GOLDEN YELLOW SCJ 1006

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1996

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study according to OECD protocol without deviations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

92/69/EEC

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

Identification: FAT 40543/A
Description: Red powder
Batch: TV RN 196-200
Purity: 90%
Test substance storage: At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: November 01, 2000
Vehicle: 1% aqueous carboxymethyl cellulose
Stability in vehicle: Stable for at least 4 hours

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System: Rat: Wistar Crl:(WI) BR (outbred, SPF-Quality), Recognised by international guidelines as the recommended test system (e.g. EPA, FDA, OECD, EEC). Females were nulliparous and non-pregnant. Source : Charles River, Sulzfeld, Germany
Age at Start of Treatment: Approximately 6 weeks.
Number of animals: 30 males, 30 females
Randomisation: At least 5 days before study start, by computer-generated random algorithm according to body weight, with all animals within + 20% of the sex mean. A health inspection was performed prior to commencement of treatment to ensure that the animals were in a good state of health.
Identification: Earmark and tattoo
ALLOCATION
1 Main group (vehicle, Dose Level 0 mg/Kg bw/ d): 5 males and 5 females
1 Recovery group (Dose Level 0 mg/Kg bw/ d): 5 males and 5 females
2 Main group (Dose Level 50 mg/Kg bw/ d): 5 males and 5 females
3 Main group (Dose Level 200 mg/Kg bw/ d): 5 males and 5 females
4 Main group (Dose Level 1000 mg/Kg bw/ d): 5 males and 5 females
4 Recovery group (Dose Level 1000 mg/Kg bw/ d): 5 males and 5 females

Dose levels were selected on the basis of a 5-day dose range finding study (NOTOX Project 162269). A summary of results is included in this report.
ANIMAL HUSBANDRY
Conditions: Air-conditioned room with approximately 15 air changes per hour and the environment controlled with optimal conditions considered as being a temperature of 21°C and a relative humidity of 50%. Fluctuations from these optimal conditions were noted, but were considered not to have affected study integrity. Lighting was 12 hours artificial fluorescent light and 12 hours dark per day.
Accommodation: Group housing of 5 animals per sex per cage in stainless steel suspended cages with wire mesh floors. Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.
Diet: Free access to standard pelleted laboratory animal diet (from Carfil Quality BVBA, Oud-Turnhout, Belgium). Each batch is analysed for nutrients and contaminants are analysed on a regular basis. Results are examined and archived.
Water: Free access to tap-water. Certificates of analysis (performed quarterly) were examined and archived.

Administration / exposure

Route of administration:

oral: unspecified

Vehicle:

other: 1 % aqueous carboxymethylcellulose

Details on oral exposure:

TREATMENT
Method: Oral gavage, using a stainless steel stomach tube.
Frequency: Once daily for at least 28 days, approximately the same time each day, 7 days per week.
Dose volume: 5 ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.
RECOVERY
Duration: At least 14 days.

Details on analytical verification of doses or concentrations:

ANALYSIS OF DOSE PREPARATIONS
Test substance formulations in 1% aqueous carboxymethyl cellulose were noted as stable for at least 4 hours and formed a homogeneous suspension at the concentrations tested. Analysis of the accuracy of dose preparations revealed values within the range of 97 % to 100 % of nominal, which was considered to represent an acceptable level of accuracy for formulations of this type.
Test substance FAT 40543/A was a mixture and consisted of 2 components. Quantitative analyses were based on the largest peak in the HPLC chromatogram of FAT 40543/A. (See NOTOX PROJECT Project 162811: "Development and validation of an analytical method for FAT 40543/A").
Calibration solutions: Two independently prepared calibration solutions of FAT 40543/A in dimethyl sul foxide were used each day of analysis to calibrate the analytical method.

