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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
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report Date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: crystalline
Details on test material:
- Name of test material: Nicotinamide

Test animals

Species:
rat
Strain:
other: Bor: VISV (SPFCpb)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: Males 6 weeks; Females 7 weeks
- Weight at study initiation: Males 146 - 186 g; Females 125 - 165 g
- Housing: Macrolon cages, type II
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: Approx. 2 weeks under test conditions before administration of the test substance. Veterinary supervision of the animals before start of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45 - 70
- Photoperiod (hrs dark / hrs light): 6 a.m. - 6 p.m. CET artificial lighting / 6 p.m. - 6 a.m. CET natural light-dark rhythm

IN-LIFE DATES:
- From: 3 June 1991
- To: 1 July 1991

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Stability of the test item in administrated form: Before start of the study stability of the test substance in the solutions for administration at concentrations of 46.4 mg/ml and 215 mg/ml were examined by the analytical department (CFA). According to information from CFA the test substance was stable in the administration form at room temperature for 7 days.
- Concentration of the test item: A determination of the concentration of the test substance in the solutions for administration was done by the analytical department (CFA). Therefore in weeks 1 and 3 one sample each was taken from each solution (each test substance concentration), including pure vehicle, which are prepared for administration to the animals on the respective day and despatched to the sponsor for analysis.

VEHICLE
- Concentration in vehicle: 0, 46.4, 215 mg/mL
- Amount of vehicle (if gavage): 4.64 mg/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- HPLC/UV verification of doses. All tested samples proved to be colourless, clear solutions. Crystallizations or precipitations of the active ingredient were not observed in any of the solutions. In the solutions designated sample 1 (group 1), representing the pure vehicle no active ingredient was detectable under the chosen HPLC test conditions. Sample 2 (nominal content: 46.4 mg/mL) showed contents of nicotinamide of 100.3 and 100.9 % of the declared value (beginning of the test) and 101.7 and 101.4 % of the declared value (end of the test). Sample 3 (declared content: 215 mg/ml) showed values of the test item determined by HPLC of 101.1 and 100.7 % in the solutions of the beginning and 102.1 and 102.5 % in the solutions of the end of the test. Thus, all tested samples corresponded to the required contents.
Duration of treatment / exposure:
4 week administration, 6 week recovery period
Frequency of treatment:
Once daily a.m. (7 days per week)
Doses / concentrations
Remarks:
Doses / Concentrations:
Group 1: 0 mg/kg bw/day, Group 2: 215 mg/kg bw/day, Group 3: 1000 mg/kg bw/day
Basis:
nominal in water
No. of animals per sex per dose:
Total Number of Animals: 50 animals, 25 males and 25 females
Number of Animals per Dose Group: 10 male and 10 female animals in groups 1 and 3 each. The last 5 animals of each sex of these two groups were used as recovery animals. 5 male and 5 female animals in group 2.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The test system was selected on basis of international recommendations. According to these, rats are suitable for detecting potential toxic properties of test substances in rodents. For more than 5 years the rat strain Bor: WISW (SPFCpb) is being used in the Institute of Toxicology, so that historic control data of a number of toxicological studies are available.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- The animals were observed for the occurrence of toxicity symptoms as well as their severity and duration.
- Mortality was checked twice daily (a.m. and p.m.), on Saturdays, Sundays, and on national and business holidays only once daily (a.m.).

BODY WEIGHT: Yes
- Time schedule for examinations: Once a week, starting with pretest period
- 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 INTAKE (if drinking water study): Yes
- Time schedule for examinations: Food consumption was determined once a week, starting with pretest period, expressed as g/animal/day.

EXAMINATIONS OF REFLEXES, EYES, HEARING, AND TEETH: Yes
- Time schedule: Prior to first substance administration and in week 4
- Dose groups that were examined: All groups.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: In week 4 a.m. as well as in week 10 from recovery animals
- Anaesthetic used for blood collection: Yes (CO2 anesthesia)
- Animals fasted: No
- How many animals: All animals
- Parameters checked: Erythrocytes (RBC), hematocrit (Hct), hemoglobin (Hb), leucocytes (VBC), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), thrombocytes (Platelets).
Performance of examinations with COULTER COUNTER S Plus II supplied by Coulter Electronics GmbH, 0-4150 Krefeld, according to the methods of the manufacturer. Differential leucocyte count staining of the blood smears according to PAPPENHEIM and microscopical evaluation (% and quantity per mL). Reticulocytes staining of the blood smears with Brillantkresyl-blue. Staining of the blood smears with Brillantkresyl-blue; microscopical evaluation (Retic %0).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: In week 4 as well as in week 10 from recovery animals
- Animals fasted: No
- How many animals: All animals
- Parameters checked: albumin, alkaline phosphatase (AKP), aspartate aminotransferase (ASAT), blood urea, calcium (Ca), chloride (CI), cholinesterase (CHE) , creatine kinase (CK), creatinine, y-glutamyltransferase (Gamma-GT), glucose, glutamate dehydrogenase (GLDH), inorganic phosphate (P), potassium (K), serum electrophoresis, sodium (Na), total bilirubin (Tot.Bili), total cholesterol (Chol), total protein (Tot.Prot), triglycerides (Triglyc).

