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Description of key information

Based on the findings of the 28-day repeated dose toxicity study in male and female Wistar rats by oral gavage, the No Observed Adverse Effect Level (NOAEL) of Reactive Yellow 214 was set at 1000 mg/kg bw/day.

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 July 2006 to 20 November 2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
not specified
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Qualifier:
according to guideline
Guideline:
other: Japanese Guidelines for Screening, Toxicity Testing of Chemicals: Testing Methods for new Substances, enacted July 13, 1974, amended December 5, 1986
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
Identity: FAT 40826/A
Batch no.: TZ 5604 BOP 01/06
Expiration date: February 01, 2011
Purity: Content of organic part (Na-salt): approx. 78 %; Oligomers: 13 %; Main component: approx. 48 %
Solubility in water: Approx. >50 g/L at room temperature
Stability in water: Max. 7 days at room temperature
pH: 7.6 (1 g/L)
Aggregate state/physical form at room temperature: Solid (orange powder)
Storage conditions: At room temperature at about 20 °C, away from direct sunlight
Specific instructions: Store in desiccator
Species:
rat
Strain:
Wistar
Details on species / strain selection:
Rationale Recognized by the international guidelines as the recommended test system.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services, CH-4414 Füllinsdorf / Switzerland
- Age at delivery: 6 weeks
- Weight at acclimatization: Males: 135.4-159.3 grams (mean 148.1 grams), Females: 113.3- 131.1 grams (mean 121.3 grams)
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('Lignocel' Schill AG, CH-4132 Muttenz/Switzerland).
- Diet: Pelleted standard Provimi Kliba 3433 (batch nos. 23/06 and 36/06) rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst/ Switzerland), ad libitum.
- Water: Community tap-water from Itingen was available ad libitum in water bottles.
- Acclimation period: Under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Details on route of administration:
Rationale: Administration by gavage is a common and accepted route of exposure for studies of this type.
Vehicle:
water
Remarks:
bidistilled water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The dose formulations were prepared weekly. FAT 40826/A was weighed into a glass beaker on a tared Mettler balance and the vehicle added. The mixtures were prepared weekly using a magnetic stirrer and stored at room temperature (20±5 °C) in glass beakers. Homogeneity of the test item in the vehicle was maintained during the daily administration period using a magnetic stirrer.