Duration of treatment / exposure:

Test duration: 28 days

Frequency of treatment:

Dosing regime: 7 days/week

Doses / concentrations

Remarks:

Doses / Concentrations:
0, 50, 200 and 1000 mg/kg bw/d
Basis:
actual ingested

No. of animals per sex per dose:

Male: 10 animals at 0 mg/kg bw/day Male: 5 animals at 50 mg/kg bw/day Male: 5 animals at 200 mg/kg bw/day Male: 10 animals at 1000 mg/kg bw/day Female: 10 animals at 0 mg/kg bw/day Female: 5 animals at 50 mg/kg bw/day Female: 5 animals at 200 mg/kg bw/day Female: 10 animals at 1000 mg/kg bw/day

Control animals:

yes, concurrent vehicle

Details on study design:

Based on the results of a 5-day range finding study, the dose levels for the 28-day toxicity study were selected to be 0, 50, 200 and 1000 mg/kg/day. The study was based on the following guidelines: EEC Directive 92/69/EEC, B.7 Sub-acute Toxicity - Oral, 1992 and Substance Law 1987, Notification of Dec. 9 1986 by EA, MHW and MITI.
The test substance 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. The following parameters were evaluated: clinical signs daily; body weight and food consumption weekly; ophthalmoscopy at weeks 4 and 6; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

OBSERVATIONS
Mortality / Viability: Twice daily.
Clinical signs: At least once daily from day 1 onwards. The time of onset, degree and duration were recorded.
Body weights: During treatment period days 1, 8, 15, 22 and 28 and during recovery period days 1, 8 and 14.
Food consumption: Weekly.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Ophthalmoscopic examinations: Both eyes were examined following instillation of tropicamide solution (5 mg/ml) during weeks 4 (all groups) and 6 (recovery groups).
CLINICAL LABORATORY INVESTIGATIONS
Blood samples were collected under light ether anaesthesia, between 7.30 and 9.30 a.m. The animals were fasted overnight before blood sampling, but water was provided. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes prepared with EDTA for haematological parameters (0.6 ml), with citrate for clotting tests (1.0 ml) and untreated tubes for clinical biochemistry parameters (>2.0 ml).
HAEMATOLOGY
The following haematology parameters were determined from blood prepared with EDTA as an anti-coagulant: Erythrocyte count/RBC, Haemoglobin/HB, Haematocrit/HCT, Mean corpuscular volume/MCV, Mean corpuscular haemoglobin/MCH, Mean corpuscular haemoglobin concentration/MCHC, Platelet count, Red cell distribution width/RDW, Total leucocyte count/WBC, Differential leucocyte count/SEG (Neutrophils) EO (Eosinophils), BASO (Basophils), LYMPH (Lymphocytes), MONO (Monocytes)
The following haematology parameters were determined from blood prepared with citrate as an anti-coagulant:
Prothrombin time/PT, Partial thromboplastin time/PTT
CLINICAL BIOCHEMISTRY
The following clinical biochemistry parameters were determined from serum samples :
Alanine aminotransferase/(ALAT/GPT), Aspartate aminotransferase/(ASAT/GOT), Bilirubin, total/BILI T., Cholesterol, total/CHOLEST. T., Triglycerides/TRIGL., Creatinine, Glucose, Urea, Protein, total/PROTEIN T., Protein, albumin/ALBUMIN, Protein, globulin/GLOBULIN, Albumin Globulin ratio A/G RATIO, Alkaline phosphatase/ALP, Sodium, Potassium, Chloride, Calcium, Phosphorus/INORG. PHOSPH .