URINALYSIS: Yes
- Time schedule for collection of urine: In week 4 as well as in week 10 from recovery animals
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters checked: Bilirubin, glucose, hemoglobin/erythrocytes, ketones, leucocytes, nitrite, osmolality, pH-value, protein, urobilinogen. Microscopical examinations of the urine sediment were done in animals whose urine state showed pathological changes in hemoglobin/erythrocytes, leucocytes, or protein.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals, including the intercurrently killed animal, were subjected to full gross necropsy which included examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents. All gross lesions, adrenal glands (r/l), bone marrow (as present in sternum), bone marrow smear (from femur), brain (cerebrum, cerebellum, brain stem), cecum, colon, duodenum, heart, ileum, jejunum, kidneys (r/l), liver, lungs (including main bronchi), ovaries (r/l), rectum, spleen, stomach, testes (r/l).

HISTOPATHOLOGY: Yes
- All above mentioned organs and tissues except for bone marrow smears were fixed in a 4% neutral buffered formaldehyde solution (10% formalin) according to LILLIE. The bone marrow smears were air dried. All fixed organs and tissues of group 1 animals (and group 3 animals as well as of the intercurrently killed animal, and all organs with macroscopic findings were trimmed, embedded in paraffin wax, sectioned at approximately 4 um and stained with Hematoxylin and Eosin (H&E). Liver and spleen of group 2 animals and of the recovery animals of group 1 and group 3 were additionally examined histopathologically. All slides prepared with exception of bone marrow smears (no respective changes in peripheral blood parameters) were examined microscopically.

ORGAN WEIGHTHS
Adrenals (r/l), brain, heart, kidneys (r/l), liver, ovaries (r/l), spleen, testes (r/l). The organ weights (wet weights) were expressed as absolute values and relative to body weight after exsanguination (organ/body weight ratios). No organ weights were taken from the intercurrently killed animal.
Statistics:
Mean values of all parameters and where indicated - standard deviations were calculated separately for each group and sex. For statistical evaluation of food consumption, body weights, and organ weights the DUNNETT-Test was used. For values of hematological and clinical chemistry examinations the DUNNETT-Test was used in case of normal distribution, otherwise the STEEL-Test was employed. Significant differences between mean values of control group 1 and dose groups 2 and 3 were marked with * or ** (significance level of p < 0.05 or p < 0.01 according to DUNNETT) or + (significance level p < 0.05 according to STEEL), respectively.

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):
Males only, females were not affected. During the recovery period the difference in body weight gain disappeared.
Ophthalmological findings:
no effects observed
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:
effects observed, treatment-related
Description (incidence and severity):
Slight signs of an adaptively increased activity of the livers which disappeared after the end of the treatment period.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Details on results:
CLINICAL SIGNS AND MORTALITY
During daily observation of the animals only minor changes were detected. Two rats (1 male, 1 female) showed slight to moderate alopecia beginning in weeks 2 or 3. Two additional rats exhibited eschar at the neck starting in weeks 3 or 4 of the study and one rat had eschar on the head during the recovery period. Findings were recorded in treated as well as control groups without any detectable dose relation.
Two rats were wounded at the eyes during blood sampling in week 4. One animal (No. 9) was killed afterwards for reasons of animal welfare, the other was sacrificed as scheduled at the end of week 4.
All these findings were considered as non test substance related.

FOOD CONSUMPTION AND BODY WEIGHT
Both of these parameters were affected only in male rats. Food consumption was reduced significantly in animals of both treated groups (3 and 2) during the first 2 weeks of administration, but there was no difference between high and low dose animals.
During the first 2 weeks of the recovery period the difference between treated and control animals disappeared. Afterwards treated animals consumed even more than controls. The body weight gain of male rats of both treated groups was reduced during the whole treatment period. The difference in body weight appeared in a dose dependent manner in week 2 in the high dose and in week 3 in the low dose group. In high dose animals the difference reached a maximum of 38 g (about 13 % of control weight) at the beginning of the recovery period. Afterwards the difference remained almost constant.

