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Toxicological information

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
Study period:
02 January 2008 to 30 January 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: 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:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
other: Crl:WI(Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Approximately 6 weeks
- Weight at study initiation: males: 148-184 grams; females: 122-141 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: 19.9 – 21.5C),
- Humidity (%): 30-70% (actual range: 26 - 63%)
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
IN-LIFE DATES: From: 02 January 2008 to: 30 January 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 formed a homogeneous suspension at the concentrations tested (coefficient of variation <10%). Analysis of the accuracy of dose preparations revealed values within the range of 102-106% of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.
Duration of treatment / exposure:
28 days
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
Remarks:
Doses / Concentrations:
0 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
100 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
300 mg/kg bw/day
Basis:
actual ingested
Remarks:
Doses / Concentrations:
1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5
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
- Anaesthetic used for blood collection: Yes (iso-flurane anaesthesia)
- Animals fasted: Yes
- How many animals: 40
- 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
- Parameters examined: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase, Total Protein, Albumin, Total Bilirubin, Urea, Creatinine,
Glucose, Cholesterol, Sodium, Potassium, Chloride, Calcium, Inorganic Phosphate
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes:
At the end of week 1, 2, 3 and 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 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:
no effects observed
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
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
Red staining of various body parts (shoulder, head, flank, back, neck, cervical region, chest
and/or generalized staining) and red faeces were noted among all groups treated with the test
substance.
Red staining of various body parts, red faeces and reddish contents of the gastro-intestinal tract
noted among all groups treated with the test substance was considered to be related to staining
properties of the test substance, and not to represent signs of systemic toxicity. No correlation
histopathological abnormalities were noted.

Incidental findings that were noted included rales, alopecia, scabs and a wound on the cheek.
These findings were considered to be within the normal range of clinical signs in rats of this age
and strain which are housed and treated under the conditions in this study. At the incidence
observed, these were considered signs of no toxicological significance.

No toxicologically significant changes in body weights and body weight gain were noted.
The statistically significant higher body weight gain of females at 300 and 1000 mg/kg/day in
week 2 occurred in the absence of a time- and dose-related response, and was of a very slight
nature. No toxicological relevance was ascribed to this change.

No toxicologically relevant changes occurred in haematological parameters of treated rats.
Any statistically significant changes in haematological parameters were considered to be of no
toxicological significance as they occurred in the absence of a treatment-related distribution,
remained within the range considered normal for rats of this age and strain and occurred in the
absence of supportive haematological changes. These changes consisted of higher reticulocyte
counts in males at 1000 mg/kg/day, higher methaemoglobin levels in females at 100 mg/kg/day
and higher, and lower mean corpuscular volume in females at 100 and 1000 mg/kg/day.

No toxicologically relevant changes occurredin clinical biochemistry parameters of treated rats.
Any statistically significant changes in clinical biochemistry parameters were considered to be of
no toxicological significance as they occurred in the absence of a treatment-related distribution
and remained within the range considered normal for rats of this age and strain. These changes
consisted of lower albumin levels in males at 300 mg/kg/day, higher cholesterol levels in males
at 100 mg/kg/day, higher total protein levels and lower urea levels in females at 100 mg/kg/day,
and lower inorganic phosphate levels in females at 100 and 1000 mg/kg/day.
A noticeably high aspartate aminotransferase activity level (ASAT) and glucose level was noted
for male no. 10 and 12 respectively. The incidence of these high levels was unrelated to the
dose, and therefore considered to be of no toxicological significance.

Reddish contents of the gastro-intestinal tract were observed among all animals treated with the
test substance.
Red staining of various body parts, red faeces and reddish contents of the gastro-intestinal tract
noted among all groups treated with the test substance was considered to be related to staining
properties of the test substance, and not to represent signs of systemic toxicity. No correlation
histopathological abnormalities were noted.

Incidental findings included a gray-white focus on the right lateral lobe of the liver, fluid in the
uterus and scab formation on the skin. These findings are occasionally seen among rats used in
these types of studies. In the absence of a treatment-related distribution they were considered
changes of no toxicological significance.

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
(NO(A)EL) for PIGMENT RED 112 of 1000 mg/kg/day was established.

Effect levels

Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: No toxicologically significant changes were noted in any of the parameters investigated in this study.

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The toxicity of the test item when given by oral administration (gavage) to groups of 5 rats per sex per dose for 28 consecutive days at dosages of 0, 100, 300 or 1000 mg/kg bw/day, for 7 days/week have been investigated. 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). Red staining of various body parts, red faeces and reddish contents of the gastro-intestinal tract noted among all groups treated with the test substance was considered to be related to staining properties of the test substance, and not to represent signs of systemic toxicity. No correlating histopathological abnormalities were noted. On the basis of these results, the high dose level of 1000 mg/kg/day was considered to be the No Observed Adverse Effect Level (NOAEL) for the test substance.