Sacrifice and pathology:

PATHOLOGY/NECROPSY
All animals surviving to the end of the observation period were deeply anaesthetised using ether vapour and subsequently exsanguinated. All animals assigned to the study 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 neutral phosphate buffered 4% formaldehyde solution:
Adrenal glands, Aorta, Brain, Caecum, Cervix, Clitoral gland, Colon, Duodenum, Epididymides, Eyes with optic nerve and Harderian gland, Female mammary gland area, Femur including joint, Heart, Ileum, Jejunum, Kidneys, Larynx, Lacrimal gland, exorbital, Liver, Lung, infused with formalin, Lymph nodes - mandibular, mesenteric, Nasopharynx, Oesophagus, Ovaries, Pancreas, Pituitary gland, Preputial gland, Prostate gland, Rectum, Salivary glands - mandibular, sublingual, Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Sternum with bone marrow, Stomach, Testes, Thymus, Thyroid including parathyroid, Tongue, Trachea, Urinary bladder, Uterus, Vagina, All gross lesions.
ORGAN WEIGHTS
The following organ weights (and terminal body weight) were recorded from the surviving animals on the scheduled day of necropsy:
Adrenal glands, Brain, Heart, Kidneys, Liver, Ovaries, Spleen, Testes.
HISTOTECHNOLOGY
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin.
HISTOPATHOLOGY
Slides of adrenals, heart, kidneys, liver, spleen, stomach and testes, collected at the scheduled sacrifice from all main group animals of the control and the highest dose group, and all gross lesions of all animals were examined by a pathologist. All abnormalities were described and included in the report.

Statistics:

STATISTICAL ANALYSIS
The following statistical methods were used to analyse the data:
Univariate one-way analysis of variance was used to assess the significance of intergroup differences.
If the variables could be assumed to follow a normal distribution, the Dunnett-test (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-test (many-one rank test) was applied when the data could not be assumed to follow a normal distribution.
The exact Fisher-test was applied to the ophthalmoscopic examination 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. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

very slight reduction in body weight gainof females in HD group

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:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

slight effect on Bilirubin levels in females of HD group

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

only reddish discolouration of the gastro-intestinal tract was noted in HD group without otxicological significance.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

OBSERVATIONS
MORTALITY
No mortality occurred during the study period.
CLINICAL SIGNS
There were no clinical signs of toxicity or behavioural changes over the 28-day treatment period or the 14-day recovery period that were considered to be related to treatment.
Red staining of the faeces, urine, fur and/or tail was noted in animals treated at 1000 mg/kg/day, and red urine was also seen in all animals receiving 50 or 200 mg/kg/day. These findings were ascribed to the physico-chemical properties of the test substance without any toxicological significance.
Other findings noted in control and treated animals were considered to be within the range of biological variation for rats of this age and strain or were related to blood sampling procedures.
BODY WEIGHT
A very slight reduction in body weight gain was observed in females receiving 1000 mg/kg/day over the treatment period. No effects on body weights were seen among males receiving 1000 mg/kg/day and males and females treated 50 or 200 mg/kg/day.
FOOD CONSUMPTION
There were no clear differences in food consumption before or after allowance for body weight between treated and control animals.
OPHTHALMOSCOPIC EXAMINATION
There were no ophthalmology findings at weeks 4 and 6 that were attributed to treatment with FAT 40543/A. Abnormalities that were observed were considered to be within the normal range of background alterations for rats in this type of study.
CLINICAL LABORATORY INVESTIGATIONS
HAEMATOLOGY
After 4 and 6 weeks, haematological parameters of treated rats were considered not to have been affected by treatment. Minor statistically significant differences arising between controls and animals receiving 200 or 1000 mg/kg/day were considered to have arisen by chance and not to represent a change of toxicological significance as values remained within the historical range or no dose response relationship was established.
CLINICAL BIOCHEMISTRY
Total bilirubin levels were increased in females treated at 1000 mg/kg/day. Bilirubin levels in male of this dose group were within the historical range. Other values in treated males and females, achieving a level of statistical significance when compared to controls, were considered to have arisen as a result of slightly high or low control values and in the absence of treatment-related distribution or corroborative findings in the opposite sex, considered to be of no toxicological significance.
PATHOLOGY
MACROSCOPIC EXAMINATION
Macroscopic observations at necropsy did not reveal any alterations that were considered to have arisen as a result of systemic toxicity. Reddish discolouration of the gastro-intestinal tract was noted at the end of the treatment period in all male and female rats receiving 1000 mg/kg/day. These discolourations were considered to result from the staining properties of FAT 40543/A and not to represent a toxicological effect. These findings were no longer detected at the end of the 14-day recovery period. Other findings noted among treated animals were considered to be within the range of biological variation for rats of this age and strain.
ORGAN WEIGHTS
There were no changes in organ weights that were considered to represent a clear sign of toxicity. Among females of the 1000 mg/kg/day group a statistically significant decrease in adrenal:body weight ratios was noted after 4 weeks. For adrenals absolute organ weights are more relevant for evaluation as the weight of this organ generally does not vary with body weight. Moreover, for this organ the opposite reaction might be expected (i.e. adrenal enlargement) as a result of stress-induced effects. A statistically significant decrease in absolute liver and spleen weights and an increase in testes:body weight ratios, observed among males treated at 200 mg/kg/day after 4 weeks, were attributed to the slightly lower body weights at autopsy and therefore considered not to represent a toxicological change. After 6 weeks, kidney weights and kidney:body weight ratios were increased in males previously treated at 1000 mg/kg/day. As no such finding was present after 4 weeks nor in the treated females, this was considered to have occurred by chance.
MICROSCOPIC EXAMINATION
There were no microscopic findings noted that were considered to be treatment-related. The small number of changes recorded in treated animals were within the normal range of biological variation for rats of this age and strain.
CONCLUSION
Test substance preparations in 1% aqueous carboxymethyl cellulose appeared to be stable and homogeneous and sufficiently accurate concentrations were encountered for the purpose of this study. Slight effects were noted in 1000 mg/kg treated females only and consisted of slightly lower body weight gain and increased bilirubin levels in the blood. In the absence of any concurrent changes, no mechanism could be evaluated for these findings. From the results presented in this report a definitive No Observed Effect Level (NOEL) of 200 mg/kg/day was established. However, as no indication of altered organ morphology was seen or any clinical manifestation of adverse effects, a No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg/day may be concluded.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No indication of altered organ morphology was seen or any clinical manifestation of adverse effects.

