Registration Dossier

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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 May 2015 to 10 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-referenceopen allclose all
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:
27 May 2015 to 10 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same 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)
Deviations:
yes
Remarks:
See below under 'Priniciples of method...'
Qualifier:
according to guideline
Guideline:
other: OECD 474 adopted on September 2014
Deviations:
yes
Remarks:
See below under 'Priniciples of method...'
Principles of method if other than guideline:
Protocol deviations:
Uterus of the female humanely killed on Day 3 post coitum was not immersed in a 10-20% solution of ammonium sulphide to reveal evidence of implantation
and pregnancy was therefore not established in this animal.
Animal no. 41 was not observed on Day 4 post partum and recovery males and females were not observed on Day 47.
Animals of the positive control group were not weighed prior to sacrifice.
Parturition check for animal no. 3 started on Day 19 of gestation instead of Day 20.
Slides for micronucleus test were not dried overnight as indicated in the study protocol but as necessary to obtain complete dehydration.
These deviations were not considered to have compromised the purpose or conduct of the study.
No other deviations occurred.
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
A total of 125 Sprague Dawley [Crl:CD(SD)BR] rats (65 males and 60 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females, were ordered from and supplied by Charles River Italia S.p.A., Calco (Lecco), Italy.After arrival, on 21 May 2015, the weight range for each sex was determined (189-216 g for females, 226-250 g for males, slightly outside the range at order) and the animals were temporarily identified within the cage by means of a coloured mark on the tail.A health check was then performed by a veterinarian. An acclimatisation period of at least 14 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C +/- 2°C and 55% +/- 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study.There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
Route of administration:
oral: gavage
Details on route of administration:
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.
Vehicle:
other: Sesame oil
Details on oral exposure:
The required amount of Vat Black 25 was dissolved/suspended in the vehicle. The formulations were prepared daily (concentrations of 12,5, 50 and 200 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Analysis was not performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory as the test item is neither extractable in hydrophilic nor in lipophilic solvents. The correct concentration preparation 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 through the day before necropsy (Days 43 and 44 of study). They were treated for a total of 42 or 43 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 approximately 42 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 Day1 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 6 consecutive weeks. No treatment was given during the recovery period.

Positive Control group (Group 7):
Animals received a single dose of the positive control (for genotoxicity endpoint) approximately 24 hours before sacrifice.
Frequency of treatment:
Once daily
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
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 which were sacrificed after 2 weeks of recovery (Groups 5 and 6). For genotoxicity endpoint, a satellite control group (Positive Control group) comprised 5 male rats (Group 7).
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels have been selected in consultation with the Sponsor based on information from previous studies.
- Rationale for animal assignment (if not random): random
- Rationale for selecting satellite groups: A recovery group was included to assess recovery from any delayed toxicity or persistence of adverse effects observed during the dosing phase. In addition, the micronucleus test was included (5 male rats 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.
- Post-exposure recovery period in satellite groups: 2 weeks
Positive control:
Positive control treatments for genotoxicity endpoints used the well-known clastogen Mitomycin-C (Sigma-Aldrich, batch no. SLBH9906V, expiry date: January 2018).
Observations and examinations performed and frequency:
Main and Recovery groups:
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded.
Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions (15-45 minutes after treatment).

Observations of the cage tray
Observations of the cage tray were performed during mating period for all main groups and were recorded daily.

Positive Control group
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded.

Neurotoxicity assessment (removal of animals from the home cage and open arena).
Once before commencement of treatment and at least once per 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 (for at least 3 minutes). The test 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, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded.

Grip strength and sensory reaction to stimuli
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reaction to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli); an assessment of grip strength was also performed. Measurements were performed using a computer generated random order (for the main groups). For males (main groups), the tests were performed on Day 40 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 41 of the study (during treatment) and once during Week 2 of recovery (Day 10).

Motor activity assessment (MA)
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order (for the main groups). For males (main groups) the tests were performed on Day 41 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 42 of the study (during treatment) and once during Week 2 of recovery (Day 13).

Body weight
Main groups
Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.
Recovery groups
Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.
Positive Control group
Animals were weighed on the day of allocation and on the day of dosing.

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.

Clinical pathology investigations
During the necropsy procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters) randomly selected from each group under conditions of food deprivation for haematological, coagulation and clinical chemistry evaluations.

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

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

Parental males (Main groups)
The males were killed after the mating of all females or 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 which did not give birth 25 days after positive identification of mating werekilled shortly after.

Males and females (Recovery groups)
Animals were killed after 2 weeks of recovery.

Positive Control group
Animals were killed approximately 24 hours after treatment.

Necropsy (Main and Recovery groups)
The clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed (excluding the animal sacrificed for humane reasons) and the required tissue samples preserved in fixative and processed for histopathological examination.

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

Organ weights (Main and Recovery groups)
From all animals completing the scheduled test period, the following organs were dissected free of fat and weighed:
Adrenal glands
Brain (cerebrum, cerebellum, medulla/pons)
Epididymides
Heart
Kidneys
Liver
Ovaries with oviducts
Parathyroid glands
Prostate gland
Seminal vesicles with coagulating glands
Spleen
Testes
Thymus
Thyroid
Uterus including cervix

The ratios of organ weight to body weight were calculated for each animal.

Tissues fixed and preserved (Main and Recovery groups)
Samples of all the tissues listed below in were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).

Abnormalities
Adrenal glands
Bone marrow (from sternum)
Brain (cerebrum, cerebellum, medulla/pons)
Caecum
Clitoral gland
Colon
Duodenum
Epididymides
Heart
Ileum (including Peyer’s patches)
Jejunum
Kidneys
Liver
Lungs (including mainstem bronchi)
Lymph nodes – cervical
Lymph nodes – mesenteric
Nasal cavity
*Oesophagus
*Ovaries with 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 including cervix
Vagina

*not examined as no signs of toxicity or target organ involvement were observed.

After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS).
The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was also performed.
The examination was restricted as detailed below:
i Tissues specified above from 5 males and 5 females (main groups) randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term.
ii Tissues specified above from all animals killed or dying during the treatment period.
iii All abnormalities in all main groups.
Other examinations:
No data
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. The nonparametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups was assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p < 0.05.
Clinical signs:
no effects observed
Description (incidence and severity):
No signs of toxicological relevance were observed during the study.
Slight orange staining in the cage tray was observed during the mating phase in some cages of mid- and high dose animals.
Observation of animals at removal from the cage and in an open arena did not reveal changes attributable to the test item.
No alterations in motor activity, grip strength and sensory reaction to stimuli were observed in any treatment group at the examination performed at the end of treatment.
At the end of recovery period, motor activity, grip strength and sensory reaction to stimuli were comparable between control and treated groups of both sexes.
Mortality:
no mortality observed
Description (incidence):
One female of the high dose group, receiving 1000 mg/kg bodyweight/day was killed for humane reasons on Day 3 of the gestation phase. On the day of sacrifice, hunched posture and marked swelling of the fore and hind limbs were recorded.
Macroscopic findings observed at post mortem examination in this animal were represented by swollen and oedematous consistency of hindlimbs and right forelimb, as well as unilateral pelvic dilatation.
Marked arthritis of hindlimbs and one forelimb, observed at histopathology, could be considered as a factor contributory to the illness status of the animal sacrificed for humane reasons. Therefore, this death was considered to be unrelated to treatment.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Throughout the study, no difference in body weights was recorded in animals of both sexes compared to the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effects on food consumption were observed in both sexes.
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):
No relevant changes were observed. The statistically significant differences between controls and treated males (mean corpuscular volume, neutrophils and eosinophils percentages), were of minimal severity and/or not dose-related, therefore considered of no toxicological significance.

Coagulation: no changes were recorded.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No relevant changes were observed. The statistically significant differences recorded (urea, glucose, chloride, phosphorus in males) were not dose-related, therefore considered unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Main and recovery groups
Observation of animals at removal from the cage and in an open arena did not reveal changes attributable to the test item.
No alterations in motor activity, grip strength and sensory reaction to stimuli were observed in any treatment group at the examination performed at the end of treatment.
At the end of recovery period, motor activity, grip strength and sensory reaction to stimuli were comparable between the control and treated group in both sexes.
Immunological findings:
not specified
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Absolute and relative organ weights were comparable in all groups.
The following statistically significant difference was noted in the organ weights of the main groups:
– Decreased relative uterus weight in females receiving 250 and 1000mg/kg body weight/day (-30 % and -25 %, respectively). Absolute uterus weight was also decreased in all groups when compared to controls. This difference was without dose-dependency and not accompanied by histological findings. Therefore, it was considered not to be treatment-related. No toxicological significance was attributed to the other statistically significances observed in the weight of organs.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no compound-related effects.
No relevant changes were noted in study animals sacrificed at the end of the treatment period or after 2 weeks of recovery period.
Changes such as pelvic dilatation, swollen liver and small size of thymus in the control and treated groups, both in final and recovery animals, were considered to be incidental.
Neuropathological findings:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No microscopic observation was observed in treated animals that could be considered treatment-related.
A limited number of lesions, reported in control and/or treated animals, were considered to be an expression of spontaneous and/or incidental pathology, seen in this species and age of untreated animals.
In addition seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.
Histopathological findings: neoplastic:
no effects observed
Other effects:
not specified
Details on results:
No signs of genotoxicity were detected by measuring the micronuclei in polychromatic erythrocytes.

