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REACH

Anthra[2,1,9-mna]naphth[2,3-h]acridine-5,10,15(16H)-trione

EC number: 221-897-7 | CAS number: 3271-76-9

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Description of key information

Repeated dose toxicity via oral route

NOEL 1000 mg/kg bw in male and female Sprague-Dawley rats (OECD 422)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

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:

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:

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:

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	1^a
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

(10)

No significant signs

38.9

43.5

42.2

APPEARANCE

Hair loss

30.0

0.0

6.5

EYE – EAR – MOUTH

Damaged ear

16.0

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

(10)

No significant signs

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

(10)

REMOVAL

Removal easy

7.0

HANDLING REACTIVITY

Handling reactivity normal

7.0

LACHRYMATION

Lachrymation absent

7.0

PALPEBRAL CLOSURE

Palpebral closure absent

7.0

SALIVATION

Salivation absent

7.0

PILOERECTION

Piloerection absent

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

(10)

REMOVAL

Removal easy

6.9

6.8

6.9

6.6

HANDLING REACTIVITY

Handling reactivity normal

6.9

6.8

6.9

6.6

LACHRYMATION

Lachrymation absent

6.9

6.8

6.9

6.6

PALPEBRAL CLOSURE

Palpebral closure absent

6.9

6.8

6.9

6.6

SALIVATION

Salivation absent

6.9

6.8

6.9

6.6

PILOERECTION

Piloerection absent

6.9

6.8

6.9

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

(10)

REARING

Rearing absent

Rearing 1-3

Rearing 4-7

Rearing 8-10

Rearing 11-14

Rearing 15-20

Rearing 21-30

0.0

1.5

2.6

1.7

3.0

1.8

0.0

2.0

1.8

2.8

1.6

0.0

2.0

3.0

1.7

2.0

2.2

2.4

0.0

1.0

2.7

2.8

1.9

1.0

SPASMS

Spasms absent

7.0

MYOCLONIA

Myoclonia absent

7.0

GAIT

Normal gait

7.0

MOBILITY IMPAIRMENT

Mobility impairment absent

7.0

AROUSAL

Arousal normal

Arousal slow

7.0

0.0

7.0

0.0

6.8

2.0

7.0

0.0

VOCALISATION

Vocalisation absent

7.0

STEREOTYPIES

Stereotypies absent

7.0

UNUSUAL RESPIRATION

Unusual respiration absent

7.0

BIZARRE BEHAVIOUR

Bizarre behaviour absent

7.0

URINATION

Urination absent

Urination 1-3

Urination 4-6

Urination 7-9

Urination more than 10

4.0

3.0

1.5

1.0

0.0

4.8

1.7

1.2

1.0

2.0

5.0

2.2

1.2

0.0

1.0

5.1

1.3

2.7

1.0

0.0

DEFECATION

Defecation absent

Defecation 1-3

Defecation 4-6

4.9

2.5

2.0

6.0

1.2

1.0

6.2

1.4

1.0

5.8

1.7

1.0

TREMORS

Tremors absent

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

(10)

REARING

Rearing 11-14

Rearing 15-20

Rearing 21-30

Rearing more than 30

2.2

3.6

2.3

1.3

2.4

3.6

3.2

1.0

1.6

4.4

3.3

1.4

2.7

3.6

2.2

1.0

SPASMS

Spasms absent

6.9

6.8

6.9

6.6

MYOCLONIA

Myoclonia absent

6.9

6.8

6.9

6.6

GAIT

Normal gait

6.9

6.8

6.9

6.6

MOBILITY IMPAIRMENT

Mobility impairment absent

6.9

6.8

6.9

6.6

AROUSAL

Arousal normal

6.9

6.8

6.9

6.6

VOCALISATION

Vocalisation absent

6.9

6.8

6.9

6.6

STEREOTYPIES

Stereotypies absent

6.9

6.8

6.9

6.6

UNUSUAL RESPIRATION

Unusual respiration absent

6.9

6.8

6.9

6.6

BIZARRE BEHAVIOUR

Bizarre behaviour absent

6.9

6.8

6.9

6.6

URINATION

Urination absent

Urination 1-3

Urination 4-6

Urination 7-9

Urination more than 10

5.1

1.7

1.0

5.6

1.5

1.0

0.0

5.2

1.6

1.4

1.0

0.0

4.0

2.1

1.8

0.0

DEFECATION

Defecation absent

Defecation 1-3

6.7

1.0

6.5

1.0

6.8

1.0

6.3

1.0

TREMORS

Tremors absent

6.9

6.8

6.9

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”