EXAMINATIONS OF REFLEXES, EYES, HEARING, AND TEETH
No substance related effects were detected.

HAEMATOLOGY
During the hematological examination in week 4 males of the high dose group showed a minimally reduced number of platelets and an equally marginal increase in the relative number of neutrophils.
At the same time in females a reduced number of red blood cells and an increased number of white cells were found. Additionally relative and absolute numbers of neutrophils were increased, while the number of lymphocytes was reduced. All of these changes were only minimal and well within the normal range for rats of this strain and age.
At the end of the recovery period males showed none of the changes observed in week 4 but a marginal increase in hemoglobin content. Females exhibited minimal increases in erythrocytes, hemoglobin, and hematocrit. As in week 4 the deviations from the control values are considered as incidental.

CLINICAL CHEMISTRY
After 4 weeks of treatment in the low dose group only females showed small deviations from the control values in clinical chemistry. The activity of alanine aminotransferase and the concentration of calcium were slightly increased. In the high dose group male and female rats exhibited alterations in week 4.
In males the activities of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase as well as the concentrations of blood urea, albumin, and alpha2-globulins were statistically significant increased. The concentration of alpha1-globulins was slightly reduced.
In females of this group increased values were found for the activity of alanine aminotransferase and the concentrations of blood urea, total bilirubin, cholesterol, and calcium. In week 10 none of these findings had remained.

URINALYSIS
No differences between treated and non-treated animals were detected in weeks 4 and 10.

ORGAN WEIGHTS
At the end of the treatment period in the low dose group the only statistically significant finding was the increase of the relative brain weights in males, which is considered as an incidental finding because of the missing dose/response relation.
In the high dose males the absolute weights of spleens were minimally reduced and the relative weights of livers, kidneys, and adrenals were increased. These findings probably were due to the reduced body weights of these animals. In females of this group the absolute as well as the relative weights of livers were increased. After the 6-week recovery period in males the weights of right kidneys and right testes and in females the weight of hearts were reduced.

GROSS PATHOLOGY
All noted macroscopical findings are considered as incidental or artificial changes, e.g. caused by blood collection, and were not related to the treatment.

HISTOPATHOLOGY
Treatment related changes were seen in the liver and spleen. All other findings are considered to be non treatment related incidental changes.
- Liver: The liver of treated animals reacted with an adaptive response visible as mild hepatocellular induction/hypertrophy. The lesion was distributed diffusely throughout the cut surface. In the lobule it was restricted to the central areas. The cytoplasm of affected cells was characterized by an increased eosinophilia with deep eosinophilic clumps and the absence of glycogen deposits. Occasionally the cells appeared multinucleated and enlarged. This lesion was found in 1/5 group 1, 4/5 group 2, and 4/5 group 3 males. In 2/5 group 3 males it was graded slight, in all other cases it was graded minimal. In females hepatocellular induction occurred only in 2/5 group 3 animals, each one case graded minimal and slight. In the recovery animals hepatocellular induction/hypertrophy was detected in 1/5 control males and 2/5 group 3 males, graded as minimal. No female of the recovery groups has been affected. These results indicate a nearly complete regression of hepatocellular induction/hypertrophy after the recovery period.
The finding of minimal to mild hepatocellular induction/hypertrophy correlates with the increased liver weights of high dose males and females.
- Spleen: The amount of extramedullary hematopoiesis was relatively reduced in treated animals compared with controls. This became obvious in a higher incidence and severity of the extramedullary hematopoiesis in the control animals.
In females an increased extramedullary hematopoiesis was detected in each 4/5 groups 1 and 2 animals and in 2/5 group 3 rats. It was graded slight in 3/5 control and 1/5 low dose animals, moderate in 1/5 control females. In all other cases it was graded minimal. In the males the effect was less pronounced. Each 4 rats of groups 1 and 2 as well as 3 of group 3 had this finding, graded slight in 2, 2, and 1 animal of groups 1, 2, and 3, respectively, and minimal in
the remaining cases.
In the recovery groups an effect of the treatment was seen only in females, indicating a tendency towards regression. The increased extramedullary hematopoiesis was detected in 5/5 control and 2/5 group 3 rats, graded minimal with the exception of 1 group 3 animal.
All other histopathological findings are considered to be incidental changes or artificial, e.g. caused by blood collection or sacrifice procedure, or are agonal alterations without any relation to the treatment.

