Registration Dossier

Administrative data

Endpoint:
acute toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Principles of method if other than guideline:
Groups of 20 male and 20 female rats were exposed via the diet to 0%, 5%, 10%, or 20% DR, seven days per week (mean daily intake of 0.0, 3.6, 7.6, and 15.0 g/kg body weight/day in males and 0.0, 4.4, 8.5, and 15.7 g/kg body weight/day in females), for 13 consecutive weeks. To adapt the rats to high dietary levels of DR, and to lessen the initial growth retardation, all rats in the 20% DR group were fed a diet containing 10% DR and 10% potato starch during the first three days of treatment. Thereafter, they were maintained on a diet containing 20% DR. Dose selection was based on results from a 14-day pilot study in rats (5/sex/dose) at the same
dosage levels. Cage-side clinical observations were performed each morning on all animals; an additional afternoon check was performed most days for dead or moribund animals. Body weights were recorded at study Day 0, weekly during the study, and at scheduled necropsy. Food consumption was assessed on a daily basis by weighing the feeders and expressed as grams per rat per day. Food conversion efficiency was calculated and expressed as grams of weight gain per gram of food consumed. The intake of DR was calculated on a weekly basis from the nominal dietary DR concentration, the food consumption, and the mean body weight for the week in question. Water consumption was measured, per cage, during study Weeks 1, 6, and 12, by weighing the drinking water bottles daily for four or five consecutive days during each of these weeks. Water consumption was expressed as grams consumed per animal per day. Ophthalmoscopic examinations were performed on all animals prior to the start of the study (Day-5), and again during the last week of the study on all control and high-dose animals.
A complete battery of hematological, clinical chemistry, and urinalysis measurements were taken at baseline and during the final week of the study on the same 10 animals per sex from each dose group (i.e., 2–3 animals from each cage). Beginning on study Day 85, the selected animals were placed in stainless-steel metabolism chambers (one rat per chamber) for three days, during which time urine was collected on ice, first from non-fasted, and then, from fasted (24 h) animals. Blood was also collected from the tip of the tail of the fasted animals for fasting glucose analysis. Urinalysis parameters assessed in both fasted and non-fasted animals included urine volume, pH, creatinine, uric acid, and glucose; additional assessments in fasted rats only included urine density, appearance, dipstick measurements (e.g., occult blood, ketones, protein, bilirubin), sediment microscopy (e.g., red blood cells (RBC), white blood cells (WBC), crystals, casts, bacteria), and electrolytes (i.e., sodium, potassium, chloride). At scheduled necropsy for the selected animals (study Days 91–94), blood was collected from the abdominal aorta following laparotomy with appropriate anticoagulation. Hematological parameters evaluated included hemoglobin, packed cell volume, erythrocyte count, leukocyte count (total and differential), reticulocytes, prothrombin time, thrombocyte count, and activated partial thromboplastin. Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated and reported. Clinical chemistry parameters evaluated on separated plasma samples included alkaline phosphatase (ALP), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), gamma glutamyl transferase (GGT), total protein, albumin, albumin/globulin ratio, urea, creatinine, uric acid, total bilirubin, cholesterol, triglycerides, phospholipids, inorganic phosphate, calcium (Ca), chloride (Cl), potassium (K), and sodium (Na).
All surviving test and control animals were sacrificed at study termination (study Days 91–95) by exsanguination via the abdominal aorta under CO2/O2 anesthesia and subjected to a complete necropsy. The
organs listed in Table 1 were harvested and weighed (paired organstogether), and relative organ weights (g/kg body weight) were calculated using terminal body weights.
GLP compliance:
yes
Test type:
other: Sub-chronic oral toxicity

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Dry powder, white to slightly yellow

Test animals

Species:
rat
Strain:
Wistar
Remarks:
(Crl:(WI)WU BR SPF)
Sex:
male/female
Details on test animals and environmental conditions:
Male and female albino Wistar rats (Crl:(WI)WU BR SPF) were
obtained from Charles River Deutschland (Sulzfeld, Germany) and
acclimated for 13 days prior to study treatment initiation. Animals were
housed (5/sex/cage) in polycarbonate cages with stainless steel grid covers
and wood shavings (Woody Clean; Type 3/4). Room climate was maintained
at a temperature of 22 ± 3 C and 30–70% relative humidity, with
10 air changes per hour and a 12-h light/dark cycle. Feed and drinking
water were available ad libitum. Animals were approximately five weeks of
age on the first day of dosing. The studies were conducted in accordance
with good laboratory practices (GLP) (OECD, 1988), the US Food and
Drug Administration (FDA, 1982), and the Organisation for Economic
Cooperation and Development (OECD) guidelines (OECD, 1998).

