following:
– external and internal abnormalities;
– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals).
Uteri of females with no visible implantationswere immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

ORGAN WEIGHTS
From all animals completing the scheduled test period, the following organs were dissected free of fat and weighed. The ratios of organ weight to body weight were calculated for each animal:
Adrenal glands
Brain (cerebrum, cerebellum, medulla/pons)
Epididymides
Heart
Kidneys
Liver
Ovaries and oviducts
Parathyroid glands
Prostate gland
Seminal vesicles with coagulating glands
Spleen
Testes
Thymus (where present)
Thyroid
Uterus – cervix

TISSUES Tissues fixed and preserved -Main and Recovery groups:
Samples of all the following tissues (all parental animals) were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves, testes and epididymides which were fixed inModified Davidson’s fluid and preserved in 70% ethyl alcohol):
Abnormalities
Adrenal glands
Bone marrow (from femur)
Bone marrow (from sternum)
Brain (cerebrum, cerebellum, medulla/pons)
Caecum
Clitoral gland
Colon
Duodenum
Epididymides
Heart
Ileum
Jejunum (including Peyer’s patches)
Kidneys
Liver
Lungs (including mainstem bronchi)
Lymph nodes – cervical
Lymph nodes – mesenteric
Nasal cavity*
Oesophagus*
Ovaries and oviducts
Parathyroid glands
Pituitary gland
Penis
Prostate gland
Rectum
Sciatic nerve
Seminal vesicles with coagulating glands
Spinal column*
Spinal cord (cervical, thoracic, lumbar)
Spleen
Stomach (forestomach and glandular)
Testes
Thymus (where present)
Thyroid
Trachea
Urinary bladder
Uterus – cervix
Vagina

*Not examined since no signs of toxicity or target organ involvement were noted

Histopathological examination -Main and Recovery groups:
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of theseminiferous epithelium (staging of spermatogenic cycle) was also performed.
The examination was restricted as detailed below:
1. Tissues specified above from 5 males and 5 females randomly selected (animals
evaluated for clinical pathology) in the control and high dose groups
killed at term.
2. All abnormalities in all groups.

Statistics:

Main and Recovery groups:
Standard deviationswere 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.
The non-parametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters.
The criterion for statistical significance was p < 0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Main groups:
Green staining of the tail was the main clinical sign recorded in all high dose animals and occasionally in the mid-dose group.
At observation of the cage tray, green staining was observed in the high dose group. This staining was explained by the colour of the test item which was likely excreted in the urine and faeces.

Recovery groups:
Green staining of the tail was recorded in treated animals throughout the study, during treatment and recovery periods.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups:
No differences in body weight were recorded between groups of both sexes throughout the study.
Some significant differences in body weight gain were recorded in high dose females before pairing (decreased and increased mean values on Days 8 and 15, respectively), compared to the control group.

Recovery groups:
No differences in body weight were recorded between groups of both sexes throughout the study.
Minimal but stastically significant decreased body weight gain was found in males of the high dose group, when compared to controls at the end of treatment (Day 29).
During recovery, minimal but statistically significant increased body weight gain was found in males of the high dose group compared to the control group on Day36, reflecting the lower body weight gain on Day 29.
These slight variations in body weight gain of main and recovery groups, were considered incidental findings.

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:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups:
Slight reticulocytosis was recorded in males dosed at 1000 mg/kg body weight/day (27% above controls), with no related changes in the other red blood cell parameters. In addition, leucocytosis was recorded in two high dose males. Leucocytes were approximately 47% above controls. The other statistically significant differences between control and treated males (mean corpuscular volume and mean corpuscular haemoglobin) were of minimal severity (up to 7%), therefore they were considered to be incidental findings.

Recovery groups:
Changes recorded during the dosing phase showed reversibility. Treated males showed slight leucopenia (26% below mean control data). However, since this finding was not observed during the dosing phase, it was considered unrelated to treatment. The statistically significant difference of mean corpuscular haemoglobin concentration recorded between control and treated females (2%) was of minimal severity, therefore it was considered to be an incidental finding.

