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REACH

Reaction product of 3,9-dibromobenzanthrone condensed with 2 equivalents of 1-aminoanthraquinone, subsequently further condensed under oxidative conditions

EC number: 944-232-9 | CAS number: -

Life Cycle description
Uses advised against

Toxicological information

Endpoint summary

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

Toxicity to reproduction via oral route

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.

Link to relevant study records

Reference

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

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:

not specified

GLP compliance:

yes

Limit test:

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.

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:

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.

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).

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.

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: 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.

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.

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

Key result

Critical effects observed:

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

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

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

Key result

Dose descriptor:

NOEL

Generation:

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:

Key result

Reproductive effects observed:

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Treatment related:

Relation to other toxic effects:

not specified

Dose response relationship:

Relevant for humans:

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

X0160021

X0160023

X0160025

X0160027

X0160029

X0160031

X0160033

X0160035

X0160037

X0160029

X0160041

X0160043

X0160045

X0160047

X0160049

X0160051

X0160053

X0160055

X0160057

X0160059

X0160061

X0160063

X0160065

X0160067

X0160069

X0160071

X0160073

X0160075

X0160077

X0160079

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)

Mean

18.30

2.87

17.40

2.46

4.61

4.28

16.30

1.70

110

5.74

6.95

22.10

0.32

Mean

15.70

4.14

15.30

4.32

3.70

6.84

14.60

4.33

110

4.39

6.64

22.20

0.42

Mean

16.38

2.67

16.38

2.67

0.00

15.88

2.53

2.88

4.41

22.38

0.52

Mean

15.11

1.17

15.11

1.17

0.00

14.22

1.20

5.71

6.91

22.00

0.00

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)

Mean

16.30

1.70

16.20

1.81

0.67

2.12

106.16

18.56

6.87

0.70

13.40

4.88

16.29

29.78

129.70

50.81

9.20

1.84

Mean

14.60

4.33

14.40

4.27

1.26

2.66

103.16

26.81

7.29

0.88

14.20

4.10

2.41

3.15

138.17

33.24

10.04

1.35

Mean

15.88

2.53

14.13

5.11

12.18

27.18

97.63

38.48

7.24

0.94

13.25

5.26

17.36

29.71

133.74

51.32

10.30

1.35

Mean

14.22

1.20

14.22

1.20

0.00

99.81

10.15

7.21

0.61

13.44

1.74

5.41

9.44

132.79

20.00

9.88

0.72

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

Total

%males

Total

%males

Mean

7.00

1.70

9.30

2.67

16.30

1.70

43.60

12.14

6.00

2.67

7.40

3.27

13.40

4.88

41.28

18.56

Mean

7.70

2.26

6.90

3.63

14.60

4.33

56.81

19.68

7.40

2.12

6.80

3.61

14.20

4.10

56.31

19.89

Mean

8.00

1.85

7.88

3.64

15.88

2.53

52.05

15.49

6.63

3.07

6.63

3.70

13.25

5.26

50.71

13.38

Mean

7.00

1.50

7.22

1.79

14.22

1.20

49.43

10.92

6.67

1.66

6.78

1.79

13.44

1.74

49.68

10.89

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

Abdominal cavity: all organs autolysed

No milk in stomach

X0160037

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

No milk in stomach

X0160003

1, 2, 3, 4, 5

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

X0160005

1, 2, 3, 5, 6, 8

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

X0160007

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

3, 10, 11, 12, 13, 14

X0160009

1, 2, 3, 4, 5, 6

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

X0160011

1, 2, 3, 4

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

X0160013

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

8, 9, 10, 11, 12, 13

No milk in stomach

X0160015

1, 2, 3, 4, 6, 7

8, 9, 11, 12

X0160017

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

10, 11, 12, 13, 14, 15

X0150019

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

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

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

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

9, 10, 11, 12, 13

X0160023

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

9, 10, 12, 13, 14, 15

X0160025

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

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

X0160027

1, 2, 3, 4

X0160029

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

11, 12, 13, 14, 15

X0160031

1, 2, 3, 4

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

X0160033

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

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

X0160035

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

9, 10, 11, 12, 13, 14

X0160037

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

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

X0160039

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

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

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

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

9, 10, 11, 14

X0160043

1, 2, 3, 4, 5, 6

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

X0160045

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

9, 10, 11, 12, 13, 14

X0160047

X0160049

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

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

X0160051

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

12, 13, 14, 15, 16, 17

X0160053

X0160055

1, 2, 4, 5, 6

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

X0160057

X00160059

1, 2, 3, 4, 5, 6

7, 8, 9, 10, 11

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

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

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

X0160063

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

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

X0160065

1, 2, 3, 5, 6

7, 8, 9, 10, 11, 12

X0160067

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

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

X0160071

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

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

Tail: not evident

X0160073

1, 3, 4, 5, 8

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

X0160075

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

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

X0160077

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

10, 11, 12, 13, 14

X0160079

1, 2, 3, 4

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

N = No abnormalities detected

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 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.

Effect on fertility: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 1 000 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: Klimisch 1 - GLP accredited study conducted in accordance with OECD Guideline 422.

Effect on fertility: via inhalation route

Endpoint conclusion:: no study available

Effect on fertility: via dermal route

Endpoint conclusion:: no study available

Additional information

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.

Effects on developmental toxicity

Effect on developmental toxicity: via oral route

Endpoint conclusion:: no adverse effect observed
Dose descriptor:: NOAEL
: 1 000 mg/kg bw/day
Study duration:: subacute
Species:: rat
Quality of whole database:: Klimisch 1 - GLP accredited study conducted in accordance with OECD Guideline 422.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:: no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:: no study available

Additional information

Based on the results of the screening study conducted in accordance with OECD Guideline 422, 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.

Justification for classification or non-classification

Based on the results of this study, the test substance is not classified for reproductive/developmental toxicity in accordance with the CLP Regulation.

Additional information

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