No treatment-related effects indicating systemic toxicity were observed in male or female animals at any of the dose levels investigated (0, 62.5, 250 and 1000 mg/kg body weight/day).
Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects of toxicological significance at highest dose tested: 1000 mg/kg bw/day
Key result
Critical effects observed:
no

FATE OF FEMALES – GROUP INCIDENCE

Group

1

2

3

4

Initial group size

10

10

10

10

Not pregnant

0

0

2

0

Unilateral implantation

0

1

0

0

Humane killed

0

0

0

1a

With live pups on Day 4 post partum

10

10

8

9

a= pregnancy not detected

 

CLINICAL SIGNS OF MALES – MAIN GROUPS – GROUP INCIDENCE

Interval: 1! – 30” Days

Group

Observation

 

1

(10)

 

2

(10)

 

3

(10)

 

4

(10)

 

a

b

a

b

a

b

a

b

No significant signs

10

38.9

10

43.5

10

43.5

10

42.2

APPEARANCE

               Hair loss

 

1

 

30.0

 

0

 

0.0

 

0

 

0.0

 

2

 

6.5

EYE – EAR – MOUTH

               Damaged ear

 

1

 

16.0

 

0

 

0.0

 

0

 

0.0

 

0

 

0.0

Key:        () = Number of animals alive at start of interval

               a = Number of animal affected

               b = Number of days with clinical sign/animal

Note:      ! = Premating phase; “ = Mating phase

 

CLINICAL SIGNS OF FEMALES BEFORE PAIRING – MAIN GROUPS – GROUP INCIDENCE

Interval: 1 - 15 Days

Group

Observation

 

1

(10)

 

2

(10)

 

3

(10)

 

4

(10)

 

a

b

a

b

a

b

a

b

No significant signs

10

15.0

10

15.0

10

15.0

10

15.0

Key:        () = Number of animals alive at start of interval

               a = Number of animal affected

               b = Number of days with clinical sign/animal

 

CLINICAL SIGNS OF FEMALES –POST COITUMANDPOST PARTUMPERIODS - MAIN GROUPS – GROUP INCIDENCE

Interval: 0! – 4” Days

Group

Observation

 

1

(10)

 

2

(10)

 

3

(10)

 

4

(10)

 

a

b

a

b

a

b

a

b

No significant signs

10

27.1

10

27.2

10

27.1

10

24.1

APPEARANCE

               Cyphosis

               Swollen

 

0

0

 

0.0

0.0

 

0

0

 

0.0

0.0

 

0

0

 

0.0

0.0

 

1

1

 

1.0

1.0

Key:        () = Number of animals alive at start of interval

               a = Number of animal affected

               b = Number of days with clinical sign/animal

Note:      ! = Gestation phase phase; “ = Postpartum phase

 

CLINICAL OBSERVATIONS DURING TREATMENT – BEHAVIOURAL EXAMINATION – REMOVAL OF ANIMALS FROM THE HOME CAGE - MAIN GROUPS – GROUP INCIDENCE

MALES

Interval: 1! – 6” Weeks

Group

Observation

 

1

(10)

 

2

(10)

 

3

(10)

 

4

(10)

 

a

b

a

b

a

b

a

b

REMOVAL

               Removal easy

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

HANDLING REACTIVITY

               Handling reactivity normal

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

LACHRYMATION

               Lachrymation absent

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

PALPEBRAL CLOSURE

               Palpebral closure absent

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

SALIVATION

               Salivation absent

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

PILOERECTION

               Piloerection absent

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

Key:        () = Number of animals alive at start of interval

               a = Number of animals affected

               b = Number of week with clinical sign/animal

Note:      ! = Pretest phase; “ = Dosing phase

 

CLINICAL OBSERVATIONS DURING TREATMENT – BEHAVIOURAL EXAMINATION – REMOVAL OF ANIMALS FROM THE HOME CAGE - MAIN GROUPS – GROUP INCIDENCE

FEMALES

Interval: 1! – 6” Weeks

Group

Observation

 

1

(10)

 

2

(10)

 

3

(10)

 

4

(10)

 

a

b

a

b

a

b

a

b

REMOVAL

               Removal easy

 

10

 

6.9

 

10

 

6.8

 

10

 

6.9

 

10

 

6.6

HANDLING REACTIVITY

               Handling reactivity normal

 

10

 

6.9

 

10

 

6.8

 

10

 

6.9

 

10

 

6.6

LACHRYMATION

               Lachrymation absent

 

10

 

6.9

 

10

 

6.8

 

10

 

6.9

 

10

 

6.6

PALPEBRAL CLOSURE

               Palpebral closure absent

 

10

 

6.9

 

10

 

6.8

 

10

 

6.9

 

10

 

6.6

SALIVATION

               Salivation absent

 

10

 

6.9

 

10

 

6.8

 

10

 

6.9

 

10

 

6.6

PILOERECTION

               Piloerection absent

 

10

 

6.9

 

10

 

6.8

 

10

 

6.9

 

10

 

6.6

Key:        () = Number of animals alive at start of interval

               a = Number of animals affected

               b = Number of week with clinical sign/animal

Note:      ! = Pretest phase; “ = Dosing phase

 

CLINICAL OBSERVATIONS DURING TREATMENT – BEHAVIOURAL EXAMINATION – OPEN AREA – MAIN GROUPS – GROUP INCIDENCE

MALES

Interval: 1! – 6” Weeks

Group

Observation

 

1

(10)

 

2

(10)

 

3

(10)

 

4

(10)

 

a

b

a

b

a

b

a

b

REARING

               Rearing absent

               Rearing 1-3

               Rearing 4-7

               Rearing 8-10

               Rearing 11-14

               Rearing 15-20

               Rearing 21-30

 

0

2

5

9

10

5

0

 

0.0

1.5

2.6

1.7

3.0

1.8

0.0

 

0

1

4

9

10

5

0

 

0.0

2.0

1.8

2.8

2.8

1.6

0.0

 

1

2

3

8

10

8

0

 

2.0

3.0

1.7

2.0

2.2

2.4

0.0

 

0

1

2

9

10

7

2

 

0.0

1.0

1.0

2.7

2.8

1.9

1.0

SPASMS

               Spasms absent

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

MYOCLONIA

               Myoclonia absent

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

GAIT

               Normal gait

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

MOBILITY IMPAIRMENT

               Mobility impairment absent

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

AROUSAL

               Arousal normal

               Arousal slow

 

10

0

 

7.0

0.0

 

10

0

 

7.0

0.0

 

10

1

 

6.8

2.0

 

10

0

 

7.0

0.0

VOCALISATION

               Vocalisation absent

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

STEREOTYPIES

               Stereotypies absent

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

UNUSUAL RESPIRATION

               Unusual respiration absent

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

BIZARRE BEHAVIOUR

               Bizarre behaviour absent

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

URINATION

               Urination absent

               Urination 1-3

               Urination 4-6

               Urination 7-9

               Urination more than 10

 

10

8

2

3

0

 

4.0

3.0

1.5

1.0

0.0

 

10

7

6

1

1

 

4.8

1.7

1.2

1.0

2.0

 

10

6

5

0

1

 

5.0

2.2

1.2

0.0

1.0

 

10

7

3

2

0

 

5.1

1.3

2.7

1.0

0.0

DEFECATION

               Defecation absent

               Defecation 1-3

               Defecation 4-6

 

10

6

3

 

4.9

2.5

2.0

 

10

6

3

 

6.0

1.2

1.0

 

10

5

1

 

6.2

1.4

1.0

 

10

6

2

 

5.8

1.7

1.0

TREMORS

               Tremors absent

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

 

10

 

7.0

Key:        () = Number of animals alive at start of interval

               a = Number of animals affected

               b = Number of weeks with clinical sign/animal

Note:      ! = Pretest phase; “ = Dosing phase

 

CLINICAL OBSERVATIONS DURING TREATMENT – BEHAVIOURAL EXAMINATION – OPEN AREA – MAIN GROUPS – GROUP INCIDENCE

FEMALES

Interval: 1! – 6” Weeks

Group

Observation

 

1

(10)

 

2

(10)

 

3

(10)

 

4

(10)

 

a

b

a

b

a

b

a

b

REARING

               Rearing 11-14

               Rearing 15-20

               Rearing 21-30

               Rearing more than 30

 

5

10

8

4

 

2.2

3.6

2.3

1.3

 

5

10

6

1

 

2.4

3.6

3.2

1.0

 

7

7

6

5

 

1.6

4.4

3.3

1.4

 

6

10

6

1

 

2.7

3.6

2.2

1.0

SPASMS

               Spasms absent

 

10

 

6.9

 

10

 

6.8

 

10

 

6.9

 

10

 

6.6

MYOCLONIA

               Myoclonia absent

 

10

 

6.9

 

10

 

6.8

 

10

 

6.9

 

10

 

6.6

GAIT

               Normal gait

 

10

 

6.9

 

10

 

6.8

 

10

 

6.9

 

10

 

6.6

MOBILITY IMPAIRMENT

               Mobility impairment absent

 

10

 

6.9

 

10

 

6.8

 

10

 

6.9

 

10

 

6.6

AROUSAL

               Arousal normal

 

10

 

6.9

 

10

 

6.8

 

10

 

6.9

 

10

 

6.6

VOCALISATION

               Vocalisation absent

 

10

 

6.9

 

10

 

6.8

 

10

 

6.9

 

10

 

6.6

STEREOTYPIES

               Stereotypies absent

 

10

 

6.9

 

10

 

6.8

 

10

 

6.9

 

10

 

6.6

UNUSUAL RESPIRATION

               Unusual respiration absent

 

10

 

6.9

 

10

 

6.8

 

10

 

6.9

 

10

 

6.6

BIZARRE BEHAVIOUR

               Bizarre behaviour absent

 

10

 

6.9

 

10

 

6.8

 

10

 

6.9

 

10

 

6.6

URINATION

               Urination absent

               Urination 1-3

               Urination 4-6

               Urination 7-9

               Urination more than 10

 

10

7

3

1

1

 

5.1

1.7

1.7

1.0

1.0

 

10

6

3

0

0

 

5.6

1.5

1.0

0.0

0.0

 

10

5

5

2

0

 

5.2

1.6

1.4

1.0

0.0

 

10

9

4

0

0

 

4.0

2.1

1.8

0.0

0.0

DEFECATION

               Defecation absent

               Defecation 1-3

 

10

2

 

6.7

1.0

 

10

3

 

6.5

1.0

 

10

1

 

6.8

1.0

 

10

3

 

6.3

1.0

TREMORS

               Tremors absent

 

10

 

6.9

 

10

 

6.8

 

10

 

6.9

 

10

 

6.6

Key:        () = Number of animals alive at start of interval

               a = Number of animals affected

               b = Number of weeks with clinical sign/animal

Note:      ! = Pretest phase; “ = Dosing phase

 

BODY WEIGHT (g) OF MALES – BEFORE PAIRING – MAIN GROUPS – GROUP MEAN DATA

Group(s)

 

Day of Phase

1!