Coagulation:
Main groups
Prothrombin time and activated thromboplastin time were slightly decreased in two males dosed at 250 mg/kg body weight/day. Due to the absence of dose-relation, these changes were considered incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Main groups:
Compared with mean control data, one high dose male (1000 mg/kg body weight/day) showed increase of alanine and aspartate aminotransferases (approximately 2.1 fold), glucose (1.6 fold) and bile acids (6.5 fold). Due to the minimal incidence, this change cannot be conclusively attributed to treatment.
Increased glucose was also recorded in other males dosed at 62.5 and 1000 mg/kg body weight/day. Compared with mean control data, the increments were 1.3 and 1.4 fold, respectively.

Recovery groups:
Glucose was still higher than controls in treated males (1.2 fold). Due to the slight severity, this change was considered not adverse.
The other statistically significant differences between control and treated animals (creatinine, globulin, chloride and potassium in males, phosphorus and sodium in females) were not recorded during the dosing phase, therefore they were considered incidental.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Main and recovery groups:
Observation of treated animals at removal from the cage and in an open arena did not reveal significant changes when compared to controls.
Motor activity, grip strength and sensory reaction to stimuli measurements recorded at the end of treatment did not show differences of toxicological relevance between control and treated groups of both sexes. The significant differences of lower grip strength in Group 2 or reduced landing food splay in high dose females (Group 4) were considered incidental.
No significant differences were recorded in the recovery groups of both sexes at the end of the recovery phase.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

No relevant changes were found in terminal body weight or organ weights between groups.
Significant reduction in relative weights to terminal body weight of prostate in the low and high dose groups, and spleen in high dose males of recovery group were obtained. All these findings were not considered of toxicological importance.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Final sacrifice:
Animals killed at termination did not show relevant macroscopic changes that could be treatment-related. The changes observed such as green staining of tail or green content of stomach were attributed to the colour of the test item, while enlarged liver in rats of both sexes or reduced thymus in control and treated females are suggested to be incidental and/or due to physiological pregnancy changes, often seen in this kind of study in some treated Sprague Dawley rats of the same age.

Recovery sacrifice:
No relevant changes were noted at post mortem examination in treated animals, when compared with controls. Green staining was still observed in some treatedanimals of both sexes.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Final sacrifice:
No treatment-related changes were noted.
The sporadic lesions reported in control and treated animals were considered to be an expression of spontaneous and/or incidental pathology or are commonly seen in this species and age.

Spermatogenic cycle:
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.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

> 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In conclusion, no toxic effects of Vat Green 1 were seen after repeated dose and on reproduction/development in Sprague Dawley rats.
On the basis of the results obtained in this study, the NOAEL (No Observed Adverse Effect Level) for both general toxicity and reproduction/developmental toxicity was considered to be above 1000 mg/kg body weight/day for males and females of the parental and F1 generation.

Executive summary:

A Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity screening test was conducted with Vat Green 1 in Sprague Dawley SD rats up to Day 4 post partum. The effects of the test item systemic or local toxicity as well as any possible effects on male and female reproductive performance, such as gonadal function, mating behaviour, conceptuses, parturition and early lactation of the offspring, were investigated at the dose levels of 62.5, 250 and 1000 mg/kg body weight/day. The reversibility of toxic effects after repeated dosing with Vat Green 1 up to 4 consecutive weeks followed by a 2 week treatment-free period were also investigated, in order to assess recovery from any adverse effects observed during the dosing phase. The animals were assigned to four treatment main groups (Groups 1 to 4) of 10 animals/sex and two treatment recovery groups (Groups 5 and 6) of five animals/sex. The animals were administered orally by gavage at a constant volume of 5 mL/kg body weight. The test item was formulated in sesame oil at the final concentrations of 12.5, 50 and 200 mg/mL for treatment of Groups 2, 3 and 4/6 respectively. Control animals (Groups 1/5) received the vehicle alone at the same dose volume. Furthermore, genotoxicity assessment was also evaluated in males of the main groups, in order to assess the ability of the test item to induce cytogenetic damage in rat bone marrow, as measured by the induction of micronuclei in polychromatic erythrocytes.

