7,16-dichloro-6,15-dihydroanthrazine-5,9,14,18-tetrone

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

6,15-dihydroanthrazine-5,9,14,18-tetrone

EC Number:

201-375-5

EC Name:

6,15-dihydroanthrazine-5,9,14,18-tetrone

Cas Number:

81-77-6

Molecular formula:

C28H14N2O4

IUPAC Name:

6,15-dihydrodinaphtho[2,3-a:2',3'-h]phenazine-5,9,14,18-tetrone

Test material form:

solid: particulate/powder

Details on test material:

- Analytical purity: The test substance has been characterized by analyses.
- Stability: The stability under storage conditions over the study period was guaranteed by the sponsor.
- Homogeneity: The test substance is homogeneous by visual inspection. Additionally, homogeneity / correctness of the concentrations were confirmed by analysis.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS- Source: Charles River Laboratories, Research Models and Services, Germany GmbH - Age at study initiation: 10 - 12 weeks (males/females)- Weight at study initiation: mean(males): 304 g; mean(females): 201 g- Housing: individually in Makrolon type M III cages supplied by Becker & Co., Castrop-Rauxel, Germany, with the following exceptions: During overnight matings, male and female mating partners were housed together in Makrolon type M III cages, pregnant animals and their litters were housed together until PND 4 (end of lactation) with nesting material, for motor activity (MA) measurements the animals were housed individually in polycarbonate cages supplied by TECNIPLAST, Hohenpeißenberg, Germany and small amounts of bedding material.- Diet: Ground Kliba maintenance diet mouse-rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland; ad libitum- Water:drinking water (from water bottles); ad libitum- Acclimation period: 6 daysENVIRONMENTAL CONDITIONS- Temperature (°C): 20 - 24°C - Humidity (%): 30 - 70% - Air changes (per hr): 15/hr- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:Blue CAS 81-77-6 was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a high speed homogenizer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week. The administration volume was 10 mL/kg body weight.VEHICLE- Concentration in vehicle: 1 (100 mg/kg bw), 3 (300 mg/kg bw), 10 g/100 mL (1000 mg/kg bw)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The analyses confirmed the stability of the test substance in drinking water at room temperature over a period of 7 days, the homogeneous distribution of the test substance in drinking water, and the correctness of the prepared concentrations of the test substance preparations in drinking water (the values were in the expected range of the target concentrations, i.e. were always in a range of about 102-109% of the nominal concentrations).

Duration of treatment / exposure:

The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation in females followed by an additional treatment until one day before sacrifice (males: 34 / 35 days; females: 49 days)

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes- Time schedule: A check for moribund and dead animals was made twice daily on working days and once daily on Saturdays, Sundays and public holidays. If animals were in a moribund state, they were sacrificed and necropsied. A cageside examination was conducted at least once daily for any signs of morbidity, pertinent behavioral changes and signs of overt toxicity. Abnormalities and changes were documented daily for each affected animal.DETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: Before initial test substance administration and thereafter at weekly intervals.BODY WEIGHT: Yes - Time schedule for examinations: Body weights of F0 parents were determined on study day 0 (start of the administration period) and thereafter once a week, in males throughout the study and in females during premating and mating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, after the day of parturition (postnatal day [PND] 0) and on PND 4.FOOD CONSUMPTION AND COMPOUND INTAKE:- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes - Compound intake calculated as time-weighted averages from the consumption and body weight gain data: NoFOOD EFFICIENCY:- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: NoOPHTHALMOSCOPIC EXAMINATION: No HAEMATOLOGY: Yes - Time schedule for collection of blood: towards the end of the administration period- Anaesthetic used for blood collection: Yes (isoflurane (Isoba®, Essex GmbH Munich, Germany)) - Animals fasted: Yes - How many animals: 5 animals per sex and group- Parameters checked: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes, Prothrombin time (Hepato Quick’s test; HQT);CLINICAL CHEMISTRY: Yes - Time schedule for collection of blood: towards the end of the administration period- Animals fasted: Yes- How many animals: 5 animals per sex and group- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), gamma-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Magnesium (MG);URINALYSIS: Yes - Time schedule for collection of urine: 5 animals per sex and group towards the end of the administration period- Metabolism cages used for collection of urine: Yes - Animals fasted: Yes - Parameters checked: pH, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Blood, Specific gravity, Sediment, Color / Turbidity, VolumeNEUROBEHAVIOURAL EXAMINATION: Yes - Time schedule for examinations: Towards the end of the administration period - Dose groups that were examined: 5 animals per sex and test group.- Battery of functions tested: passive observations without disturbing the animals, followed by removal from the home cage, open field observations in a standard arena and sensorimotor tests as well as reflex tests, motor activityOTHER: Reproduction data and pup observation (See chapter "Toxicity to reproduction")

Sacrifice and pathology:

