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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:
04 Aug - 03 Sep 1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report date:
1993

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food, U.S.A./F.D.A./ Bureau of Foods, 1982
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Vinasses, residue of fermentation
EC Number:
932-215-9
Molecular formula:
Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance).
IUPAC Name:
Vinasses, residue of fermentation
Details on test material:
- Name of test material (as cited in study report): Lucullus 235/02
- Physical state/ appearance: brown viscous liquid
- Analytical purity: 100%
- Composition of test material, percentage of components: 75.6% active substance (dry matter) in water
- Lot/batch No.: 235/04
- Storage condition of test material: 4 °C

Test animals

Species:
rat
Strain:
other: Sprague-Dawley rats of the Crl CD (SD) BR strain
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Centre d'Elevage Charles River, 76410 Saint-Aubin-lès-Elbeuf, France
- Age at study initiation: approximately 6 weeks old
- Weight at study initiation: mean body weight of 176 g for males and 160 g for females
- Fasting period before study: none
- Housing: in suspended wire-mesh cages (43.0 x 21.5 x 18.0 cm)
- Diet (ad libitum): AO4 C pelleted diet (U.A.R., 91360 Villemoisson-sur-Orge, France)
- Water (ad libitum): tap water filtered with a 0.22 micron filter (Millipore S.A., 78140 Vélizy, France)
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2
- Humidity (%): 50 +/- 20
- Air changes (per hr): 13
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: AO4 C pelleted diet (U.A.R., 91360 Villemoisson-sur-Orge, France)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): beige powder, AO4 pelleted diet (U.A.R., 91360 Villemoisson-sur-Orge, France)
- Storage temperature of food: 4 °C
Homogeneity was obtained by mixing in the Lödige FM 50 mixer with cooling system

VEHICLE
- Concentration in vehicle: 500, 5000 and 50 000 ppm (concentrations are expressed in terms of active substance)
- Lot/batch no. (if required): batch Nos. 20206, 20305, 20415 and 20605
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
50 g of each dietary mixture (control group included) were sampled on weeks 1 and 4.
The concentration of the dietary mixtures was found to be satisfactory.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
daily ad libitum
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
48, 486 and 5221 mg/kg bw/day (females)
Basis:

Remarks:
Doses / Concentrations:
50, 498 and 5110 mg/kg bw/d (males)
Basis:

Remarks:
Doses / Concentrations:
0, 500, 5000 and 50 000 ppm
Basis:
nominal in diet
No. of animals per sex per dose:
10; in addition 8 males in a satellite group for immunological examinations
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: the concentrations were determined by the sponsor
- Rationale for selecting satellite groups: 8 males of each group were used for the Plaque Forming Cell assay and amino-acids determination

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once a day, at the same approximate daily time
BODY WEIGHT: Yes
- Time schedule for examinations: once before allocation of the animals into groups, on the first day of treatment, then once a week until the end of the study

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: Yes
Achieved dosage (mg/kg bw/day) = Concentration (ppm) x mean food consumption (g/animal/day) / mean body weight (g)

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
Food conversion ratio = mean food consumption (g/animal/week) / mean body weight gain (g/animal/week)

WATER CONSUMPTION: Yes
- Time schedule for examinations: one a week until week 3 inclusive

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before the beginning of the treatment period and on week 4
- Dose groups that were examined: control group and high dose level group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on week 4
- Anaesthetic used for blood collection: Yes (light ether)
- Animals fasted: Yes, overnight
- How many animals: all surviving animals
- Parameters checked: Leucocytes, erythrocytes, haemoglobin, packed cell volume, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, thrombocytes, differential white cell count with cell morphology (neutrophils, eosinophils, basophils, lymphocytes, monocytes), prothrombine time, activated partial thromboplastin time, fibrinogen

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on week 4
- Animals fasted: Yes
- How many animals: all survivng animals
- Parameters checked: sodium, potassium, chloride, calcium, inorganic phosphorus, glucose, urea, creatinine, total bilirubin, total proteins, albumin, albumin/globulin ratio, cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase, alanine aminotransferase, immunoglobulins G, immunoglobulins M, free-thyroxine, free-triiodothyronine, free aminoacids

URINALYSIS: Yes
- Time schedule for collection of urine: on week 4
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: Volume, pH, specific gravity, proteins, glucose, ketones, bilirubin, nitrites, blood, urobilinogen, leucocytes, erythrocytes, cylinders, magnesium ammonium phosphate crystals, calcium phosphate crystals, calcium oxalate crystals, cells, appearance