1”

8

15#

1

(n)

Mean

SD

10

291.27

8.04

10

351.93

14.40

10

391.82

19.31

10

418.66

24.10

2

(n)

Mean

SD

10

290.91

6.82

10

343.76

11.97

10

375.38

16.31

10

400.39

20.38

3

(n)

Mean

SD

10

290.21

8.48

10

344.05

15.02

10

378.62

15.50

10

404.02

17.39

4

(n)

Mean

SD

10

291.32

7.25

10

351.16

12.67

10

387.61

16.39

10

416.80

17.10

Note:      ! = Pretest phase; “ = Premating phase; # = Start of pairing phase

* = mean value of group is significantly different from control at p<0.05

** = mean value of group is significantly different from control at p<0.01

Statistical analysis:Dunnett’s test if group variances are homogenous

                                               Modified t test is group variances are inhomogenous ($)

 

BODY WEIGHT (g) OF MALES – FROM PAIRING PERIOD TO SACRIFICE – MAIN GROUPS – GROUP MEAN DATA

Group(s)

 

Day of Phase

8

15

22

29

1

(n)

Mean

SD

10

440.52

28.74

10

463.91

35.21

10

484.62

32.37

10

491.88

43.22

2

(n)

Mean

SD

10

424.16

21.21

10

445.27

27.57

10

467.04

31.46

10

474.21

35.30

3

(n)

Mean

SD

10

424.73

19.19

10

449.58

19.40

10

470.60

23.64

10

479.52

34.21

4

(n)

Mean

SD

10

436.14

16.77

10

463.39

22.11

10

484.57

21.84

10

492.04

28.45

Note:      Data for Mating phase

* = mean value of group is significantly different from control at p<0.05

** = mean value of group is significantly different from control at p<0.01

Statistical analysis:Dunnett’s test if group variances are homogenous

                                               Modified t test is group variances are inhomogenous ($)

 

BODY WEIGHT (g) OF FEMALES – BEFORE PAIRING – MAIN GROUPS – GROUP MEAN DATA

Group(s)

 

Day of Phase

1!

1”

8

15#

1

(n)

Mean

SD

10

214.83

8.35

10

236.18

5.69

10

242.60

6.15

10

2.53.35

12.29

2

(n)

Mean

SD

10

215.15

9.37

10

234.15

8.25

10

239.03

15.65

10

253.72

14.16

3

(n)

Mean

SD

10

214.46

8.79

10

235.66

8.48

10

244.87

12.84

10

256.33

12.30

4

(n)

Mean

SD

10

214.42

8.98

10

231.99

9.54

10

243.20

9.19

10

254.36

12.53

Note:      ! = Pretest phase; “ = Premating phase; # = Start of pairing phase

* = mean value of group is significantly different from control at p<0.05

** = mean value of group is significantly different from control at p<0.01

Statistical analysis:Dunnett’s test if group variances are homogenous

                                               Modified t test is group variances are inhomogenous ($)

 

BODY WEIGHT (g) OF PREGNANT FEMALES –POST COITUMANDPOST PARTUMPERIODS – MAIN GROUPS – GROUP MEAN DATA

Group(s)

 

Day of Phase

 

0!

7

14

20

1”

4

1

(n)

Mean

SD

10

262.25

21.09

10

294.87

15.77

10

334.16

15.23

10

424.81

25.24

10

314.68

19.39

10

315.23

35.00

2

(n)

Mean

SD

10

256.72

13.40

10

294.21

15.86

10

332.61

19.14

10

414.23

33.26

10

316.05

20.48

10

308.52

20.76

3

(n)

Mean

SD

8

258.81

16.48

8

297.51

16.70

8

336.34

20.84

8

429.21

31.50

8

321.90

24.58

8

310.40

31.87

4

(n)

Mean

SD

9

356.60

6.76

9

288.48

9.48

9

323.19

8.28

9

402.31

12.52

9

311.44

9.82

9

300.98

20.89

Note:      ! = Gestation phase; “ =Post partumphase

* = mean value of group is significantly different from control at p<0.05

** = mean value of group is significantly different from control at p<0.01

Statistical analysis:Dunnett’s test if group variances are homogenous

                                               Modified t test if group variances are inhomogenous ($)

 

MACROSCOPIC OBSERVATIONS – MAIN GROUP – UNSCHEDULED DEATH – GROUP INCIDENCE

 

Group:

Number in group:

--Females--

4

1

Kidneys

               Pelvic dilation

 

1

Forelimbs

               Abnormal shape

               Abnormal consistency

 

1

1

Hindlimbs

               Abnormal shape

               Abnormal consistency

 

1

1

 

MACROSCOPIC OBSERVATIONS – MAIN GROUPS – FINAL SACRIFICE – GROUP INCIDENCE

 

Group:

Number in group:

--Males--

--Females--

1

10

2

10

3

10

4

10

1

10

2

10

3

10

4

9

Adrenals

               Abnormal size

 

1

 

0

 

0

 

0

 

0

 

0

 

1

 

0

Cervical nodes

               Abnormal colour

 

0

 

0

 

1

 

0

 

0

 

0

 

0

 

0

Kidneys

               Abnormal area (s)

               Pelvic dilation

 

0

0

 

0

1

 

1

3

 

0

3

 

0

0

 

0

0

 

0

0

 

0

0

Liver

               Abnormal shape

               Abnormal size

 

0

1

 

2

0

 

0

0

 

1

0

 

0

2

 

0

0

 

0

0

 

0

0

Ovaries

               Abnormal size

 

 

 

 

 

1

 

0

 

0

 

0

Thymus

               Abnormal area (s)

               Abnormal colour

               Abnormal size

 

0

0

0

 

1

0

0

 

0

1

0

 

1

0

0

 

0

0

0

 

0

0

1

 

0

0

0

 

0

0

2

Uterus

               Not pregnant

               Unilateral implantation

 

 

 

 

 

0

0

 

0

1

 

2

0

 

0

0

Ears

               Abnormal area (s)

 

1

 

0

 

0

 

0

 

0

 

0

 

0

 

0

Whole animal

               No abnormalities detected

 

8

 

6

 

6

 

5

 

7

 

8

 

7

 

7

 

MICROSCOPIC OBSERVATIONS – UNSCHEDULED DEATH – GROUP INCIDENCE

 

Controls from group (s): 1

 

Tissues With Diagnoses

 

Animal Sex:

Dosage group:

No. in group:

--Animals Affected--

--Females--

4

1

Kidneys

               HYDRONEPHROSIS

Number examined:

1

1

Lungs

               EMPHYSEMA

               INFLAMMATORY CELL FOCI

Number examined:

1

1

1

Forelimbs

               ARTHRITIS

Number examined:

1

1

Hindlimbs

               ARTHRITIS

Number examined:

1

1

 

MICROSCOPIC OBSERVATIONS – FINAL SACRIFICE – GROUP INCIDENCE

 

Controls from group (s): 1

 

Tissues With Diagnoses

 

--Animals Affected--

Animal Sex:

--Males--

--Females--

Dosage group:

No. in group:

Ctls

10

2

10

3

10

4

10

Ctls

10

2

10

3

10

4

10

Adrenals

               ACCESSORY NODULE

               CORTICAL CELL VACUOLIZATION

               CYTS/S

Number examined:

5

1

1

0

0

0

0

0

0

0

0

0

5

0

1

0

5

0

0

0

0

0

0

0

1

0

0

0

5

0

0

1

Cervical nodes

               CONGESTION

Number examined:

5

0

0

0

1

1

5

0

5

0

0

0

0

0

5

0

Heart

               INFLAMMATORY CELL FOCI

Number examined:

5

4

0

0

0

0

5

2

5

0

0

0

0

0

5

2

Kidneys

               INFLAMMATORY CELL FOCI

               NEPHROPATHY

               HYDRONEPHROSIS

Number examined:

5

1

0

0

1

0

0

1

4

1

2

1

5

0

1

2

5

0

1

0

0

0

0

0

0

0

0

0

5

0

0

0

Liver

               INFLAMMATORY CELL FOCI

               HEPATOCYTIC VACUOLATION

               CLEAR CELL CHANGE

               EXTRAMEDULLARY HAEMOPOIESIS

Number examined:

6

4

1

1

0

2

1

0

2

0

0

0

0

0

0

6

4

0

1

0

7

0

0

2

2

0

0

0

0

0

0

0

0

0

0

5

1

0

0

0

Lungs

               AGGREGATION OF ALVEOLAR MACROPHAGES

Number examined:

5

1

0

0

0

0

5

2

5

4

0

0

0

0

5

4

Prostate

               INFLAMMATORY CELL FOCI

Number examined:

5

1

0

0

0

0

5

2

 

 

 

 

Testes

               TUBULAR CELL DEGENERATION

Number examined:

5

1

0

0

0

0

5

0

 

 

 

 

Thymus

               ATROPHY

               CONGESTION

Number examined

5

0

0

1

0

0

1

1

1

5

0

0

5

0

0

1

1

0

0

0

0

6

1

0

Thyroid

               ECTOPIC THYMIC TISSUE

Number examined:

5

1

0

0

0

0

5

2

5

0

0

0

0

0

5

0

Ears

               CHRONIC INFLAMMATION

Number examined:

1

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Note: Entries flagged with a – (minus) are significantly higher than control at the 0.05 level using the Kolmogorov-Smirnov one-tailed test.