2

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Based on slight effects noted in 1000 mg/kg treated females only and consisting of slightly lower body weight gain and increased bilirubin levels in the blood.

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Clinical observations: No deaths occurred. Slightly decreased body weight gain was observed in females at 1000 mg/kg/day. After recovery, bodyweights were comparable to controls. Substance-related red staining of the faeces, urine, fur and/or tail was noted at 1000 mg/kg/day (reversed after 2 days recovery) and red coloured urine at 50 and 200 mg/kg/day.

Laboratory findings: A slight increase in bilirubin was noted in females at 1000 mg/kg, but not in recovery animals. No effect on haematological parameters were observed.

Effects in organs: In females at 1000 mg/kg/day, a slight increase in body-relative adrenal weight noted at 4 weeks, was not observed in recovery females. Red staining of gastro-intestinal tract was observed at 1000 mg/kg/day, but was reversed in recovery animals. No microscopic findings were observed.

Applicant's summary and conclusion

Conclusions:

Result of subacute oral study with FAT 40543/A: NOAEL: 1000 mg/kg bw/d, NOEL: 200 mg/kg bw/d

Executive summary:

Test substance preparations in 1% aqueous carboxymethyl cellulose appeared to be stable and homogeneous and sufficiently accurate concentrations were encountered for the purpose of this study. Slight effects were noted in 1000 mg/kg treated females only and consisted of slightly lower body weight gain and increased bilirubin levels in the blood. In the absence of any concurrent changes, no mechanism could be evaluated for these findings. From the results presented in this report a definitive No Observed Effect Level (NOEL) of 200 mg/kg/day was established. However, as no indication of altered organ morphology was seen or any clinical manifestation of adverse effects, a No Observed Adverse Effect Level (NOAEL) of 1000 mg/kg/day may be concluded.