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Repeated dose toxicity: oral

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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
Remarks:
The study has been performed according to OECD guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
2,20-dichloro-13,31-diethyl-4,22-dioxa-13,18,31,36-tetraazanonacyclo[19.15.0.0³,¹⁹.0⁵,¹⁷.0⁶,¹⁴.0⁷,¹².0²³,³⁵.0²⁴,³².0²⁵,³⁰]hexatriaconta-1(36),2,5,7(12),8,10,14,16,18,20,23(35),24(32),25,27,29,33-hexadecaene
EC Number:
606-790-9
Cas Number:
215247-95-3
Molecular formula:
C34H22Cl2N4O2
IUPAC Name:
2,20-dichloro-13,31-diethyl-4,22-dioxa-13,18,31,36-tetraazanonacyclo[19.15.0.0³,¹⁹.0⁵,¹⁷.0⁶,¹⁴.0⁷,¹².0²³,³⁵.0²⁴,³².0²⁵,³⁰]hexatriaconta-1(36),2,5,7(12),8,10,14,16,18,20,23(35),24(32),25,27,29,33-hexadecaene
Test material form:
solid: nanoform

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: males: 142-169 grams; females: 126-142 grams
- Fasting period before study: none
- Housing: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm; during overnight activity monitoring individual housing in MIII type; height 15 cm.) with sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd), Surrey, United Kingdom). No cage-enrichment was provided during overnight activity monitoring. Certificates of analysis were examined and then retained in the NOTOX archives.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.0 ± 3.0°C (actual range: 19.3 - 21.5°C),
- Humidity (%):30-70% (actual range: 30 - 65%)
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
IN-LIFE DATES: From: 28 January 2008 to: 10 March 2008

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 4 hours prior to dosing and were homogenised to a visually acceptable level.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Test substance formulations in propylene glycol were noted as stable for at least 5 hours and formed a homogeneous suspension at the concentrations tested (coefficient of variation <10%). Analysis of the accuracy of dose preparations revealed mean values within the range of 91-93% of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.
Duration of treatment / exposure:
28 days treatment with additonal 14-day treatment-free recovery groups for the control and high dose
Frequency of treatment:
Once daily for 28 days, 7 days per week, approximately the same time each day with a maximum of 4 hours difference between the earliest and latest dose. Animals were dosed up to the day prior to necropsy.
Doses / concentrationsopen allclose all
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 (with 5 additional recovery animals/sex in the control and high dose group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected on the basis of a 5-day dose range finding study
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily
- Cage side observations included: mortality, viability
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily after dosing
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not applicable
FOOD 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: Yes
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: subjective appraisal
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment and recovery
- Anaesthetic used for blood collection: Yes (iso-flurane anaesthesia)
- Animals fasted: Yes
- How many animals: 40 (end of treatment), 20 (end of recovery)
- Parameters examined: White blood cells, Differential leucocyte count (neutrophils, lymphocytes, monocytes, eosinophils, basophils), Red blood cells, Reticulocytes, Red blood cell distribution width, Haemoglobin, Haematocrit, Mean corpuscular volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, Platelets,
Clotting potential (Prothrombin time, Activated Partial thromboplastin time)
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment
- Animals fasted: Yes
- How many animals: 40 (end of treatment), 20 (end of recovery)
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine,
Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate
URINALYSIS: Yes
- Time schedule for collection of blood: end of treatment
- Animals fasted: Yes
- How many animals: 40 (end of treatment), 20 (end of recovery)
- Parameters examined: Volume, Colour, Clarity, Specific gravity , pH, Protein, Glucose, Ketone, Bilirubin, Blood, Leucocytes , Nitrite, Urobilinogen, Sodium, Potassium, Calcium, Sediment, (white blood cells, red blood cells, casts, epithelial cells, crystals, bacteria, other)
NEUROBEHAVIOURAL EXAMINATION: Yes:
During week 4 of treatment, the following tests were performed on all animals:
- hearing ability, pupillary reflex, static righting reflex and grip strength.
- motor activity test (recording period: 12 hours during overnight for individual animals, using a computerized monitoring system, Pearson Technical Services, Debenham, Stowmarket, England).
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All animals were necropsied and descriptions of all macroscopic abnormalities recorded.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in 10% buffered formalin (neutral phosphate buffered 4% formaldehyde solution):
Identification marks: not processed
Ovaries, Adrenal glands, Pancreas, Aorta, Peyer's patches [jejunum, ileum] if detectable, Brain [cerebellum, mid-brain, cortex], Pituitary gland, Caecum,
(Preputial gland), Cervix, Prostate gland, (Clitoral gland), Rectum, Colon, (Salivary glands - mandibular, sublingual), Duodenum, Sciatic nerve, Epididymides, Seminal vesicles, (Eyes with optic nerve [if detectable] and (Skeletal muscle), Harderian gland), (Skin) , (Female mammary gland area), Spinal cord -cervical, midthoracic, lumbar, (Femur including joint), Spleen, Heart, Sternum with bone marrow, Ileum, Stomach, Jejunum, Testes, Kidneys, Thymus, (Larynx), Thyroid including parathyroid [if detectable], (Lacrimal gland, exorbital), (Tongue), Liver, Trachea, Lung, infused with formalin, Urinary bladder, Lymph nodes - mandibular, mesenteric, Uterus, (Nasopharynx), Vagina, Oesophagus, All gross lesions