Conclusions:
Based on the results of the present study, the NOEL (No Observed Effect Level) for general toxicity, reproductive and developmental toxicity was considered to be 1000 mg/kg bw/day for males and females.
Executive summary:

Repeated dose oral toxicity study performed in male & female Sprague-Dawley rats in accordance with OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test).

 

Male animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 43 and 44 of study). They were treated for a total of 42 or 43 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

 

Females 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 approximately 42 days). Dose volumes were adjusted once per week for each animal according to the last recorded bodyweight. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.

 

Recovery group animals were dosed once a day, 7 days a week, for a minimum of 6 consecutive weeks. No treatment was given during the recovery period.

 

Each main group comprised 10 male and 10 female rats. Two groups (control and high dose levels) included 5 animals per sex which were sacrificed after 2 weeks of recovery.

 

One female of the high dose group, receiving 1000 mg/kg bodyweight/day, was killed for humane reasons on Day 3 of the gestation phase.

Marked arthritis of hind limbs and one forelimb, observed at histopathology, could be considered as a factor contributory to the illness status of the animal sacrificed for humane reasons. Therefore, this death was considered to be unrelated to treatment.

 

No clinical signs of toxicological relevance or signs of neurotoxicity (weekly observations at removal from the cage and in an open arena, alterations in motor activity, grip strength and sensory reaction to stimuli) were observed during the study in males and females. Slight orange staining in the cage tray was observed during the mating phase in some cages of mid- and high dose animals.

 

No differences in body weight and food consumption were observed in treated animals compared to the control group.

 

No changes of toxicological significance were recorded at haematological, clinical chemistry investigations or at coagulation tests in animals sacrificed at the end of the study.

 

No relevant changes were detected at post mortem examination (terminal body weight, organ weights, macroscopic and microscopic examinations) in treated animals, when compared with controls.

 

Based on the results of the present study, the NOEL (No Observed Effect Level) for general toxicity was considered to be 1000 mg/kg bw/day for males and females.

Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
27 May 2015 to 10 March 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to same study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
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 because there is ample experience
and background data on this species and strain.
Sex:
male
Details on test animals or test system and environmental conditions:
A total of 125 Sprague Dawley [Crl:CD(SD)BR] rats (65 males and 60 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females, were ordered from and supplied by Charles River Italia S.p.A., Calco (Lecco), Italy.After arrival, on 21 May 2015, the weight range for each sex was determined (189-216 g for females, 226-250 g for males, slightly outside the range at order) and the animals were temporarily identified within the cage by means of a coloured mark on the tail.A health check was then performed by a veterinarian. An acclimatisation period of at least 14 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C +/- 2°C and 55% +/- 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
Route of administration:
oral: gavage
Vehicle:
The vehicle for the test item was sesame oil.
The vehicle for the positive control item was sterile water for injection.
Test item
The required amount of Vat Black 25 was dissolved/suspended in the vehicle. The formulations were prepared daily (concentrations of 12,5, 50 and 200mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.
Positive Control item
The required amount of positive control item was dissolved in the vehicle. The formulation was prepared on the day of dosing at a concentration of0.2 mg/mL.
Details on exposure:
The test item was administered orally by gavage at a dose volume of 5 mL/kg body weight. Control animals received the vehicle alone at the same dose volume. The 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.
Analysis was not performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory as the test item is neither extractable in hydrophilic nor in lipophilic solvents. The correct concentration preparation was monitored in the weighing record of the test item in each formulation process.
Positive Control
Animals received a single dose approximately 24 hours before sacrifice. Mitomycin-C (positive control) was administered once by intraperitoneal injection at the dose volume of 10 mL/kg body weight. 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.
Duration of treatment / exposure:
Animals were dosed once a day, 7 days a week, for a minimum of 2 consecutive weeks prior to pairing, through the mating period and thereafter through the day before necropsy (Days 43 and 44 of study). They were treated for a total of 42 or 43 days. Dose volumes were adjusted once per week for each animal according to the last recorded body weight.
Frequency of treatment:
Once a day
Dose / conc.:
62.5 mg/kg bw/day
Dose / conc.:
250 mg/kg bw/day
Dose / conc.:
1 000 mg/kg bw/day
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 which were sacrificed after 2 weeks of recovery (Groups 5 and 6). For genotoxicity endpoint, a satellite control group (Positive Control group) comprised 5 male rats (Group 7).
Control animals:
yes, concurrent vehicle
Positive control(s):
For genotoxicity endpoint, a satellite control group (Positive Control group) comprised 5 male rats (Group 7).The required amount of positive control item was dissolved in the vehicle (sterile water for injection). The formulation was prepared on the day of dosing at a concentration of 0.2 mg/mL. No documents on Mitomycin-C are included in the report. Determination of the stability and concentration of solutions of the positive control item was not undertaken.Mitomycin-C (positive control) was administered once by intraperitoneal injection at the dose volume of 10 mL/kg body weight. 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.Positive Control group animals were killed under carbon dioxide asphyxiation.
Tissues and cell types examined:
Bone marrow from one femur of males only.
Details of tissue and slide preparation:
Extraction of bone marrow
Samples of bone marrow were collected approximately 24 and 48 hours after the 2 final treatments, from the same 5 males of the main groups randomly selected for clinical pathology investigation (see section 4.4). Samples of bone marrow were also collected approximately 24 hours after the single treatment from all males of Group 7 (Positive Control group). One femur of each animal was rapidly dissected out and cleaned of surrounding tissue. In order to extract the bone marrow, the bone was cut at the proximal end and irrigated with foetal calf serum using a syringe. The suspension of cells was aspirated, and this procedure was repeated several times.Preparation of the smearsThe suspension thus obtained was centrifuged at 1000 rpm for at least 5 minutes and the supernatant was completely removed. The cells of the sediment were resuspended and transferred onto clean microscope slides as smear preparations. They were air-dried and then fixed with methanol for 10 minutes. Subsequently, slides were stained with haematoxylin and eosin solutions. Finally, the slides were rinsed in distilled water and allowed to dry.Scoring of the slides and data analysisFor each animal, at least three slides were prepared. These slides were randomised and coded by staff not subsequently involved in the scoring.The adequate quality and the sufficient number of cells were evaluated before scoring. Scoring was performed using a microscope and high-power objective. Immature polychromatic erythrocytes (PCEs) stain a pink-purple colour (since they retain basic ribosomal material for approximately 24 hours after enucleation), and can be distinguished from the pink normochromatic erythrocytes (NCE).Erythrocytes lack nuclei, making micronuclei obvious when present; the criteria of Schmid (1976) were used to score micronuclei. At least four thousand polychromatic erythrocytes per animal were scored for the presence of micronuclei. At the same time the number of normochromatic erythrocytes was recorded, as well as the number of micronucleated NCE. The proportion of immature erythrocytes among total erythrocytes gives an indication of the toxicity of the treatment; a reduction in the proportion indicates inhibition of cell division. Finally, the incidence of micronucleated PCE provides an index of induced genetic damage.
Evaluation criteria:
Acceptance criteria
The assay was considered valid if the following criteria were met:
– The incidence of micronucleated PCEs of the vehicle control group fell within the historical negative control range.
– The positive control item results fell within the historical control range and were significantly increased, at statistical analysis, when comparedwith the concurrent negative control.
– 5 males per group were available for slide analysisEvaluation of results
The test item was considered to induce micronuclei if a statistically significant increase in the micronucleus incidence of polychromatic erythrocytes(at p< 0.05) was observed in any treatment group and a dose-effect relationship was demonstrated. Where statistically significant increases in the incidence of micronucleated PCEs were observed, but all results were inside the distribution of negative control values within this laboratory, then historical control data were used to demonstrate that these increases did not have any biological significance.
Statistics:
Only counts from polychromatic cells were subjected to statistical analysis. Using the original observations (and not the micronucleus frequencies per 1000 cells), a modified chi-squared calculation was employed to compare treated and control groups. The degree of heterogeneity within each group was first calculated and where significant it was taken into account in the comparison between groups. If there was no significant within-group heterogeneity, the chi-squared test was used to compare treated groups with the controls. If there was significant within-groups heterogeneity, then that group was compared with the controls using a variance ratio (F) value calculated from the between-group and within-group chi-squared values.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Incidence of micronucleated cells
Following treatment with the test item, a slight increase in the number of micronucleated PCEs over the concurrent negative control was observed for animals from the low and high dose groups; however, the incidences of micronucleated PCEs were comparable to RTC historical control data for negative control animals. A marked increase in the frequency of micronucleated PCEs was observed in the positive control group.Bone marrow cell toxicityThe ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no relevant inhibitory effect on erythropoietic cell division was observed at any dose level.
Analysis of resultsFollowing treatment with the test item, statistically significant increases in the incidence of micronucleated PCEs over the negative control value were observed for animals from the low and high dose levels (p<0.01). A significant dose effect relationship was found after a trend test evaluation (p<0.05). The dose-related and statistically significant increases of micronucleated PCEs could be attributable to the low incidence of micronucleated cells in the vehicle control group, which fell in the lower part of the distribution range of historical control data. In addition, the frequency of micronucleated immature erythrocytes observed at all dose levels was inside the distribution of historical negative control data (95% upper confidence limit = 2.0), therefore the observed increases were not considered biologically meaningful.