Main groups: According to the study design, males were treated for 2 weeks prior to pairing and during pairing with females until the day before necropsy, up to 45 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 approximately 41-54 days, depending on the female’s performance. The following investigations were performed in all groups: mortality check, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), body weight, food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology and clinical chemistry), litter data, macroscopic observations and organ weights. Evaluation of body weight, clinical signs and macroscopic observations of pups were also performed. Routine histopathological examination was performed on control and high dose groups (five animals/sex/group randomly selected). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was also included.

Recovery groups: Animals of the recovery groups were treated for a total of 4 consecutive weeks and sacrificed after 2 weeks of recovery (Groups 5, 6). The following parameters were evaluated in these animals: mortality, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reaction to stimuli), body weight, food consumption, clinical pathology investigations (clinical chemistry and haematology with the exception of coagulation), macroscopic observations and organ weights. As no adverse effects were noted in main group animals, histopathology was not performed in recovery animals.

Mortality and fate of females: No mortality occurred throughout the study. Non-pregnant females were found in the mid- and in the high dose groups. One mid-dose female had unilateral implantation. The number of females with live pups on Day 4 post partum was: 10 in each of the control and low dose groups, 7 in the mid-dose group and 9 in the high dose group.

Clinical signs and observation of cage tray: Green staining of tail was the main clinical sign recorded during treatment and recovery period in the high dose animals. At observation of the cage tray, green staining was also recorded in the high dose group during the treatment period. Moreover, treated animals of the recovery group (Group 6) showed green staining in the cage tray also during the recovery period. This discolouration was due to the colour of the test item and its properties as textile dye.

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

Body weight: Body weight and body weight gain were unaffected by treatment both in males and females throughout the main or recovery phases.

Food consumption: No changes in food consumption were recorded in male and female animals throughout the main or recovery phases.

Clinical pathology: No changes of toxicological relevance were recorded in clinical pathology investigation at the end of dosing or recovery periods.

Reproductive performance: Oestrous cycle, pre-coital intervals, copulatory and fertility indices did not show intergroup differences.

Implantation, pre-birth loss data and gestation length of females: No significant differences were observed in the number of implantations, corpora lutea, total litter size, pre-implantation loss and pre-birth loss between control and treated groups. The majority of dams gave birth on Day 22 post coitum.

Litter data: Reproductive outcomes of dams which included the number and body weight of pups and the percentage of live pups on Days 1 and 4 post partum did not differ between groups. Sex ratio of pups was also comparable between groups.

Clinical signs of pups: There were no treatment-related effects at clinical observation of pups.

Necropsy findings in deceased pups and in pups sacrificed on Day 4 post partum: No treatment-related findings were recorded at necropsy of deceased pups or those sacrificed at term.

Terminal body weight and organ weights: No relevant differences of toxicological significance were seen in terminal body weight or organ weights between controls and treated animals, both in main and recovery groups.

Macroscopic observations: Animals killed at termination did not show relevant macroscopic changes that could be treatment-related. No relevant changes were noted at post mortem examination in recovery animals, when compared with controls.

Microscopic observations: No microscopic alterations were noted in treated animals that could be considered treatment related.

Spermatogenic cycle: 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.

 

Conclusion: No treatment-related effects indicating local or systemic toxicity were observed in male or female animals at any of the dose levels investigated. No effects on sexual function and fertility or in developmental parameters and lactation were observed at any of dose level investigated.

Based on the results obtained in the study, the NOAEL (No Observed Adverse Effect Level) for both general toxicity and reproduction/developmental toxicity was considered to be above 1000 mg/kg body weight/day for males and females of the parental and F1 generation.