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Toxicological information

Repeated dose toxicity: oral

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Administrative data

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:
2012-1013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose:
reference to same study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
BASF SE Experimental Toxicology and Ecology 67056 Ludwigshafen, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Name of test substance: Pigment Yellow 155
Physical state/ appearance: solid/ yellow
Expiry date: 23 Mar 2021
Storage conditions: room temperature
Specific details on test material used for the study:
- Lot/batch No.: DEB2 146870
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the manufacturer, and the manufacturer holds this responsibility.
- Storage condition of test material: room temperature

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: 10-11 wks
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

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

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Pigment Yellow 155 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.

VEHICLE
- Concentration in vehicle: 1, 3, or 10 g/100 mL, respectively
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
males treated 41 days (pre-mating, post-mating)
females treated 52 days (pre-mating, gestation, 4 days lactation)
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 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:
10
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked: any signs of morbidity, pertinent behavioral changes and signs of overt toxicity, littering and lactation behavior of the dams, parturition behavior of the dams

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals

BODY WEIGHT: Yes
- Time schedule for examinations: Before the start of the administration period. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).

FOOD CONSUMPTION:
Food consumption was determined once a week for male and female parental animals, except during mating period, during gestation.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: no data
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: 5 of each sex
- Parameters checked: leukocyte count, erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, platelet count, differential blood count, reticulocytes, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: no data
- Animals fasted: Yes
- How many animals: 5 of each sex
- Parameters checked: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), gamma-Glutamyltransferase (GGT), sodium, potassium, chloride, inorganic phosphate, calcium, urea, creatinine, glucose, total bilirubin, total protein, albumin, globulins, triglycerides, cholesterol, bile acids

URINALYSIS: Yes
- Time schedule for collection of urine: males on day 35 and females on day 51
- 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: Functional observational battery was performed in the first five parental males and females (with litter) per group at the end of the administration period. The motor activity assessment (MA) was carried out in the first five parental males and females (with litter) per group at the end of the administration period.
- Dose groups that were examined: all
- Battery of functions tested: functional observational battery / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (Adrenal glands, Aorta, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Eyes with optic nerve, Esophagus, Extraorbital lacrimal gland, Epididymides, 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, 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, Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina)

HISTOPATHOLOGY: Yes (All gross lesions, Adrenal glands, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides, Heart, Ileum, Jejunum, Kidneys, Liver, Lung, Lymph nodes (mesenteric and axillary lymph nodes), Ovaries, Oviducts, Peyer’s patches, Prostate, Rectum, Sciatic nerve, Seminal vesicles, Spinal cord (cervical, thoracic and lumbar cords), Spleen, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina)

Organn weights: Epididymides, Testes, Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus
Statistics:
Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter where analysed by simultaneous comparison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity where analysed by 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.
Blood parameters were analysed by non-parametric one-way analysis using KRUSKAL-WALLIS test for parameters with bidirectional changes. 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 hypothesis of equal medians. For parameters with unidirectional changes pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians was used.
Urinalysis parameters were analysed by pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
Weight parameters were analysed by non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pair wise comparison of each dose group with the control group was performed using the WILCOXON test for the hypothesis of equal medians.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
yellow discolored feces, no mortality
Mortality:
mortality observed, treatment-related
Description (incidence):
yellow discolored feces, no mortality
Body weight and weight changes:
no effects observed
Description (incidence and severity):
The body weight in male animals was significantly decreased in test group 1 (100 mg/kg bw/d) on post-mating day 0 (-4.4%). This is however within the biological variation and not dose-related.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
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:
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:
CLINICAL SIGNS AND MORTALITY: No animal died prematurely in the present study. All male and female animals of test group 2 (300 mg/kg bw/d) and 3 (1000 mg/kg bw/d) showed yellowish discolored feces towards the end of the study.

BODY WEIGHT AND WEIGHT GAIN: Mean body weights and mean body weight gain of the F0 males in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control throughout the entire study period. The body weight in male animals was significantly decreased in test group 1 (100 mg/kg bw/d) on post-mating day 0 (-4.4%). Mean body weight and mean body weight gain of the F0 females in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control throughout the entire premating, gestation and lactation periods.