 

Dose level

(mg/kg/day)

Incidence in micronucleated PCEs

PCE/s(PCEs+NCEs) %

Over the mean control value

Mean

SE

Range

0.00

62.5

250

1000

Mitomycin-C 2.00 mg/kg

0.2

0.9

0.3

0.9

8.2

0.1

0.3

0.1

0.2

1.2

0.0 – 0.5

0.3 – 1.8

0.0 – 0.5

0.5 – 1.5

5.5 – 12.5

100

101

97

96

97

 

SUMMARY OF INCIDENCE OF MICRONUCLEATED CELLS

INCIDENCE OF MICRONUCLEATED CELLS/1000 CELLS

Dose level

mg/kg/day

Scored cells

NCE/PCE Ratio

PCE/(PCE+NCE) Ratio

% over the Mean control value

Polychromatic

Normochromatic

PCE

NCE

Mean

SE

Min

Max

Mean

SE

Min

Max

0.00

62.5

250

1000

20000

20000

20000

20000

22374

21840

23814

24165

1.12

1.09

1.19

1.21

0.47

0.48

0.46

0.45

100

101

97

96

0.2

0.9

0.3

0.9

0.1

0.3

0.1

0.2

0.0

0.2

0.0

0.5

0.5

1.8

0.5

1.5

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.0

0.2

0.0

0.2

Mitomycin C 2.00

20000

23531

1.18

0.46

97

8.2

1.2

5.5

12.5

0.0

0.0

0.0

0.0

NCE/PCE ratio: The ratio of NCE/PCE calculated as the mean of the ratio values for the individual animals.

PCE/(PCE+NCE) ratio: The ratio of PCE/(PCE+NCE) calculated as the ratio of the total PCEs over the total erythrocytes scored

% over the mean control: Percentage of the PCE/(PCE+NCE) ratio of each treated group value over the negative control value

MEAN:    The group mean incidence of micronucleated PCEs and NCEs

SE:          The standard error of the mean incidence

MIN:       Minimum value observed in an individual animal

MAX:      Maximum value observed in an individual animal

 

HISTORICAL CONTROL DATA

INCIDENCES OF MICRONUCLEATED PCEs (%)

(1991 – 2015)

Male animals

 

Vehicle controls

Positive controls

Mean

0.7

16.4

SD (σn-1)

0.70

8.41

n

165

164

Upper confidence limit (95%)

2.0

NC

Maximum

3.5

47.5

Minimum

0.0

0.5

SD = standard deviation

n = number of animals

NC = not calculated

Conclusions:
Interpretation of results (migrated information): negative
On the basis of the results obtained, it is concluded that Vat Black 25 does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.
Executive summary:

The purpose of this study was to provide information on toxic effects on male and female rats after repeated dosing with the test item, the micronucleus test was included 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.

 

Experimental procedures were based on the following guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

 

Incidence of micro nucleated cells

Following treatment with the test item, a slight increase in the number of micro nucleated PCEs over the concurrent negative control was observed for animals from the low and high dose groups; however, the incidences of micro nucleated PCEs were comparable to RTC historical control data for negative control animals.

A marked increase in the frequency of micro nucleated PCEs was observed in the positive control group.

 

Bone marrow cell toxicity

The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes among total erythrocytes were analysed to evaluate the bone marrow cell toxicity. Based on these results, no relevant inhibitory effect on erythropoietic cell division was observed at any dose level.

 

Analysis of results

Following treatment with the test item, statistically significant increases in the incidence of micro nucleated PCEs over the negative control value were observed for animals from the low and high dose levels (p<0.01). A significant dose effect relationship was found after a trend test evaluation (p<0.05). The dose-related and statistically significant increases of micro nucleated PCEs could be attributable to the low incidence of micro nucleated cells in the vehicle control group, which fell in the lower part of the distribution range of historical control data. In addition, the frequency of micro nucleated immature erythrocytes observed at all dose levels was inside the distribution of historical negative control data (95% upper confidence limit = 2.0), therefore the observed increases were not considered biologically meaningful.

 

Conclusions

On the basis of the results obtained, it is concluded that Vat Black 25 does not induce micronuclei in the polychromatic erythrocytes of treated rats, under the reported experimental conditions.

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:
not specified
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reaction product of 3,9-dibromobenzanthrone condensed with 2 equivalents of 1-aminoanthraquinone, subsequently further condensed under oxidative conditions
EC Number:
944-232-9
Molecular formula:
Not available - UVCB substance
IUPAC Name:
Reaction product of 3,9-dibromobenzanthrone condensed with 2 equivalents of 1-aminoanthraquinone, subsequently further condensed under oxidative conditions
Test material form:
solid: particulate/powder

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 because 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:
A total of 125 Sprague Dawley [Crl:CD(SD)BR] rats (65 males and 60 virgin females), 7 to 8 weeks old and weighing 200 to 225 g for males and 175 to 200 g for females, were ordered from and supplied by Charles River Italia S.p.A., Calco (Lecco), Italy.After arrival, on 21 May 2015, the weight range for each sex was determined (189-216 g for females, 226-250 g for males, slightly outside the range at order) and the animals were temporarily identified within the cage by means of a coloured mark on the tail.A health check was then performed by a veterinarian. An acclimatisation period of at least 14 days was allowed before the start of treatment, during which time the health status of the animals was assessed by thorough observations.The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C +/- 2°C and 55% +/- 15% respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: Sesame oil
Details on exposure:
The required amount of Vat Black 25 was dissolved/suspended in the vehicle. 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.
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. 5). The subsequent pairing was monitored for mating as described above.
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
Analysis was not performed to confirm that the proposed formulation procedure was acceptable and that the stability of the formulation was satisfactory as the test item is neither extractable in hydrophilic nor in lipophilic solvents. The correct concentration preparation 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 through the day before necropsy (Days 43 and 44 of study). They were treated for a total of 42 or 43 days. Dose volumes were adjusted once per week for each animalaccording 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 approximately 42 days). Dose volumes were adjusted once per week for each animal according to the last recorded bodyweight. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Day 1 post partum. Thereafter individual dose volumes remained constant.

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

Positive Control group (Group 7)
Animals received a single dose approximately 24 hours before sacrifice.
Frequency of treatment:
Once daily
Details on study schedule:
Dose levels (0, 62.5, 250 and 1000 mg/kg body weight/day) were selected in consultation with the Sponsor based on information from previous studies.
Doses / concentrationsopen allclose all
Dose / conc.:
62.5 mg/kg bw/day (nominal)
Dose / conc.:
250 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
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 which were sacrificed after 2 weeks of recovery (Groups 5 and 6). For genotoxicity endpoint, a satellite control group (Positive Control group) comprised 5 male rats (Group 7).
Control animals:
yes, concurrent no treatment
Details on study design:
The test item was administered orally by gavage. The oral route was selected as it is a possible route of exposure of the test item in man.
Dose levels of 62.5, 250 and 1000 mg/kg bodyweight/day were selected by the Sponsor based on information from previous studies.
Positive control:
For genotoxicity endpoint, a satellite control group (Positive Control group) comprised 5 male rats (Group 7).

Examinations

Parental animals: Observations and examinations:
Throughout the study, all animals were checked early in each working day in the morning and in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

Clinical signs
Main and Recovery groups:
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded.Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions (15-45 minutes after treatment).

Observations of the cage tray:
Observations of the cage tray were performed during mating period for all main groups and were recorded daily.

Positive Control group:
Once before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded.

Neurotoxicity assessment (removal of animals from the home cage and open arena)
Once before commencement of treatment and at least once per 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 (for at least 3 minutes). The test 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, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded. Animals were examined in an open arena for a minimum of 3 minutes.

Grip strength and sensory reaction to stimuli
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group for evaluation of sensory reaction to stimuli of different modalities (e.g. auditory, visual and proprioceptive stimuli); an assessment of grip strength was also performed. Measurements were performed using a computer generated random order (for the main groups). For males (main groups), the tests were performed on Day 40 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 41 of the study (during treatment) and once during Week 2 of recovery (Day 10).

Motor activity assessment (MA)
Once during the study, towards the end of treatment, 5 males and 5 females were randomly selected from each group and the motor activity was measured (for approximately 5 minutes) by an automated activity recording device. Measurements were performed using a computer generated random order (for the main groups). For males (main groups) the tests were performed on Day 41 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 42 of the study (during treatment) and once during Week 2 of recovery (Day 13).

Body weight
Main groups: Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1 and 4 post partum.
Recovery groups: Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy.
Positive Control group: Animals were weighed on the day of allocation and on the day of dosing.

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.