Tissues/organs mentioned in parentheses were not examined by the pathologist since there were no changes in macroscopic appearance indicative of (potential) toxicity.

The following organ weights (and terminal body weight) were recorded from the animals on the scheduled day of necropsy: Adrenal glands, Brain, Epididymides, Heart, Kidneys, Liver, Spleen, Testes, Thymus

HISTOPATHOLOGY:
- all tissues collected at the scheduled sacrifice from all Main group 1 and 4 animals,
- all gross lesions.
Other examinations:
None
Statistics:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test1 (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test2 (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test3 was applied to frequency data.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Red staining of various body parts and red faeces were noted among all groups treated with the test substance. This was considered to be related to staining properties of the test substance (a violet powder).
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
The statistically significant, lower white blood cell counts (WBC) of males at 1000 mg/kg/day at the end of the recovery phase was absent at the end of the treatment phase. The statistically significant lower mean corpuscular haemoglobin concentration (MCHC) values of females at 50 and 250 mg/kg/day occurred in the absence of a dose-related trend. Therefore, these changes were considered to be of no toxicological significance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Calcium levels of males at 1000 mg/kg/day were slightly reduced at the end of the treatment period, achieving a level of statistical significance. At the end of the recovery phase calcium levels were similar to control levels.
The statistically significant lower alanine aminotransferase activity level (ALAT) and cholesterol level of females at 50 mg/kg/day occurred in the absence of a dose-related trend. The statistically significant lower glucose levels of females at 1000 mg/kg/day at the end of the recovery phase was absent at the end of the treatment phase. Therefore, these changes were considered to be of no toxicological significance.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
At the end of the treatment period, the following statistically significant differences in urine between treated and control animals were noted:
- Slightly reduced sodium excretion (mmol/TPV) in males at 1000 mg/kg/day,
- Slightly reduced potassium excretion (mmol/TPV) in males at 250 and 1000 mg/kg/day (without a clear dose-related trend).
These changes achieved a level of statistical significance. At the end of the recovery phase, no statistically significant changes in these parameters were observed.
The nature of the statistically significant lower number of crystals in females at 1000 mg/kg/day was considered not to represent toxicity. The statistically significant lower urinary pH of females at 1000 mg/kg/day at the end of the recovery phase was absent at the end of the treatment phase, and hence considered to be of no toxicological significance.

Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No toxicologically significant changes were noted in organ weights and organ to body weight ratios. The statistically significant higher testes weight and testes to body weight ratio at 1000 mg/kg/day at the end of the recovery phase was absent at the end of the treatment period. Also, mean testes weights remained within the range encountered at the end of the treatment period. The statistically significant lower spleen weight and higher spleen to body weight ratio in males and females at 250 mg/kg/day respectively occurred in the absence of a dose-related trend. These changes were therefore considered not to be a sign of toxicity. Other organ weights and organ to body weight ratios among the dose groups were similar to control levels
Gross pathological findings:
no effects observed
Description (incidence and severity):
no effects (except for purple contents of the caecum, stomach and/or colon, and/or purple discolouration of the tail and/or hairs of various body parts were noted among groups treated with the test substance). Purple discolouration of the tail remained present in most animals at the end of the recovery period. These findings were considered to be related to staining properties of the test substance (a violet powder). Histologically, the presence of the purple pigment in the caecum, stomach and skin of the tail was not associated with any pathological alteration. Therefore, these findings were considered to be of no toxicological relevance.
Other necropsy findings were considered to be of no toxicological significance since they occurred in the absence of a treatment-related distribution, and are occasionally seen among rats used in these types of studies. These findings consisted of an enlarged tail of the epididymides, a yellowish-son nodule on the epididymides, red discolouration of the mandibular lymph node and fluid in the uterus. No macroscopic abnormalities were noted in control males at the end of the treatment period.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There was no morphologic evidence of toxicity.
Purple contents were seen in the cecum of two animals of group 2 and all animals of groups 3 and 4 (main) correlating to the purple contents recorded at necropsy. In the stomach of one male and female of group 3 and one male of group 4 purple pigment was seen, correlating to the purple contents recorded at necropsy. Microscopically purple pigment was present on the keratin layer of the skin of the tail of some treated animals of groups 3 and 4 (main and recovery) correlating to the purple discolouration recorded at necropsy.
The remaining microscopic findings recorded were within the normal range of background pathology encountered in Wistar rats of this age and strain
Histopathological findings: neoplastic:
no effects observed
Other effects:
not examined