Effect levels

Dose descriptor:
NOAEL
Effect level:
215 mg/kg bw (total dose)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
The results of this study indicate the very low toxicity of the test item. Even at a dose of 1000 mg/kg daily administered for 4 weeks only livers were slightly affected. All changes were reversible after the end of exposure to the unphysiologically high levels of nicotinamide.
Executive summary:

A study according to EU Method B.7 and OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents) was carried out. The toxicity after 28 days of oral administration to Wistar rats and the reversibility of possible effects were examined. The study comprised 3 groups of male and female animals. While group 2 (low dose) consisted of 5 rats of each sex, the groups 1 (control) and 3 (high dose) contained 10 animals of each sex. Half of the animals of groups 1 and 3 were kept after the end of the administration period for 6 weeks to determine the reversibility of possible effects. The test material, a white crystalline powder, was dissolved in deionized water at concentrations of 46.4 mg/mL and 215 mg/mL. Animals of the control group (group 1) were treated with tap water. The doses were selected from a previous dose finding study as 215 mg/kg and 1000 mg/kg bw/day. The administration volume was 4.64 mL/kg.

RESULTS

- Clinical Investigations: During the treatment period neither behaviour nor general condition of the animals were altered due to the test substance. None of the animals died as a consequence of the test substance administration. Food consumption as well as body weight gain of females were not affected by the test material. In males of both dose groups a reduction of food consumption as well as body weight gain was detected. Food consumption was reduced significantly during the first 2 weeks and the body weight gain increasingly fell behind the control animals during the whole treatment period. During the recovery period the difference in body weight gain disappeared. At examinations of reflexes, eyes, hearing, and teeth no substance related effects were recorded.

- Clinical Pathology: The examination of hematology parameters in weeks 4 and 10 revealed no test substance related changes of toxicological relevance. Among the clinical chemistry parameters examined in week 4 the activities of aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase and the concentrations of blood urea and albumin were increased in males of group 3. Electrophoresis revealed relative increases of albumin and alpha2-globulins while alpha1-globulins were reduced in these animals. In females similarly the values of alanine aminotransferase, blood urea, total bilirubin, and calcium were increased in high dose rats. Alanine aminotransferase as well as calcium were increased in group 2 females also. All changes were only minimal to slight and were still within the normal range for rats of this strain and age. At the end of the 6-week recovery period all of the changes had disappeared. Urinalysis did not reveal any significant changes.

- Pathology: Significant differences from control were present only in animals of group 3. The absolute weights of spleen were minimally reduced in males, while the liver weights were increased in females. Relative organ weights were increased only in males. The organs affected were liver, kidneys, adrenals, and brain. After the end of the recovery period males of group 3 showed minimal reductions in the absolute weights of kidneys and testes. In females relative as well as absolute weights of hearts seemed slightly reduced. Macroscopical examination revealed no test substance related findings. During microscopical investigations changes in liver and spleen were detected. Livers, predominantly of high dose males, showed a minimal to mild hepatocellular induction (hypertrophy, which was almost completely reversible within the recovery period). In the spleens of treated animals the extramedullary hematopoiesis was minimally to slightly reduced as compared to the control animals. The finding was recorded in both dose groups.

EVALUATION

During the present 4-week oral toxicity study the test substance caused only few mild changes even at a dose of 1000 mg/kg/day. Clinically only a slight and fully reversible reduction of body weight gain and food consumption was recorded in male rats. Females did not show any test substance related effect. Hematology parameters, although statistically different, were within the normal range of variability of this strain of rats. Clinical chemistry as well as the determination of organ weights revealed slight signs of an adaptively increased activity of the livers which disappeared after the end of the treatment period. This view is also supported by the microscopical findings in this organ. The relatively reduced extramedullary hematopoiesis of treated animals compared with controls may be an adaptive response, possibly related to the abundance of administered test item. Because no effects were seen in the cellularity of the bone marrow and in the hematological parameters a toxicological significance of this finding is not to be assumed.

CONCLUSION

The results of this study indicate the very low toxicity of the test item. Even at a dose of 1000 mg/kg daily administered for 4 weeks the liver as the main target organ is only slightly affected. All changes are reversible after the end of exposure to unphysiologically high levels of test item.