Dose volume: 10 mL/kg body weight
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment.
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Control
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Low dose
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Middle dose
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
High dose
No. of animals per sex per dose:
30 males and 30 females;
Groups 0 mg/kg/day and 1000 mg/kg/day: 10 males; 10 females
Groups 50 mg/kg/day and 200 mg/kg/day: 5 males; 5 females
Control animals:
yes, concurrent vehicle
Details on study design:
- In this subacute toxicity study, FAT 40826/A was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, bidistilled water, only. The groups comprised 5 animals per sex that were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14 day treatment-free recovery period after which they were sacrificed.
- Rationale for dose level selection: Based upon the results of a non-GLP 5-day dose range-finding study (RCC Study Number A73697) in which FAT 40826/A was administered by gavage to 2 rats per group and sex.
Observations and examinations performed and frequency:
- Mortality/viability: Observations for mortality/viability were recorded twice daily.
- Cage side observations: The animals were observed for clinical signs once before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28 and once daily during days 29-42 (recovery).
- Clinical observations: The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter.
- Food consumption: The food consumption was recorded once during the pretest period and weekly thereafter.
- Body weight: Body weights were recorded weekly during the pretest, treatment and recovery and before necropsy.
- Functional observational battery: During week 4, relevant parameters from a modified Irwin screen test were evaluated in all animals. Grip strength: Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded. Locomotor activity: Locomotor (decreased or increased) activity was measured quantitatively with AMS Föhr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded. Low beams count was reported in 10-minute intervals as well as the total activity of the measuring period.
- Clinical laboratory investigations: Blood samples were drawn from the retro-orbital plexus from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. The samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Urine was collected during the 18 hours fasting period into a specimen vial, using a metabolism cage. The following hematology parameters were determined: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Platelet count, Reticulocyte count, Reticulocyte maturity index, Leukocyte count, Differential leukocyte count, Heinz bodies, Methemoglobin, Thromboplastin time, Activated partial thromboplastin time. The following clinical biochemistry parameters were determined: Glucose, Urea, Creatinine, Bilirubin, Cholesterol, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase, Creatine kinase, Alkaline phosphatase, Gamma-glutamyl-transferase, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic, Protein, Albumin, Globulin, Albumin/Globulin ratio. The following urinalysis parameters were determined: Volume (18 hours), Specific gravity (relative density), Color, Appearance, pH, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes.
Sacrifice and pathology:
- All animals were weighed and necropsied. Descriptions of all macroscopic abnormalities were recorded.
- All animals surviving to scheduled necropsy were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution (unless otherwise indicated): Adrenal glands, Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain (4 levels), Cecum, Colon, Duodenum, Epididymites (fixed in Bouin's solution), Esophagus, Eyes with optic nerve (fixed in Davidson's solution), Harderian gland (fixed in Davidson's solution), Heart, Ileum with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Larynx, Lacrimal gland (exorbital), Liver, Lungs (infused with formalin at necropsy), Lymph nodes (mesenteric, mandibular), Mammary gland area, Nasal cavity, Ovaries, Pancreas, Pituitary gland, Prostate gland (incl. coagulating gland), Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, mid-thoracic, lumbar), Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland), Tongue, Trachea, Urinary bladder (infused with formalin at necropsy), Uterus, Vagina, Gross lesions.
-The following organ weights were recorded on the scheduled dates of necropsy: Brain, Heart, Liver, Thymus, Kidneys, Adrenals, Spleen, Testes, Epididymites, Ovaries. The organ to terminal body weight ratios as well as organ to brain weight ratios were determined. The determination of the terminal body weight was performed immediately prior to necropsy.
- Slides of organs and tissues that were collected at scheduled sacrifice from the animals of control and high-dose groups were examined by a pathologist. Due to findings in the high dose group, forestomach from the animals of the low and middle dose groups were examined to establish a no-effect level.
Statistics:
The following statistical methods were used to analyze the grip strength, locomotor activity, body weight, clinical laboratory investigations, organ weights and ratios and macroscopic findings as well as:
• The Dunnett-test (many to one t-test) based on a pooled variance estimate were applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
• The Steel-test (many-one rank test) was applied instead of the Dunnetttest when the data could not be assumed to follow a normal distribution.
• Fisher's exact-test was applied to the macroscopic findings.
• Student's t-test was applied to grip strength and locomotor activity data.
Clinical signs:
no effects observed
Description (incidence and severity):
However, alopecia on the left shoulder was found in one male (no. 18) treated with 200 mg/kg/day during the last three days of treatment. One female (no. 47) treated with 200 mg/kg/day transiently had crusts around the left eye during days 14-17 of treatment.
Mortality:
no mortality observed
Description (incidence):
However, One female of the control group (No. 35) died in anesthesia after blood sampling on the scheduled day of necropsy.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
In test item treated males, the mean body weight was slightly higher than in controls during treatment and recovery. The difference was statistically significant in weeks 2 and 3 in males treated with 1000 mg/kg/day and in week 4 in males treated with 200 mg/kg/day. Mean body weight gain tended to be increased during weeks 1 to 3 in all test item treated males without statistical significance. Insofar as there was no dose relation, this finding is considered not to be test item-related. In test item treated females, mean body weight and body weight gain were comparable to that of controls.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In males treated with 1000 mg/kg/day, the following statistically significant changes were found after 4 weeks of treatment: Mean cell haemoglobin and mean cell haemoglobin concentration were decreased with dose relationship. Absolute and relative number of reticulocytes and absolute number of eosinophils was increased. Absolute number of large unstained cells was increased and the prothrombin time was elevated. In females treated with 1000 mg/kg/day, the following statistically significant changes were found after 4 weeks of treatment: Mean cell haemoglobin concentration was decreased. The absolute number of large unstained cells was increased and the prothrombin time was elevated. White blood cell count, the number of lymphocytes, and the number of basophils were increased. Methemoglobin was slightly decreased. After 2 weeks of recovery, increased red cell distribution width and partial thromboplastin time were noted in males treated with 1000 mg/kg/day only. However, these findings were within the range of normal biological variation in rats of this strain and age and are considered to be of no toxicological relevance.
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
no effects observed
Description (incidence and severity):
The few findings in urinalysis after 4 weeks only were minor, inconsistent across sexes and either without dose relation or within the range of normal biological variation for rats of this strain and age.
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
Grip Strength
No test item-related differences were noted in the mean grip strength when compared with the controls. A statistically significant decrease was noted in forelimb grip strength of females treated with 1000 mg/kg/day. This observation was neither dose related nor consistent across sexes, and is therefore considered to be incidental.