Clinical pathology investigations
During the necropsy procedure, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava from 5 males and 5 females (females with viable litters) randomly selected from each group under conditions of food deprivation for haematological, coagulation and clinical chemistry evaluations.
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
Oestrous cyclicity (parental animals):
Vaginal smears were taken daily in the morning starting from two weeks before pairing until a positive identification of copulation was made. The vaginal smear data were examined to determine the following:1. anomalies of the oestrous cycle2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Sperm parameters (parental animals):
Parameters examined in [all/PF1/F2] male parental generations:Testis weight, epididymis weight, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS).The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was also performed.
Litter observations:
A parturition check was performed from Day 20 to Day 25 post coitum. Female nos. 47 and 53 (Group 3) which did not give birth after 25 days of post coitum period were sacrificed shortly after (Day 26 post coitum). These animals were found not pregnant at necropsy. In addition, female no. 69 (Group 4) was sacrificed for humane reasons on Day 3 post coitum and it was not possible to evaluate pregnancy.
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 killed or dying during the lactation period were weighed before the despatch to necropsy. Observations were performed once daily for all litters.
Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day of commencement of birth (i.e. first detected presence of offspring in the cage). The day that offspring were first detected in the cage was considered Day 0 post partum.
Postmortem examinations (parental animals):
Main and Recovery groups
One animal, sacrificed for humane reasons, was killed under carbon dioxide asphyxiation. Animals that had completed the scheduled test period and selected for blood collection, were killed by exsanguination under isofluorane anaesthesia. Animals that had completed the scheduled test period and not selected for blood collection, were killed by carbon dioxide asphyxiation.
Positive Control group
Positive Control group animals were killed under carbon dioxide asphyxiation.
Parental males (Main groups)
The males were killed after the mating of all females or 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 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.

Positive Control group
Animals were killed approximately 24 hours after treatment.

Necropsy (Main and Recovery groups)
The clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed (excluding the animal sacrificed for humane reasons) and the required tissue samples preserved in fixative and processed for histopathological examination.

Females (Main groups)
All females were also examined for the following:
– external and internal abnormalities;
– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals).

Uteri of apparently non-pregnant females or uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

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

Tissues fixed and preserved (Main and Recovery groups)
Samples of all the tissues listed below in were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).Tissues fixed and preserved (Main and Recovery groups)Samples of all the tissues listed below in were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves and Harderian glands, testes and epididymides which were fixed in modified Davidson’s fluid and preserved in 70% ethyl alcohol).

Abnormalities
Adrenal glands
Bone marrow (from sternum)
Brain (cerebrum, cerebellum, medulla/pons)
Caecum
Clitoral gland
Colon
Duodenum
Epididymides
Heart
Ileum (including Peyer’s patches)
Jejunum
Kidneys
Liver
Lungs (including mainstem bronchi)
Lymph nodes – cervical
Lymph nodes – mesenteric
Nasal cavity
*Oesophagus
*Ovaries with 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 including cervix
Vagina

*not examined as no signs of toxicity or target organ involvement were observed
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin. In addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was also performed.
The examination was restricted as detailed below:
i Tissues specified above from 5 males and 5 females (main groups) randomly selected (animals evaluated for clinical pathology) inthe control and high dose group killed at term.
ii Tissues specified above from all animals killed or dying during the treatment period.
iii All abnormalities in all main groups.
Postmortem examinations (offspring):
Pups that had completed the scheduled test period (Day 4 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal.
All pups found dead in the cage were examined for external and internal abnormalities. All live pups sacrificed at termination were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
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. The nonparametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups was assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p < 0.05.
Reproductive indices:
Group mean values were calculated for all parameters. Data from nonpregnant females were excluded from group mean calculations.
Males
Copulatory Index (%) = no: of mated x100/ no: of males paired
Fertility Index (%) = no: of males which induced pregnancy x 100 / no: of paired
Females
Copulatory Index (%) = no: of mated x 100 / no: of paired
Fertility Index (%) = no: of pregnant females x 100 / no: of females paired
Offspring viability indices:
Males and females
Copulatory Interval (Mean time to mating) = Mean number of days between pairing and mating
Females
Pre-birth loss (post-implantation loss) was calculated as a percentage from the formula:no: of visible implantations - total litter size at birth/ no: of visible implantations x 100
Pre-implantation loss was calculated as a percentage from the formula:no: of corpora lutea - no: of visible implantations/no: of corpora lutea x 100
Pup loss at birth was calculated as a percentage from the formula:Total litter size - live litter size/ Total litter size x 100
Cumulative pup loss on Day 4 post partum was calculated as a percentage from the formula:Total litter size at birth - live litter size at Day 4/ Total litter size at birth x 100
Sex ratios were calculated at birth and on Day 4 post partum and were presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs of toxicological relevance or signs of neurotoxicity (weekly observations at removal from the cage and in an open arena, alterations in motor activity, grip strength and sensory reaction to stimuli) were observed during the study in males and females. Slight orange staining in the cage tray was observed during the mating phase in some cages of mid- and high dose animals.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One female (animal no. 69) of the high dose group, receiving 1000 mg/kg bodyweight/day was killed for humane reasons on Day 3 of the gestation phase. On the day of sacrifice, hunched posture and marked swelling of the fore and hind limbs were recorded. Macroscopic findings observed at post mortem examination in this animal were represented by swollen and oedematous consistency of hindlimbs and right forelimb, as well as unilateral pelvic dilatation.Marked arthritis of hindlimbs and one forelimb, observed at histopathology, could be considered as a factor contributory to the illness status of the animal sacrificed for humane reasons.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No differences in body weight were observed in treated animals compared to the control group.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No differences in food consumption were observed in treated animals compared to the control group.
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):
No relevant changes were observed. The statistically significant differences between controls and treated males (mean corpuscular volume, neutrophils and eosinophils percentages), were of minimal severity and/or not dose-related, therefore considered of no toxicological significance.

Coagulation: No changes were recorded.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No relevant changes were observed. The statistically significant differences recorded (urea, glucose, chloride, phosphorus in males) were not dose-related,
therefore considered unrelated to treatment.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Main and recovery groups
Observation of animals at removal from the cage and in an open arena did not reveal changes attributable to the test item.
No alterations in motor activity, grip strength and sensory reaction to stimuli were observed in any treatment group at the examination performed at the end of treatment.
At the end of recovery period, motor activity, grip strength and sensory reaction to stimuli were comparable between the control and treated group in both sexes.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No microscopic observation was observed in treated animals that could be considered treatment-related.
A limited number of lesions, reported in control and/or treated animals, were considered to be an expression of spontaneous and/or incidental pathology, seen in this species and age of untreated animals.
In addition seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
No microscopic observation was observed in treated animals that could be considered treatment-related.
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Concerning reproductive parameters, no treatment-related anomalies were noted in the oestrus cycle of the treated females when compared to controls. Oestrus cycle did not show intergroup differences.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and no alterations were noted.
Reproductive performance:
no effects observed
Description (incidence and severity):
Concerning reproductive parameters, no treatment-related anomalies were noted in the oestrus cycle of the treated females when compared to controls. Oestrus cycle, pre-coital intervals, copulatory index and fertility index did not show intergroup differences.
All females mated. However, 2 females in the mid-dose group (nos. 47 and 53) were found not pregnant. One male of the control group (no. 6) did not mate.
The copulatory index was 100% for females of all dose groups and males of Groups 2, 3 and 4, while it was 90% for control males. The fertility indices were 80% in Group 3 and 100% in Groups and 1, 2, and 4 for both sexes.

Details on results (P0)

Gestation periods were similar in treated groups and controls. All dams gave birth between Days 22 and 23 post coitum. Corpora lutea, implantations and pre-implantation loss, total litter size and pre-birth loss (percentage) did not show dose-related or treatment-related differences. No significant differences in total litter size, live litter size, mean pup loss, sex ratio and mean pup weights were observed among the surviving treated dams and the controls.

Effect levels (P0)

Key result
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Other: No effects of toxicological significance at highest dose tested: 1000 mg/kg bw/day

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Cold to touch, pale aspect, apparently no food intake (milk) and small appearance were the main clinical signs noted in control and treated pups.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
Found dead pups were observed both in control and treated groups, without dose relation.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No significant differences in mean pup weights.
Food consumption and compound intake (if feeding study):
not examined
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:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy findings in deceased pups and in pups sacrificed on Day 4 post partum did not reveal any treatment-related effect.
No milk in the stomach and autolysed abdominal/thoracic organs were generally observed in pups which died during the lactation period.
Histopathological findings:
not examined
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

No significant differences in total litter size, live litter size, mean pup loss, sex ratio and mean pup weights.

