Registration Dossier

Diss Factsheets

Administrative data

Description of key information

No indication of systemic uptake after ingestion at the limit dose of 1000 mg/kg bw was observed in a GLP compliant study with rats following OECD testing guideline 422 (BASF 2013).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2011-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD 422 compliant study
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)
Limit test:
no
Species:
rat
Strain:
other: Cri :WI(Han)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services GmbH, Sulzfeld, Germany
- Age at study initiation: 11 - 12 weeks
- Weight at study initiation:
- Fasting period before study: overnight
- Housing: 1 rat per cage, except during mating
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: six days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2012-02-14 To: 2012-03-30
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
To prepare the 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 magnetic stirrer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least twice a week and were stored at room temperature.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability of the test substance in drinking water for a period of 7 days at room temperature was proven before the study.
Homogeneity and concentration control analyses of the test-substance preparations were performed in all concentrations at the start of the administration period. Samples taken toward the end of the administration period were not evaluated.

Duration of treatment / exposure:
42 days
Frequency of treatment:
daily
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw
Basis:

No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose-range-finding study
Positive control:
not required
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: No

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: No

OPHTHALMOSCOPIC 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, Volume


NEUROBEHAVIOURAL 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 activity

OTHER: Reproduction data and pup observation (See chapter "Toxicity to reproduction")


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Weight assessment was carried out on all animals. The following weights were determined:
Adrenal glands, Anesthetized animals, Brain, Epididymides, Heart, Kidneys, Liver, Testes, Spleen, Thymus

The 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.




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.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food efficiency:
no effects observed
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:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
The detailed clinical observations on study days 0, 7, 14, 21, 28, 35 in males and females and, additionally, on study days 42, 49 and 56 in female animals did not reveal any additional abnormalities to red colored feces, which were seen from study day 7 onwards, in animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d).
A red pigment, ranging from fine particles of 1 μm to crystals of approximately 50 μm in diameter was frequently seen in the contents of the gastrointestinal tract of males and females of test group 3 (1000 mg/kg bw/d). This substance was present with higher incidence in the glandular stomach, jejunum and cecum and less frequently in the duodenum and colon. The substance amount ranged from minimal to slight.
As the test item is a red pigment, this finding indicates gastrintestinal passage.

Dose descriptor:
NOEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: absence of effects up to the limit dose
Critical effects observed:
no
Conclusions:
Pigment Red 177 causes no indication of toxicity upon subacute oral dosing of rats.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Valid without restriction.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Pigment Red 177 was administered orally via gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (mg/kg bw/d; test group 0), 100 mg/kg bw/d (test group 1), 300 mg/kg bw/d(test group 2) and 1000 mg/kg bw/d(test group 3). Drinking 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 behavior, 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 followed by an additional treatment until one day before sacrifice.

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. For the 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.

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 under isoflurane anesthesia with CO2 and examined macroscopically for external and visceral findings.

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.

Clinicochemical and hematological examinations as well as urinalyses were also performed towards the end of the administration period in 5 animals per sex and test group.

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

The various 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 red pigment caused the discoloration of feces. No adverse effects were observed at any dose level. The NOEL is 1000 mg/kg bw.

This finding is consistent with prediction of lack of systemic uptake after ingestion based on the physico-chemical properties. This is described in detail in the section of the toxicokinetic properties. Therefore, testing of subchronic toxicity is not proposed.

 

 


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Valid study

Justification for classification or non-classification

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. No serious irreversible effects were observed at dose levels of less than 150 mg/kg bw upon subacute exposure. As a result the substance is not considered to be classified for repeated dose toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive2009/2/EG.

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. There were no significant toxic effects at doses of less than 300 mg/kg bw upon subacute oral exposure in rats. As a result the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the third time in Directive EC 618/2012.