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Dosage formulations were prepared in powder form using a modified
pelleted commercial rodent diet (Rat & Mouse No. 3 Breeding Diet
(RM3), SDS Special Diets Services, Witham, England), in which 20% of
the barley was replaced by test article and/or pregelatinized potato starch
(Paselli WA-4, AVEBE, Foxhol, the Netherlands). The potato starch was
added to the basal diet at a level of 20% (w/w) and the DR incorporated at
the expense of potato starch. Fresh batches of test diet were prepared four
times during the course of the study and immediately stored at 18 C in
sealed plastic packages containing daily aliquots. Animal test diets were
replaced with a fresh aliquot from storage on a daily basis. Dosage
formulations were analyzed via HPLC three times during the study; all
samples were within 10% of the targeted concentrations. DR was stable in
the test diet for one day at ambient conditions and for five weeks in a
sealed container at 18 C.
Doses:
0%, 5%, 10%, or 20% DR, seven days per week (mean daily intake of 0.0, 3.6, 7.6, and 15.0 g/kg body weight/day in males and 0.0, 4.4, 8.5, and 15.7 g/kg body weight/day in females
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
Groups of 20 male and 20 female rats were exposed via the diet to 0%, 5%, 10%, or 20% DR, seven days per week (mean daily intake of 0.0, 3.6, 7.6, and 15.0 g/kg body weight/day in males and 0.0, 4.4, 8.5, and 15.7 g/kg body weight/day in females), for 13 consecutive weeks. To adapt the rats to high dietary levels of DR, and to lessen the initial growth retardation, all rats in the 20% DR group were fed a diet containing 10% DR and 10% potato starch during the first three days of treatment. Thereafter, they were maintained on a diet containing 20% DR. Dose selection was based on results from a 14-day pilot study in rats (5/sex/dose) at the same
dosage levels. Cage-side clinical observations were performed each morning on all animals; an additional afternoon check was performed most days for dead or moribund animals. Body weights were recorded at study Day 0, weekly during the study, and at scheduled necropsy. Food consumption was assessed on a daily basis by weighing the feeders and expressed as grams per rat per day. Food conversion efficiency was calculated and expressed as grams of weight gain per gram of food consumed. The intake of DR was calculated on a weekly basis from the nominal dietary DR concentration, the food consumption, and the mean body weight for the week in question. Water consumption was measured, per cage, during study Weeks 1, 6, and 12, by weighing the drinking water bottles daily for four or five consecutive days during each of these weeks. Water consumption was expressed as grams consumed per animal per day. Ophthalmoscopic examinations were performed on all animals prior to the start of the study (Day-5), and again during the last week of the study on all control and high-dose animals.
A complete battery of hematological, clinical chemistry, and urinalysis measurements were taken at baseline and during the final week of the study on the same 10 animals per sex from each dose group (i.e., 2–3 animals from each cage). Beginning on study Day 85, the selected animals were placed in stainless-steel metabolism chambers (one rat per chamber) for three days, during which time urine was collected on ice, first from non-fasted, and then, from fasted (24 h) animals. Blood was also collected from the tip of the tail of the fasted animals for fasting glucose analysis. Urinalysis parameters assessed in both fasted and non-fasted animals included urine volume, pH, creatinine, uric acid, and glucose; additional assessments in fasted rats only included urine density, appearance, dipstick measurements (e.g., occult blood, ketones, protein, bilirubin), sediment microscopy (e.g., red blood cells (RBC), white blood cells (WBC), crystals, casts, bacteria), and electrolytes (i.e., sodium, potassium, chloride). At scheduled necropsy for the selected animals (study Days 91–94), blood was collected from the abdominal aorta following laparotomy with appropriate anticoagulation. Hematological parameters evaluated included hemoglobin, packed cell volume, erythrocyte count, leukocyte count (total and differential), reticulocytes, prothrombin time, thrombocyte count, and activated partial thromboplastin. Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC) were calculated and reported. Clinical chemistry parameters evaluated on separated plasma samples included alkaline phosphatase (ALP), aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT), gamma glutamyl transferase (GGT), total protein, albumin, albumin/globulin ratio, urea, creatinine, uric acid, total bilirubin, cholesterol, triglycerides, phospholipids, inorganic phosphate, calcium (Ca), chloride (Cl), potassium (K), and sodium (Na).
All surviving test and control animals were sacrificed at study termination (study Days 91–95) by exsanguination via the abdominal aorta under CO2/O2 anesthesia and subjected to a complete necropsy. The
organs listed in Table 1 were harvested and weighed (paired organstogether), and relative organ weights (g/kg body weight) were calculated using terminal body weights.
Statistics:
Body weight data were analyzed using a one-way analysis of covariance, followed by Dunnett’s multiple comparison tests (Dunnett, 1955, 1964; Cochran, 1957; Steel and Torrie, 1960). One-way analysis of variance (ANOVA) was used in analyzing body weight change, organ weight, food and water consumption, food efficiency, red blood cell and clotting potential variables, total white blood cell counts, absolute differential white blood cell counts, clinical chemistry values, and quantitative urinary parameters. When group means were significantly different (P < 0.05), individual pair-wise comparisons were made using Dunnett’s multiple comparison method (Dunnett, 1955, 1964; Siegel, 1956; Cochran, 1957; Steel and Torrie, 1960). Independent of the ANOVA results, the homogeneity of variances was tested using Bartlett’s test, and if significantly different (P < 0.01), Kruskal–Wallis non-parametric ANOVA was performed. For reticulocyte, relative differential white blood cell count, and semi-quantitative urinary (i.e., dipstick or sediment data) parameter data, a Kruskal–Wallis non-parametric ANOVA was performed, followed by Mann–Whitney U-tests (Steel and Torrie, 1960). Fisher’s exact test was performed on all histopathology data (Siegel, 1956).

Results and discussion

Effect levels
Key result
Sex:
male/female
Dose descriptor:
LD50
Effect level:
>= 15 000 mg/kg bw
Based on:
test mat. (total fraction)
Remarks on result:
not determinable due to absence of adverse toxic effects
Mortality:
One incidental death in the male 10% group.
Clinical signs:
No effect
Body weight:
No acute effect
Gross pathology:
No gross pathology observed

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
No acute toxicity at 15 g/kg/d for 13 weeks
Executive summary:

The present study evaluated the toxicity from sub-chronic administration of D-ribose (DR) to male and female albino Wistar rats. Groups of 20 male and 20 female rats were exposed via the diet to 0%, 5%, 10%, or 20% DR, seven days per week (mean daily intake of 0.0, 3.6, 7.6, and 15.0 g/kg body weight/day in males and 0.0, 4.4, 8.5, and 15.7 g/kg body weight/day in females), for 13 consecutive weeks.

No acute effect of D-ribose was observed.