Locomotor Activity
No test item-related differences were noted in the mean locomotor activity when compared with the controls. A statistically significant increase in locomotor activity was seen from 0-10 min. in females treated with 200 or 1000 mg/kg/day (p <0.01). As this observation was not consistent across sexes, it is considered to be incidental.
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
After 4 Weeks
In females treated with 1000 mg/kg/day, a decrease in adrenal weight was noted, and was statistically significant in absolute weight and adrenal to brain weight ratio. This is considered to be a stress response.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
After 4 weeks of treatment, one male treated with 50 mg/kg/day had enlarged bronchial lymph nodes. Discoloration of the ovaries was found in one female of the control group and watery cysts in the ovaries were found in one female treated with 1000 mg/kg/day. After the recovery period, one male and one female of the control group showed renal pelvic dilation. Foci in the thymus were found in one male of the control group and one female treated with 1000 mg/kg/day. One male treated with 1000 mg/kg/day showed discoloration of the pancreas and one female had foci in the stomach. These findings are within the range of normal findings in rats of this strain and age and are considered to be incidental.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
After 4 weeks of treatment, minimal to slight reversible focal spongiosis of the stratified squamous epithelium in the forestomach at the limiting ridge between forestomach and glandular stomach was found in all animals treated with 1000 mg/kg/day and in none of the controls. Additionally, minimal diffuse hyperkeratosis of the forestomach epithelium was found in all animals treated with 1000 mg/kg/day, in 3 of 5 males and females each treated with 50 mg/kg/day, in 3 of 5 males and 4 of 5 females treated with 200 mg/kg/day, and in 4 of 5 males and 3 of 5 females of the control group. Focal spongiosis of the stratified squamous epithelium in the forestomach is regarded as test item-related whereas minimal diffuse hyperkeratosis is regarded as treatment-related minimal local irritation. After recovery, spongiosis was not found and the incidence of hyperkeratosis in controls and test item treated animals was comparable.
All other microscopic findings noted were considered to be incidental as their morphology, severity, and incidence did not distinguish treated rats from control rats.
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: Study findings
Dose descriptor:
NOEL
Effect level:
200 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Study findings
Critical effects observed:
not specified
Conclusions:
The No Observed Adverse Effect Level (NOAEL) of FAT 40826/A in 28-day repeated dose toxicity study in Wistar rats was determined to be 1000 mg/kg bw/day.
Executive summary:

In ta GLP-compliant subacute toxicity study, FAT 40826/A was administered daily by oral gavage to Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, bi-distilled water, only. The study was carried out according to OECD 407 and EU method B.7. The groups comprised 5 animals per sex, which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed. Clinical signs, food consumption and body weights were recorded periodically during acclimatization, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals. From the animals of the low and middle dose groups, forestomach were examined to establish a no-effect level. Oral administration of the test substance for 28 days did not resulted in mortality, clinical signs, effects on grip strength or locomotor activity, effects on food consumption, and effects on body, organ weight, clinical pathology and gross pathology. There was minimal to slight focal spongiosis of the stratified squamous epithelium in the forestomach in all animals treated with 1000 mg/kg/day is regarded as test item-related but was reversible after recovery and therefore is considered not to be adverse. Minimal diffuse reversible hyperkeratosis of the forestomach epithelium present in all animals treated with 1000 mg/kg/day and in some males and females treated with 200 or 50 mg/kg/day or of the control group was regarded as treatment-related minimal local irritation. Based on the study findings 200 mg/kg bw/day of FAT 40826/A was established as the no-observed-effect-level (NOEL) and 1000 mg/kg bw/day of FAT 40826/A as the no-observed-adverse-effect-level (NOAEL).

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:
GLP-compliant guideline study

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

Repeated dose toxicity: oral

In ta GLP-compliant subacute toxicity study, FAT 40826/A was administered daily by oral gavage to Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg body weight/day for a period of 28 days. A control group was treated similarly with the vehicle, bi-distilled water, only. The study was carried out according to OECD 407 and EU method B.7. The groups comprised 5 animals per sex, which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed. From the animals of the low and middle dose groups, forestomach were examined to establish a no-effect level. Oral administration of the test substance for 28 days did not resulted in mortality, clinical signs, effects on grip strength or locomotor activity, effects on food consumption, and effects on body, organ weight, clinical pathology and gross pathology. There was minimal to slight focal spongiosis of the stratified squamous epithelium in the forestomach in all animals treated with 1000 mg/kg/day is regarded as test item-related but was reversible after recovery and therefore is considered not to be adverse. Minimal diffuse reversible hyperkeratosis of the forestomach epithelium present in all animals treated with 1000 mg/kg/day and in some males and females treated with 200 or 50 mg/kg/day or of the control group was regarded as treatment-related minimal local irritation. Based on the study findings 200 mg/kg bw/day of FAT 40826/A was established as the no-observed-effect-level (NOEL) and 1000 mg/kg bw/day of FAT 40826/A as the no-observed-adverse-effect-level (NOAEL).

Justification for classification or non-classification

Based on the findings of the repeated dose toxicity study, Reactive Yellow 214 does not warrant classification according to Regulation (EC) No. 1272/2008 (CLP).

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