Effect levels (F1)

Key result
Dose descriptor:
NOEL
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No effects of toxicological significance at highest dose tested: 1000 mg/kg bw/day

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no
Lowest effective dose / conc.:
1 000 mg/kg bw/day (nominal)
Treatment related:
no
Relation to other toxic effects:
not specified
Dose response relationship:
no
Relevant for humans:
no

Any other information on results incl. tables

OESTRUS CYCLE – BEFORE PAIRING – GROUP SUMMARY DATA

Animal Number

Group 1

Oestrus Cycles

Animal Number

Group 2

Oestrus Cycles

Animal Number

Group 3

Oestrus Cycles

Animal Number

Group 4

Oestrus Cycles

X0160001

X0160003

X0160005

X0160007

X0160009

X0160011

X0160013

X0160015

X0160017

X0150019

4

3

1

4

4

3

3

3

3

3

X0160021

X0160023

X0160025

X0160027

X0160029

X0160031

X0160033

X0160035

X0160037

X0160029

4

3

2

3

3

3

2

2

2

4

X0160041

X0160043

X0160045

X0160047

X0160049

X0160051

X0160053

X0160055

X0160057

X0160059

4

3

4

3

4

3

2

4

4

3

X0160061

X0160063

X0160065

X0160067

X0160069

X0160071

X0160073

X0160075

X0160077

X0160079

4

4

4

4

4

4

3

3

4

4

Means

3.1

 

2.8

 

3.4

 

3.8

 

REPRODUCTIVE PARAMETERS OF ANIMALS – SUMMARY

MALES

Group

1

2

3

4

Copulatory Index %

90.0

100.0

100.0

100.0

Fertility Index %

100.0

100.0

80.0

100.0

 

REPRODUCTIVE PARAMETERS OF ANIMALS – SUMMARY

FEMALES

Group

1

2

3

4

Copulatory Index %

100.0

100.0

100.0

100.0

Fertility Index %

100.0

100.0

80.0

100.0

 

IMPLANTATION, PRE-IMPLANTATION LOSS DATA, PRE-BIRTH LOSS DATA AND GESTATION LENGTH OF FEMALES – GROUP MEAN DATA

Group

 

Corpora Lutea

Implantations

Pre-Implantation loss %

Total litter size at birth

Pre-birth loss %

Gestation length (days)

1

Mean

SD

N

18.30

2.87

10

17.40

2.46

10

4.61

4.28

10

16.30

1.70

110

5.74

6.95

0

22.10

0.32

10

2

Mean

SD

N

15.70

4.14

10

15.30

4.32

10

3.70

6.84

10

14.60

4.33

110

4.39

6.64

0

22.20

0.42

10

3

Mean

SD

N

16.38

2.67

8

16.38

2.67

8

0.00

0.00

8

15.88

2.53

8

2.88

4.41

8

22.38

0.52

8

4

Mean

SD

N

15.11

1.17

9

15.11

1.17

9

0.00

0.00

9

14.22

1.20

9

5.71

6.91

9

22.00

0.00

9

Statistical analysis:              Kruskall Wallis test

                                     William’s test if group mean differences are different from control at p<0.05

                                      * = mean value of group is significantly different from control

 

LITTER DATA AT BIRTH, ON DAY 1 AND ON DAY 4POST PARTUMOF FEMALES – GROUP MEAN DATA

Group

 

At birth

On Day 1post partum

On Day 14post partum

Total litter size

Liver litter size

Pup loss (%)

Litter weight (g)

Mean

pup weight

(g)

Liver litter size

Cumulative loss

(%)

Litter weight (g)

Mean

pup weight (g)

1

Mean

SD

N

16.30

1.70

10

16.20

1.81

10

0.67

2.12

10

106.16

18.56

10

6.87

0.70

10

13.40

4.88

10

16.29

29.78

10

129.70

50.81

10

9.20

1.84

10

2

Mean

SD

N

14.60

4.33

10

14.40

4.27

10

1.26

2.66

10

103.16

26.81

10

7.29

0.88

10

14.20

4.10

10

2.41

3.15

10

138.17

33.24

10

10.04

1.35

10

3

Mean

SD

N

15.88

2.53

8

14.13

5.11

8

12.18

27.18

8

97.63

38.48

8

7.24

0.94

8

13.25

5.26

8

17.36

29.71

8

133.74

51.32

8

10.30

1.35

8

4

Mean

SD

N

14.22

1.20

9

14.22

1.20

9

0.00

0.00

9

99.81

10.15

9

7.21

0.61

9

13.44

1.74

9

5.41

9.44

9

132.79

20.00

9

9.88

0.72

9

Statistical analysis: Kruskall Wallis test

                       William’s test if group mean differences are different from control at p<0.05

                        * = mean value of group is significantly different from control

 

SEX RATIO OF PUPS – GROUP MEAN DATA

Group

 

At birth

On Day 4post partum

M

F

Total

%males

M

F

Total

%males

1

Mean

SD

N

7.00

1.70

10

9.30

2.67

10

16.30

1.70

10

43.60

12.14

10

6.00

2.67

10

7.40

3.27

10

13.40

4.88

10

41.28

18.56

10

2

Mean

SD

N

7.70

2.26

10

6.90

3.63

10

14.60

4.33

10

56.81

19.68

10

7.40

2.12

10

6.80

3.61

10

14.20

4.10

10

56.31

19.89

10

3

Mean

SD

N

8.00

1.85

8

7.88

3.64

8

15.88

2.53

8

52.05

15.49

8

6.63

3.07

8

6.63

3.70

8

13.25

5.26

8

50.71

13.38

8

4

Mean

SD

N

7.00

1.50

9

7.22

1.79

9

14.22

1.20

9

49.43

10.92

9

6.67

1.66

9

6.78

1.79

9

13.44

1.74

9

49.68

10.89

9

Statistical analysis: Kruskall Wallis test

                        William’s test if group mean differences are different from control at p<0.05

                       * = mean value of group is significantly different from control

 

CLINICAL SIGNS OF PUPS – INDIVIDUAL DATA

Group 1

Animal Number

Pup number

Sign (Day (s)post partum)

X0160001

All pups

6

1, 2, 5, 7+6, 9, 13, 16, 18

10, 14, 19

3, 4, 8, 11, 12, 15, 17

Cold to touch; Apparently no food intake (1-3); Smaller (3)

Cold to touch; Apparently no food intake; Smaller (4)

Found dead (4)

Found dead (3)

Missing (4)

X0160003

9, 16

6

Smaller than others (1, 4); Cold to touch (1)

Missing (1)

X0160005

7

4

Smaller than others; Apparently no food intake (1); Found dead (2)

Missing (3)

X0160007

All pups

N

X0160009

12

Pale (0-3)

X0160011

15

Found dead (2)

X0160013

#

14

Found dead (0)

Smaller than others (1-4); Apparently no food intake (4)

X0160015

5, 10, 14, 15, 16

13

Found dead (1)

Missing (1)

X0160017

All pups

N

X0160019

9

Smaller than others (4)

# = Pup dead prior to identification

 

CLINICAL SIGNS OF PUPS – INDIVIDUAL DATA

Group 2

Animal Number

Pup number

Sign (Day (s)post partum)

X0160021

9

13

Pale (1-4)

Smaller than others (4)

X0160023

4

Humane Kill (1)

X0160025

All pups

N

X0160027

All pups

N

X0160029

#

Found dead (0)

X0160031

All pups

N

X0160033

15

5

Smaller than others (1-4)

Smaller than others (4)

X0160035

All pups

N

X0160037

#

Found dead (0)

X0160039

All pups

4, 20

21

Smaller (0, 2, 3)

Pale (2-4); Smaller than others (4)

Missing (2)

# = Pup dead prior to identification

 

CLINICAL SIGNS OF PUPS – INDIVIDUAL DATA

Group 3

Animal Number

Pup number

Sign (Day (s)post partum)

X0160041

All pups

N

X0160043

8

14, 17

Smaller than others (1)

Pale (1)

X0160045

15

2

Missing (1)

Missing (2)

X0160049

7

Smaller than others (1-3)

X0160051

17

Smaller than others (0-4)

X0160055

#, #

16

3, 13, 15

10

Found dead (0)

Smaller than others (1)

Found dead (1)

Found dead (2)

X0160057

#, #, #, #, #, #, #, #, #, #, #

#, #

2

Found dead (0)

Cold to touch; Apparently no food intake (0)

Missing (1)

X0160059

#

Found dead (0)

# = Pup dead prior to identification

 

CLINICAL SIGNS OF PUPS – INDIVIDUAL DATA

Group 4

Animal Number

Pup number

Sign (Day (s)post partum)

X0160061

All pups

N

X0160063

10

14

Pale (1)

Pale (1-4); Smaller than others (4)

X0160065

15

4, 13, 14

Smaller than others (0); Missing (1)

Missing (2)

X0160067

All pups

N

X0160071

11

Tail missing (0-4)

X0160073

2

Missing (1)

X0160075

All pups

N

X0160077

All pups

N

X0160079

All pups

N

# = Pup dead prior to identification

 

NECROPSY FINDINGS IN HUMANE KILL PUPS – INDIVIDUAL DATA

Group 2

Animal Number

Day of sacrifice

Sex

Pup number

Description

X0160023

1

M

4

Skin: cannibalised, dorsum

 

NECROPSY FINDINGS IN DECEDENT PUPS – INDIVIDUAL DATA

Group 1

Animal Number

Day found dead

Sex

Pup number

Description

X0160001

3

3

3

4

4

4

4

4

4

4

4

F

F

F

M

M

M

F

F

F

F

F

10

14

19

1

2

5

7+6

9

13

16

18

Abdominal cavity: all organs autolysed

Abdominal cavity: all organs autolysed

Abdominal cavity: all organs autolysed

Abdominal cavity: all organs autolysed

Abdominal cavity: all organs autolysed

Abdominal cavity: all organs autolysed

Abdominal cavity: all organs autolysed

Abdominal cavity: all organs autolysed

Abdominal cavity: all organs autolysed

Abdominal cavity: all organs autolysed

Abdominal cavity: all organs autolysed

X0160005

2

M

7

Abdominal cavity: all organs autolysed

X0160011

2

F

15

N

X0160013

0

M

#

Abdominal cavity: all organs autolysed

No milk in stomach

X0160015

1

1

1

1

1

M

F

F

F

F

5

10

14

15

16

N

N

N

N

N

N = No abnormalities detected

# = Dead prior the identification

 

NECROPSY FINDINGS IN DECEDENT PUPS – INDIVIDUAL DATA

Group 2

Animal Number

Day found dead

Sex

Pup number

Description

X0160029

0

M

#

Abdominal cavity: all organs autolysed

No milk in stomach

X0160037

0

F

#

No milk in stomach

# = Dead prior the identification

 