NEUROBEHAVIOURAL EXAMINATION: No data


OTHER: Immune function tests: The immune function tests performed in this study were realized on male animals only, as CIT historical data were collected from male Sprague-Dawley rats. In addition, 2 subgroups of animals were used for these tests: satellite animals for PFC assay (for which an immunization of the animals is requested) and animals of principal groups for other assays.
Plaque-forming cells assay, preparation of cell suspensions, lymphoproliferative response to Concanavaline A (Con A), phytohemagglutinin, and Lipopolysaccharide of Salmonella typhimurium, mixed lymphocyte reaction and natural killer cell activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
adrenals, aorta*, brain including medulla/pons, cerebellar and cerebral cortex, caecum, colon, duodenum, eyes with Harderian glands*, femoral bone with articulation*, heart, ileum, jejunum, kidneys, liver, lungs with bronchi, lymph nodes, mammary glands*, oesophagus*, ovaries, pancreas, pituitary gland*, prostate*, rectum*, salivary glands*, sciatic nerve*, seminal vesicles*, skeletal muscle*, skin*, spinal cord*, spleen (before exsanguination), sternum, stomach, testes and epididymides, thymus, thyroids with parathyroids, tongue*, trachea*, urinary bladder*, uterus, horns and cervix, vagina*
HISTOPATHOLOGY: Yes
microscopic examination was performed on
-all macroscopic lesions and tissues listed above in animals of the high dose level and control groups (and in any animals that died during the study)
- all microscopic lesions and lungs, liver, kidneys, caecum, ileum, lymph nodes and spleen of all the animals of the low and intermediate dose level group
Statistics:
- The normality of the distribution of the values in each group was checked by Kolmogorov-Smirnov's test (1948)
- Homogeneity of varances between groups was assessed by Bartlett's test (1937) or Fisher's test (1934)
- Comparison between treated and control groups was performed by Dunnett's test (1955) or Dunn's test (1964) or by Mann Whitney's test (1947)
- Analysis was repeated after logarithmic transformation

Results and discussion

Results of examinations

Gross pathological findings:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
No clinical signs were noted in the control, 5000 and 50 000 ppm groups.
In the 500 ppm group, 2 males and 2 females presented with emaciation for one week (day 15 - day 22). This finding was in line with a low food and water consumption during this period. As it was noted in the low dose group only and for a short period of time, this emaciation was considered not to be treatment-related but may have been related to a technical problem in the food-hopper or water bottle which prevented the animals from normal feeding or drinking.
No deaths were noted in the treated groups. One control male was found dead after blood sampling and this was considered accidental and attributed to the traumatic nature of this procedure.

BODY WEIGHT AND WEIGHT GAIN
The body weight gain of the treated males and females was similar to that of respective controls. At the end of the treatment period (week 5), a slight loss in body weight was noted in the females of the 500 and 5000 ppm groups, but this was due to an error in the schedule of weighing: the animals were weighed after the fasting which preceded necropsy instead of being weighed before fasting. This finding is therefore unrelated to treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
The food consumption of the treated males and females was similar to that of respective controls.
The achieved intake of the test substance was similar in males and females: in average, it was respectively 50 and 48 mg/kg bw/day in the 5000 ppm group and was 5110 and 5221 mg/kg bw/day in the 50 000 ppm group.

WATER CONSUMPTION
The water consumption of the animals of the 500 or 5000 ppm groups was similar to that of controls.
The water consumption of the males and females of the 50 000 ppm group was higher than that of controls: it was in average 46 mL/day for the males and 36 mL/day for the females vs. 33 mL/day and 29 mL/day for the male and female control animals, respectively. This difference, statistically significant (p < 0.05 or p< 0.01) was considered to be treatment related and could be attributed to the exogenous supply of sodium in the 50 000 ppm preparations: i.e. a supply of 6.86 g of sodium per kg of mixture.

OPHTHALMOSCOPIC EXAMINATION
No abnormalities were noted at ophthalmological examination of the males and females of the 50 000 ppm group.

HAEMATOLOGY
On week 4, the slight difference from control noted in haemoglobin level and packed cell volume were minor and occurred only at the low dose level, therefore these differences were considered not to be treatment-related.

CLINICAL CHEMISTRY
On week 4, the slight differences from the control noted among the biochemical parameters were minor, the individual values were within the normal range of background data, therefore these findings were considered to be of no toxicological importance.
The chromatography of serum free amino acids revealed higher aspartic acid, hydroxyproline, glutamic acid, alanine and methionine in the animals given 50 000 ppm. This was considered to be due to the treatment, and was an expected finding in relation to the composition of the test substance.
No detectable values were obtained for 1-Methyl histidine in 8/8 treated animals vs. 3/8 control animals. Taking into consideration the consumption of the test substance, the relationship with the treatment was considered unlikely.

URINALYSIS
No differences from control

ORGAN WEIGHTS
kidneys:
- slight increase in relative weight (+11%) in males given 50000 ppm,
- adrenal glands: slight decrease in absolute and relative weight (-21% and -20% respectively) in males given 5000 ppm; slight decrease in absolute weight (-18%) in males given 50000 ppm
- thymus: slight decrease in absolute and relative weight (-23% and -21% respectively) in females given 5000 ppm.
They were considered to be of no toxicological importance.

Macroscopic and microscopic examinations were considered as being of no toxicological importance. (changes observed were those which are commonly recorded as spontaneous findings in the laboratory rat.

OTHER FINDINGS
The plaque froming cell assay revealed a lower response at 5000 ppm. In addition, a slight decrease in B-lymphocyte proliferation in response to lipopolysaccharide was noted at the same concentration. Due to great individual variability this was not statistically significant.
The same tendency was also noted at 500 ppm but the differences from the control were slighter. Nevertheless, as no differences from controls were noted at 50 000 ppm, that no abnormalities were noted, a relationship to treatment of these findings was considered to be very doubtful.
No differences from control were noted in the other functional tests.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
5 110 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: The test material induced only a higher water consumption.
Dose descriptor:
NOAEL
Effect level:
5 221 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No obvious signs of toxicity.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Plaque forming cell assay and lymphocyte proliferation to LPS

Concentration in ppm (active substance)

0

500

5000

50000

PFC (106cells)

Mean

1394

972

782

1537

Standard deviation

529.7

350.0

295.2

785.7

LPS (103cells)

Mean

67.76

50.52

48.97

59.12

Standard deviation

29.302

28.419

16.262

23.948

Applicant's summary and conclusion