FOOD CONSUMPTION: Food consumption of the male and female F0 generation parental animals in all test substance-treated groups (100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control group during the entire study period, covering premating, gestation and lactation.

HAEMATOLOGY: No treatment-related, adverse changes among hematological parameters were observed.
In males of test group 1 (100 mg/kg bw/d) total white blood cell (WBC) counts were higher compared to controls, but this parameter was not dose-dependently changed. Therefore, this alteration was regarded as incidental and not treatment-related.

CLINICAL CHEMISTRY: No treatment-related changes among clinical chemistry parameters were observed.
In males of test groups 1 and 2 (100 and 300 mg/kg bw/d) cholesterol levels were decreased, but this parameter was not dose-dependently changed. Therefore, this alteration was regarded as incidental and not treatment-related.

URINALYSIS: No treatment-related changes among urinalysis parameters were observed.

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. There were no significant deviations concerning the overall motor activity (summation of all intervals) in male and female animals of all test groups in comparison to the concurrent control group. Regarding single intervals in males and females no significant deviations were detected.

ORGAN WEIGHTS: The weight decrease in absolute epididymis and liver weight in males of test group 2 (300 mg/kg bw/day) was regarded to be incidental due to a missing dose response relationship and missing histopathologic findings in test group 3 (1000 mg/kg bw). The decrease in thymus weights of females in test group 3 (1000 mg/kg bw/day) was also regarded to be incidental as no histopathologic correlate could explain the weight decrease (for details see table 1 and 2).
All other mean weight parameters did not show significant differences when compared to the control groups.

GROSS PATHOLOGY: Several animals of test group 3 (1000 mg/kg bw) revealed a yellow discoloration of the contents of the glandular stomach, small and large intestines. These findings are regarded to be treatment related (see table 3).
Each one male and female of test groups 1 and 2 (100 and 300 mg/kg bw/day) (animals No. 12, 21, 111, 125) revealed either some or all of the following findings: deposition or foci on the lung, deposition on the diaphragm, discoloration and enlargement of the mediastinal lymph nodes, deposition on the thymus, and deposition on the sternum. All these findings were regarded to be treatment related by gavage errors.
All other gross lesions noted were single observations and they were regarded to have developed spontaneously and unrelated to compound and treatment.

HISTOPATHOLOGY: The discoloration of the content in the digestive tract was regarded to be a consequence to the oral intake of the test substance which is of yellow color. Therefore, the gross findings in the remaining animals were not investigated in addition.
The discoloration and depositions described for animals No. 12, 21, 111, 125 in several organs of the thoracic cavity were multifocal granulomas with intrahistiocytically located yellow particles, interpreted as test substance. The granulomas were foreign body reactions interpreted as a consequence to a gavage error. The discoloration of the mediastinal lymph nodes was regarded to be the physiologic clearing route of the lung. Particles that were located intraalveolar were transported via macrophages to the regional lymph nodes and caused there the activation of the lymph nodes and the discoloration. Therefore these depositions and discolorations were caused by the test substance due to a gavage error and were not regarded to be an adverse finding.
All other findings noted were either single observations or they were biologically equally distributed between control and treatment group. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.

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 adverse effect observed at the limit dose

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 11: Absolute weights: Male animals:

Male animals

Test groups

(mg/kg bw/day)

1

(100)

2

(300)

3

(1000)

Epididymides

101%

94%*

98%

Liver

93%

88%*

95%

* p≤ 0.05

Table 12: Relative weights: female animals

Female animals

Test groups

(mg/kg bw/day)

1

(100)

2

(300)

3

(1000)

Thymus

90%

105%

82%**

** p ≤ 0.01

Table 13: Gross lesions

Male animals

Female animals

Test groups

(mg/kg bw/day)

0

(0)

1

(100)

2

(300)

3

(1000)

0

(0)

1

(100)

2

(300)

3

(1000)

Glandular stomach

Discoloration of contents

2

7

1

6

7

Jejunum

Discoloration of contents

1

1

4

Ileum

Discoloration of contents

1

7

4

Cecum

Discoloration of contents

1

7

8

Colon

Discoloration of contents

1

5

3

Applicant's summary and conclusion

Conclusions:
The pigment showed no adverse effects upon subacute oral dosings of rats.