GROSS PATHOLOGY: YesHISTOPATHOLOGY: YesWeight assessment was carried out on all animals. The following weights were determined:Adrenal glands, Anesthetized animals, Brain, Epididymides, Heart, Kidneys, Liver, Testes, Spleen, ThymusThe following organs / tissues were preserved in neutral-buffered 4% formaldehyde or in modified Davidson’s solution:Adrenal glands, All gross lesions, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Eyes with optic nerve (modified Davidson’s solution), Esophagus, Extraorbital lacrimal gland, Epididymides (modified Davidson’s solution), Female mammary gland, Femur with knee joint, Heart, Ileum, Jejunum (with Peyer’s patches), Kidneys, Larynx, Liver, Lungs, Lymph nodes (axillary and mesenteric), Mammary gland (male and female), Nose (nasal cavity), Ovaries (modified Davidson’s solution), Oviducts, Pancreas, Parathyroid glands, Pharynx, Pituitary gland, Prostate gland, Rectum, Salivary glands (mandibular and sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Spinal cord (cervical, thoracic and lumbar cord), Spleen, Sternum with marrow, Stomach (forestomach and glandular stomach), Target organs, Testes (modified Davidson’s solution), Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina;From the liver, each one slices of the lobus dexter medialis and the lobus sinister lateralis were fixed in Carnoy’s solution and embedded in paraplast.

Other examinations:

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4.The pups were sexed and examined for macroscopically evident changes on PND 0. They were weighed on PND 1 and on PND 4. Their viability was recorded. At necropsy on PND 4, all pups were sacrificed and examined macroscopically for external and visceral findings.

Statistics:

Food consumption, body weight and body weight change: Simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided); Feces, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity, clinical pathology parameters, weight parameters (except for urine color and turbidity): Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians;

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No changes of toxicological concern with regard to body weight parameters were observed during the entire study period. The body weight changes in males of test group 1 (100 mg/kg bw/d) from week 1 to 2 (71%) and in one female of test group 3 (1000 mg/kg bw/d) were decreased.

Food efficiency:

no effects observed

Description (incidence and severity):

No changes of toxicological concern were observed during the entire study period. In males of test group 1 (100 mg/kg bw/d) food consumption was decreased from study week 1 to 2. Significantly decreased food consumption during the entire gestation period was seen in one female of test group 3 (1000 mg/kg bw/d).

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

When compared to control group 0 (set to 100%), the mean absolute weights of following organs were significantly altered: liver (males / 100 mg/kg bw: 89%; males / 1000 mg/kg bw: 94%). When compared to control group 0 (set to 100%), none of the mean relative weights determined was significantly altered. The reduction in liver weight in males of test group 1 (100 mg/kg bw) and test group 3 (1000 mg/kg bw) is regarded to be incidental due to a missing altered relative organ weight, a missing dose response relationship and missing histopathologic findings that could explain the weight reduction. All other mean weight parameters did not show significant differences when compared to the control groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No test substance-related, adverse findings were noted. The only findings were: Discoloration of contents of glandular stomach, jejunum, colon and discoloration of the tongue. These findings are regarded to be treatment related but not adverse. The macroscopically observed dilation of the uterus in three animals of test group 3 (1000 mg/kg bw) are regarded to be related to the cycle and therefore no pathologic finding.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No signs of toxicity in the respective tissues were noted. The only findings were: Discoloration of contents of glandular stomach and jejunum, blue particles on surface of forestomach, cecum, rectum and discoloration of the tongue. These findings are regarded to be treatment related but not adverse. In the forestomach of all investigated animals bluish particles were located on top of the mucosal surface. The mucosa did not show any additional findings.