15#

(n)

Mean

291.27

8.04

351.93

14.40

391.82

19.31

418.66

24.10

(n)

Mean

290.91

6.82

343.76

11.97

375.38

16.31

400.39

20.38

(n)

Mean

290.21

8.48

344.05

15.02

378.62

15.50

404.02

17.39

(n)

Mean

291.32

7.25

351.16

12.67

387.61

16.39

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

(n)

Mean

440.52

28.74

463.91

35.21

484.62

32.37

491.88

43.22

(n)

Mean

424.16

21.21

445.27

27.57

467.04

31.46

474.21

35.30

(n)

Mean

424.73

19.19

449.58

19.40

470.60

23.64

479.52

34.21

(n)

Mean

436.14

16.77

463.39

22.11

484.57

21.84

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”

15#

(n)

Mean

214.83

8.35

236.18

5.69

242.60

6.15

2.53.35

12.29

(n)

Mean

215.15

9.37

234.15

8.25

239.03

15.65

253.72

14.16

(n)

Mean

214.46

8.79

235.66

8.48

244.87

12.84

256.33

12.30

(n)

Mean

214.42

8.98

231.99

9.54

243.20

9.19

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

1”

(n)

Mean

262.25

21.09

294.87

15.77

334.16

15.23

424.81

25.24

314.68

19.39

315.23

35.00

(n)

Mean

256.72

13.40

294.21

15.86

332.61

19.14

414.23

33.26

316.05

20.48

308.52

20.76

(n)

Mean

258.81

16.48

297.51

16.70

336.34

20.84

429.21

31.50

321.90

24.58

310.40

31.87

(n)

Mean

356.60

6.76

288.48

9.48

323.19

8.28

402.31

12.52

311.44

9.82

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--

Kidneys

Pelvic dilation

Forelimbs

Abnormal shape

Abnormal consistency

Hindlimbs

Abnormal shape

Abnormal consistency

MACROSCOPIC OBSERVATIONS – MAIN GROUPS – FINAL SACRIFICE – GROUP INCIDENCE

Group:

Number in group:

--Males--

--Females--

Adrenals

Abnormal size

Cervical nodes

Abnormal colour

Kidneys

Abnormal area (s)

Pelvic dilation

Liver

Abnormal shape

Abnormal size

Ovaries

Abnormal size

Thymus

Abnormal area (s)

Abnormal colour

Abnormal size

Uterus

Not pregnant

Unilateral implantation

Ears

Abnormal area (s)

Whole animal

No abnormalities detected

MICROSCOPIC OBSERVATIONS – UNSCHEDULED DEATH – GROUP INCIDENCE

Controls from group (s): 1

Tissues With Diagnoses

Animal Sex:

Dosage group:

No. in group:

--Animals Affected--

--Females--

Kidneys

HYDRONEPHROSIS

Number examined:

Lungs

EMPHYSEMA

INFLAMMATORY CELL FOCI

Number examined:

Forelimbs

ARTHRITIS

Number examined:

Hindlimbs

ARTHRITIS

Number examined:

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

Adrenals

ACCESSORY NODULE

CORTICAL CELL VACUOLIZATION

CYTS/S

Number examined:

Cervical nodes

CONGESTION

Number examined:

Heart

INFLAMMATORY CELL FOCI

Number examined:

Kidneys

INFLAMMATORY CELL FOCI

NEPHROPATHY

HYDRONEPHROSIS

Number examined:

Liver

INFLAMMATORY CELL FOCI

HEPATOCYTIC VACUOLATION

CLEAR CELL CHANGE

EXTRAMEDULLARY HAEMOPOIESIS

Number examined:

Lungs

AGGREGATION OF ALVEOLAR MACROPHAGES

Number examined:

Prostate

INFLAMMATORY CELL FOCI

Number examined:

Testes

TUBULAR CELL DEGENERATION

Number examined:

Thymus

ATROPHY

CONGESTION

Number examined

Thyroid

ECTOPIC THYMIC TISSUE

Number examined:

Ears

CHRONIC INFLAMMATION

Number examined:

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.

Endpoint conclusion

Endpoint conclusion:: no adverse effect observed
Study duration:: subacute
Species:: rabbit
Quality of whole database:: Kllimisch 1 - GLP accredited study in accordance with OECD Guideline 422.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:: no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:: no study available

Additional information

Repeated dose toxicity via oral route

Male Sprague-Dawley rats 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.

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.

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.

Justification for classification or non-classification

The results of this study do not trigger classification for repeated dose toxicity in accordance with the CLP Regulation.

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