NECROPSY FINDINGS IN DECEDENT PUPS – INDIVIDUAL DATA

Group 3

Animal Number

Day found dead

Sex

Pup number

Description           

X0160055

0

0

1

 

1

 

1

 

2

F

F

M

 

F

 

F

 

F

#

#

3

 

13

 

15+1

 

10

No milk in stomach

No milk in stomach

Abdominal cavity: all organs autolysed

No milk in stomach

Abdominal cavity: all organs autolysed

No milk in stomach

Abdominal cavity: all organs autolysed

No milk in stomach

N

X0160057

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

0

 

M

 

M

 

M

 

M

 

M

 

M

 

M

 

M

 

F

 

F

 

F

#

 

#

 

#

 

#

 

#

 

#

 

#

 

#

 

#

 

#

 

#

Abdominal cavity: all organs autolysed

No milk in stomach

Abdominal cavity: all organs autolysed

No milk in stomach

Abdominal cavity: all organs autolysed

No milk in stomach

Abdominal cavity: all organs autolysed

No milk in stomach

Abdominal cavity: all organs autolysed

No milk in stomach

Abdominal cavity: all organs autolysed

No milk in stomach

Abdominal cavity: all organs autolysed

No milk in stomach

Abdominal cavity: all organs autolysed

No milk in stomach

Abdominal cavity: all organs autolysed

No milk in stomach

Abdominal cavity: all organs autolysed

No milk in stomach

Abdominal cavity: all organs autolysed

No milk in stomach

X0160059

0

M

#

N

N = No abnormalities detected

# = Dead prior the identification

 

NECROPSY FINDINGS IN DECEDENT PUPS – INDIVIDUAL DATA

Group 4

Animal Number

Day found dead

Sex

Pup number

Description

X0160063

2

F

10

N

N = No abnormalities detected

 

NECROPSY FINDINGS IN PUPS SACRIFICED ON DAY 4POST PARTUM– INDIVIDUAL DATA

Group 1

Animal Number

Sex

Pup Number (s)

Description

X0160001

F

6

No milk in stomach

X0160003

M

F

1, 2, 3, 4, 5

7, 8, 9, 10, 11, 12, 13, 14, 15, 16

N

N

X0160005

M

F

1, 2, 3, 5, 6, 8

9, 10, 11, 12, 13, 14, 15, 16, 17

N

N

X0160007

M

F

1, 2, 4, 5, 6, 7, 8, 9

3, 10, 11, 12, 13, 14

N

N

X0160009

M

F

1, 2, 3, 4, 5, 6

7, 8, 9, 10, 11, 12, 13, 14, 15

N

N

X0160011

M

F

1, 2, 3, 4

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17

N

N

X0160013

M

F

F

1, 2, 3, 4, 5, 6, 7

8, 9, 10, 11, 12, 13

14

N

N

No milk in stomach

X0160015

M

F

1, 2, 3, 4, 6, 7

8, 9, 11, 12

N

N

X0160017

M

F

1, 2, 3, 4, 5, 6, 7, 8, 9

10, 11, 12, 13, 14, 15

N

N

X0150019

M

F

1, 2, 3, 4, 5, 6, 7, 8, 9

10, 11, 12, 13, 14, 15, 16, 17, 18, 19

N

N

N = No abnormalities detected

 

NECROPSY FINDINGS IN PUPS SACRIFICED ON DAY 4POST PARTUM– INDIVIDUAL DATA

Group 2

Animal Number

Sex

Pup Number (s)

Description

X0160021

M

F

1, 2, 3, 4, 5, 6, 7, 8

9, 10, 11, 12, 13

N

N

X0160023

M

F

1, 2, 3, 5, 6, 7, 8, 11

9, 10, 12, 13, 14, 15

N

N

X0160025

M

F

1, 2, 3, 4, 5, 6, 7, 8, 9, 10

11, 12, 13, 14, 15, 16, 17

N

N

X0160027

F

1, 2, 3, 4

N

X0160029

M

F

1, 2, 3, 4, 5, 6, 7, 8, 9, 10

11, 12, 13, 14, 15

N

N

X0160031

M

F

1, 2, 3, 4

5, 6, 7, 8, 9, 10, 11, 12, 13, 14

N

N

X0160033

M

F

1, 2, 3, 4, 5, 6, 7, 8

9, 10, 11, 12, 13, 14, 15, 16

N

N

X0160035

M

F

1, 2, 3, 4, 5, 6, 7, 8

9, 10, 11, 12, 13, 14

N

N

X0160037

M

F

1, 2, 3, 4, 5, 6, 7, 8

9, 10, 11, 12, 13, 14, 15

N

N

X0160039

M

F

1, 2, 3, 4, 6, 7,

8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21

N

N

N = No abnormalities detected

 

NECROPSY FINDINGS IN PUPS SACRIFICED ON DAY 4POST PARTUM– INDIVIDUAL DATA

Group 3

Animal Number

Sex

Pup Number (s)

Description

X0160041

M

F

1, 2, 3, 4, 5, 6, 7, 8, 12, 13

9, 10, 11, 14

N

N

X0160043

M

F

1, 2, 3, 4, 5, 6

7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19

N

N

X0160045

M

F

1, 3, 4, 5, 6, 7, 8

9, 10, 11, 12, 13, 14

N

N

X0160047

NP

 

 

X0160049

M

F

1, 2, 3, 4, 5, 6, 7

8, 9, 10, 11, 12, 13, 14, 15, 16, 17

N

N

X0160051

M

F

1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11

12, 13, 14, 15, 16, 17

N

N

X0160053

NP

 

 

X0160055

M

F

1, 2, 4, 5, 6

7, 8, 9, 11, 12, 14, 16, 17+15+18

N

N

X0160057

M

F

1

3

N

N

X00160059

M

F

1, 2, 3, 4, 5, 6

7, 8, 9, 10, 11

N

N

N = No abnormalities detected

NP = Not pregnant

 

NECROPSY FINDINGS IN PUPS SACRIFICED ON DAY 4POST PARTUM– INDIVIDUAL DATA

Group 4

Animal Number

Sex

Pup Number (s)

Description

X0160061

M

F

1, 2, 3, 4, 5, 6, 7, 8

9, 10, 11, 12, 13, 14, 15

N

N

X0160063

M

F

1, 2, 3, 4, 5, 7, 8

9, 10, 11, 12, 13, 14, 15

N

N

X0160065

M

F

1, 2, 3, 5, 6

7, 8, 9, 10, 11, 12

N

N

X0160067

M

F

1, 2, 3, 4, 5, 6, 7, 8

9, 10, 11, 12, 13, 14, 15

N

N

X0160071

M

M

F

1, 2, 3, 4, 5, 9+6

11

6, 7, 8, 10, 11, 12, 13

N

Tail: not evident

N

X0160073

M

F

1, 3, 4, 5, 8

6, 7, 9, 10, 11, 12, 13

N

N

X0160075

M

F

1, 2, 3, 4, 5, 6, 7

8, 9, 10, 11, 12, 13, 14, 15, 16

N

N

X0160077

M

F

1, 2, 3, 4, 5, 6, 7, 8, 9

10, 11, 12, 13, 14

N

N

X0160079

M

F

1, 2, 3, 4

5, 6, 7, 8, 9, 10, 11, 12, 13, 14

N

N

N = No abnormalities detected

Applicant's summary and conclusion

Conclusions:
No treatment-related effects indicating systemic toxicity were observed in male or female animals at any of the dose levels investigated (0, 62.5, 250 and 1000 mg/kg body weight/day). No effects on sexual function and fertility or in developmental parameters and lactation were observed at any of the dose levels investigated.Based on the results of the present study, the NOEL (No Observed Effect Level) for reproductive and developmental toxicity was considered to be 1000 mg/kg bw/day for males and females.
Executive summary:

The toxicity as well as any possible effects of Vat Black 25 when administered by oral route at dose levels of 62,5, 250 and 1000 mg/kg body weight/day, on male and female Sprague Dawley rats reproductive performance (such as gonadal function, mating behaviour, conception, development of conceptuses and parturition) and recovery from any treatment-related effects during a period of 2 weeks were investigated in this study.

One female of the high dose group, receiving 1000 mg/kg bodyweight/day, was killed for humane reasons on Day 3 of the gestation phase.

This death was considered to be unrelated to treatment.

No clinical signs of toxicological relevance or signs of neurotoxicity (weekly observations at removal from the cage and in an open arena, alterations in motor activity, grip strength and sensory reaction to stimuli) were observed during the study in males and females. Slight orange staining in the cage tray was observed during the mating phase in some cages of mid- and high dose animals.

No differences in body weight and food consumption were observed in treated animals compared to the control group.

No changes of toxicological significance were recorded at haematological, clinical chemistry investigations or at coagulation tests in animals sacrificed at the end of the study.

No treatment-related anomalies were noted in the oestrus cycle of the treated females when compared to controls. Copulatory and fertility indices did not show any treatment-related differences among treated and control groups.

Parturition, lactation, implantation, litter data and sex ratio did not show any changes of toxicological relevance.

Necropsy findings in pups did not reveal any treatment-related effect.

No relevant changes were detected at post mortem examination (terminal body weight, organ weights, macroscopic and microscopic examinations) in treated animals, when compared with controls.

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 and a regular layering in the germinal epithelium was described.

Based on the results of the present study, the NOEL (No Observed Effect Level) for general toxicity, reproductive and developmental toxicity was considered to be 1000 mg/kg bw/day for males and females.