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: No animal died prematurely in the present study. No test substance-related, adverse findings were noted. All animals of test group 1 (100 mg/kg bw/d) from study week 1 onwards and all animals of test groups 2-3 (300 and 1000 mg/kg bw/d) from study week 0 onwards showed blue discoloured faeces. During gestation all animals of test group 1 and 3 (100 and 1000 mg/kg bw/d) and 9 animals of test group 2 (300 mg/kg bw/d) showed blue discoloured faeces during gestation. Nine of nine animals of test group 1, nine of nine animals of test group 2 and seven of seven animals of test group 3 (100, 300 and 1000 mg/kg bw/d) showed blue discoloured faeces during lactation period.
BODY WEIGHT AND WEIGHT GAIN: No changes of toxicological concern with regard to body weight parameters were observed during the entire study period. The body weight changes in males of test group 1 (100 mg/kg bw/d) from week 1 to 2 (71%) and in one female of test group 3 (1000 mg/kg bw/d) during gestation days 0 to 7 were decreased.
FOOD CONSUMPTION AND COMPOUND INTAKE: In males of test group 1 (100 mg/kg bw/d) food consumption was decreased from study week 1 to 2. Significantly decreased food consumption during the entire gestation period in one female of test group 3 (1000 mg/kg bw/d) was reported.
HAEMATOLOGY: No treatment-related changes among haematological parameters were measured.
CLINICAL CHEMISTRY: No treatment-related changes among clinical chemistry parameters were measured.
URINALYSIS: No treatment-related changes among urinalyses parameters were measured.
NEUROBEHAVIOUR: Deviations from "zero values" were obtained in several rats. However, as most findings were equally distributed between test-substance treated groups and controls, were without a dose-response relationship or occurred in single rats only, these observations were considered as incidental.
ORGAN WEIGHTS: When compared to control group 0 (set to 100%), the mean absolute weights of following organs were significantly altered: liver (males / 100 mg/kg bw: 89%; males / 1000 mg/kg bw: 94%). When compared to control group 0 (set to 100%), none of the mean relative weights determined was significantly altered. The reduction in liver weight in males of test group 1 (100 mg/kg bw) and test group 3 (1000 mg/kg bw) is regarded to be incidental due to a missing altered relative organ weight, a missing dose response relationship and missing histopathologic findings that could explain the weight reduction. All other mean weight parameters did not show significant differences when compared to the control groups.
GROSS PATHOLOGY: No test substance-related, adverse findings were noted. The only findings were: Discoloration of contents of glandular stomach, jejunum, colon and discoloration of the tongue. These findings are regarded to be treatment related but not adverse. The macroscopically observed dilation of the uterus in three animals of test group 3 (1000 mg/kg bw/d) are regarded to be related to the cycle and therefore no pathologic finding.
HISTOPATHOLOGY: No signs of toxicity in the respective tissues were noted. The only findings were: Discoloration of contents of glandular stomach and jejunum, blue particles on surface of forestomach, cecum, rectum and discoloration of the tongue. These findings are regarded to be treatment related but not adverse. In the forestomach of all investigated animals bluish particles were located on top of the mucosal surface. The mucosa did not show any additional findings.

Effect levels

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose tested. No adverse or toxicological relevant effects observed.

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

The NOAEL for general, systemic toxicity was found to be 1000 mg/kg bw/day in male and female rats.

Executive summary:

The test item was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg bw/day (test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/day (test group 2) and 1000 mg/kg bw/day (test group 3). Water served as vehicle.

The objective of the study was to detect possible effects of the test substance on the integrity and performance of male and female reproductive systems including gonadal function, mating behaviour, conception, gestation and parturition. Furthermore, it was intended to obtain information about the general toxicological profile including target organs and the no observed adverse effect level (NOAEL) after repeated oral administration. The duration of treatment covered a 2-week pre-mating and mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and two weeks thereafter in females.

After 2 weeks of premating treatment the F0 animals were mated to produce F1 generation pups. Mating pairs were from the same test group. Mating was discontinued as soon as sperm was detected in the vaginal smear.

A detailed clinical observation (DCO) was performed in all animals before initial test substance administration and, as a rule, thereafter at weekly intervals.

Food consumption of the F0 parents was determined once weekly during premating. In dams food consumption was determined for gestation days 0 - 7, 7 - 14, 14 - 20 and lactation days 1 - 4.

Body weights of F0 parents were determined once a week, in males throughout the study and in females during premating and mating. During gestation and lactation period, F0 females were weighed on gestation days (GD) 0, 7, 14 and 20, after the day of parturition (postnatal day [PND] 0) and on PND 4.

Clinico-chemical and haematological examinations as well as urinalyses were performed in 5 animals per sex and group towards the end of the administration period.

Towards the end of the administration period a functional observational battery was performed and motor activity was measured in 5 animals per sex and test group.

All F0 parental animals were sacrificed by decapitation, under isoflurane anaesthesia, and were assessed by gross pathology. Weights of selected organs were recorded and a histopathological examination was performed.

RESULTS

Formulation analytics confirmed the stability of the test substance in drinking water at room temperature over a period of 7 days, the homogeneous distribution of the test substance in drinking water, and the correctness of the prepared concentrations of the test substance preparations in drinking water.

In females dosed 1000 mg/kg bw/day, a slight but statistically significant lower (max. ‑13%) food consumption during the entire gestation period accompanied by slightly lower body weight gains (‑20%) during gestation days 0 to 7 resulting in slightly lower body weights compared to control females during the entire gestation period were seen. This effect is due to one female of the high-dose group (No. 138). This female started with the same body weight as all other animals and during the first week of treatment, food consumption was comparable. But during the second week of treatment, food consumption was strongly reduced (11.1 g versus 13.9 g of the group average and 14.9 of control mean; 25% less than control animals). Accordingly, animal no. 138 was the only animal that lost body weight during the second week of treatment whereas all other animals gained weight. Food consumption was even more strongly reduced during the first week of gestation (38% compared to control animals; 30% compared to animals of the same group). No adverse effects were observed in any other animals or dose groups.

No adverse effects were seen in clinical or anatomical pathology in paternal animals of all treated groups.

CONCLUSION

Thus, under the conditions of this study, the NOAEL (no observed adverse effect level) for general, systemic toxicity was found to be 1000 mg/kg bw/day.