Effect levels

Key result
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: No toxicologically significant changes were noted in any of the parameters investigated in this study. Likewise, no effects were found in the recovery group.

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
A OECD test guideline and GLP-compliant 28-day oral toxicity study including a 14-day recovery period was performed with the test item (Pigment Violet 23). Groups of 5 male and 5 female Wistar rats received doses of 0, 50, 250 and 2000 mg/kg bw by daily gavage for 28 days; an extra 5 animals per sex in the control and high dose group were allowed 14 days of recovery.
No toxicologically significant changes/observations in clinical appearance, functional observations, body weights and food intake were recorded during the in-life phase. Purple staining of various body parts and/or purple faeces among the dose groups treated with the test substance was considered to be related to staining properties of the test substance (a violet powder). The presence of the purple pigment in the caecum, stomach and skin of the tail recorded at necropsy was not associated with any pathological alteration and hence considered to be of no toxicological significance.
Calcium levels of males at 1000 mg/kg/day were slightly reduced at the end of the treatment period, achieving a level of statistical significance. At the end of the recovery phase calcium levels were similar to control levels. Urinalyses at the end of the treatment period showed slightly reduced sodium and potassium excretion in males at 1000 mg/kg/day, and for potassium excretion also in males at 250 mg/kg/day (without a clear dose-related trend). These changes had resolved at the end of the recovery phase. Also, considering the slight nature of these changes, and absence of any supportive toxicologically significant morphological findings, no toxicological significance was ascribed to these changes.
In conclusion, no toxicologically significant changes were noted in any of the parameters investigated in this study (i.e. clinical appearance, functional observations, body weight, food consumption, clinical laboratory investigations, macroscopic examination, organ weights, and microscopic examination). From the results presented in this report a definitive No Observed Adverse Effect Level (NOAEL) for the test item of 1000 mg/kg/day was established.
Executive summary:

The test substance, formulated in vehicle, was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting

of 5 males and 5 females. An extra 5 animals per sex in the control and high dose group were allowed 14 days of recovery.

Evaluated parameters

Chemical analyses of formulations preparations were conducted once during the study to assess accuracy, homogeneity and stability over 5 hours.

The following parameters were evaluated: clinical signs daily; functional observation tests in week 4; body weight and food consumption weekly; clinical pathology and macroscopy at

termination; organ weights and histopathology on a selection of tissues.

Results

Formulation analyses confirmed that formulations of test substance in propylene glycol were prepared accurately and homogenously, and were stable over at least 5 hours.

No toxicologically significant changes/observations in clinical appearance, functional observations, body weights and food intake were recorded during the in-life phase.

Purple staining of various body parts and/or purple faeces among the dose groups treated with the test substance was considered to be related to staining properties of the test substance (a

violet powder). The presence of the purple pigment in the caecum, stomach and skin of the tail recorded at necropsy was not associated with any pathological alteration and hence considered

to be of no toxicological significance.

Calcium levels of males at 1000 mg/kg/day were slightly reduced at the end of the treatment period, achieving a level of statistical significance. At the end of the recovery phase calcium

levels were similar to control levels. Urinalyses at the end of the treatment period showed slightly reduced sodium and potassium excretion in males at 1000 mg/kg/day, and for

potassium excretion also in males at 250 mg/kg/day (without a clear dose-related trend). These changes had resolved at the end of the recovery phase. Also, considering the slight nature of

these changes, and absence of any supportive toxicologically significant morphological findings, no toxicological significance was ascribed to these changes.