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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:
1988

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 (incl. certificate)
Remarks:
Hazleton UK
Limit test:
no

Test material

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

Test animals

Species:
rat
Strain:
other: Crl:CD(SD)BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Age at study initiation: approximately 28 days
- Weight at study initiation: At the start of the toxicity phase males weighed 165.1 to 211.2 g and females 118.0 to 149.1 g
- Fasting period before study: Overnight fast before laboratory investigations and necropsy
- Housing: The animals were caged in groups of 5 in stainless steel wire mesh cages suspended over cardboard-lined trays. The cardboard liners were replaced as often as was necessary to maintain hygiene.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: The animals were acclimatised in the study room for 7 days (palatability phase) or 14 days (toxicity phase) during which time their health status was reassessed and their suitability for experimental purposes confirmed.

ENVIRONMENTAL CONDITIONS
- Temperature: 19 to 25 °C
- Humidity: 40 to 70 %
- Air changes: 15 air changes/hour
- Photoperiod: Fluorescent lighting was controlled automatically to give a cycle of 12 hours light (0600 to 1800 h) and 12 hours darkness.

IN-LIFE DATES
- From: 17 July 1987
- To: 24 August 1987

Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: The control article and vehicle for the test article was powdered diet.
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Test diets were prepared weekly for each group and sex.
- Mixing appropriate amounts with (Type of food): The diet used was SQC Rat and Mouse Maintenance Diet No. 1, Expanded, Ground fine (Special Diets Services Ltd., Witham).
- Storage temperature of food: Room temperature
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Method
The analysis of the test diets was carried out using an analytical method supplied by the sponsor. The method contains a complex series of analytical procedures. The samples are acidified and extracted under pressure. Following filtration and centrifugation, the extract is submitted to ion exchange followed by HPLC.

Validation
The analytical procedure was validated by checking linearity, analytical recoveries and precision over the expected minimum and maximum concentration ranges.

Stability and Homogeneity
The stability and homogeneity of the test article in the diet was determined. Samples were taken from diet batches mixed at concentrations considered to be the highest and lowest likely in a subsequent chronic study in the rat. Stability samples were taken on the day of mixing and 7 and 14 days later and analyzed in triplicate. Homogeneity samples were taken from 3 levels (top, middle and bottom) of the bulk mix on the day of formulation and analyzed in duplicate.

Concentrations
Formulated diets from week 1 of treatment were analyzed in duplicate.
Duration of treatment / exposure:
During the toxicity phase the test article was freely available for a minimum of 28 days up to the day of necropsy.
Frequency of treatment:
Continuous
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 50, 250, 1000 mg/kg bw/day
Basis:
nominal in diet
No. of animals per sex per dose:
5 male/5 female
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: Dose levels for the toxicity phase of the study were selected by the study sponsor.
- Rationale for animal assignment (if not random): The animals were assigned to treatment groups using a randomisation procedure based on stratified body weight during the acclimatisation period.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Twice daily
- All animals were examined twice daily to detect any which were dead or moribund.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All toxicity phase animals were examined once daily for signs of ill health or overt toxicity. In addition each animal was given a detailed clinical examination at weekly intervals. An individual record was maintained of the clinical condition of each animal.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual body weights were recorded before treatment on the first day of the study, at weekly intervals thereafter and at necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The amount of food consumed by each cage of animals was determined weekly in g/animal/week.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were obtained from all animals in week 4. The samples were collected by orbital sinus puncture.
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes (overnight, about 20 h)
- How many animals: 40
- Parameters examined: The following parameters were measured on blood collected into EDTA anticoagulant:
haemoglobin
mean cell volume
red blood cell count and indices:
mean cell haemoglobin
packed cell volume
mean cell haemoglobin concentration
total and differential white blood cell count platelets

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were obtained from all animals in week 4. The samples were collected by orbital sinus puncture.
- Animals fasted: Yes (overnight, about 20 h)
- How many animals: 40
- Parameters examined: The following parameters were measured on blood collected into lithium heparin anticoagulant:
glutamate oxaloacetate transaminase
glutamate pyruvate transaminase
alkaline phosphatase
plasma (pseudo) cholinesterase
sodium
chloride
inorganic phosphorus
blood urea nitrogen
creatinine
albumin
total cholesterol
potassium
calcium
glucose
total bilirubin
total protein
albumin/globulin ratio

URINALYSIS: No
- There were no indications of treatment-related renal effects at week 4, consequently urine analysis was not carried out.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Necropsy: The animals were killed by an intraperitoneal injection of sodium pentobarbitone and exsanguination following an overnight period without food. A full internal and external examination was made under the general supervision of a pathologist and all lesions were recorded. The necropsies were carried out on 1 day.
- Organ weights: The following organs were dissected free from fat and other contiguous tissue and weighed before fixation: adrenals, kidneys, liver, testes. Paired organs were weighed separately.

HISTOPATHOLOGY: Yes
- Histology: Samples of the following tissues were fixed in neutral buffered 10 % formalin: adrenals, heart, kidneys, liver, lungs (with mainstem bronchi), spleen, all gross lesions. Samples of tissues from the control and high dose animals and gross lesions from low and intermediate dose animals were embedded in paraffin wax B.P. (mp 56 °C), sectioned at a nominal thickness of 5 mm stained with haematoxylin and eosin and examined by the study pathologist.
Statistics:
Data were processed. where appropriate. to give group mean values and standard deviations. Further statistical evaluation was considered not to be appropriate because of the small group size.

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):
Slight reduction in body weight gain at 250 and 1000 mg/kg bw/day.
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Slight increase in kidney weight at 250 and 1000 mg/kg bw/day.
Gross pathological findings:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no mortalities. There were no clinical observations recorded that were considered to be related to treatment.

BODY WEIGHT AND WEIGHT GAIN
Body weight gain in group 4 males and group 3 and 4 females was slightly reduced, resulting in decreased group mean body weight compared to controls. In group 4 male and female the reduction was ca. 7 %, in group 3 female ca. 12 %. See table 1 and 2.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were no apparent effects of treatment on the food consumption of the animals.

FOOD EFFICIENCY
Calculated test article intake was in good agreement with target dose levels.

HAEMATOLOGY
No effects of treatment on haematological parameters were apparent.

CLINICAL CHEMISTRY
Clinical chemistry parameters were apparently unaffected by treatment. Individual values for GOT, GPT and alkaline phosphatase were very variable. All data were within expected ranges and no clear trend was apparent. See tables 3 and 4.

URINE ANALYSIS
Urine analysis was not performed since clinical chemistry results did not suggest any treatment related effects.

ORGAN WEIGHTS
There were slight increases in combined relative kidney weight in group 3 female and group 4 male and female animals. Compared to their respective control groups the relative kidney weights of group 4 animals were 12 % greater. The mean relative weight of group 3 female kidneys was 8 % greater than that of the control group. See tables 5 to 8.

GROSS PATHOLOGY
There were no treatment-related findings at necropsy. There was no histopathological evidence of specific target organ toxicity in any of the tissues examined.

Effect levels

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

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

ANALYSIS OF TEST DIETS

- Stability and homogeneity: The dietary formulations were stable for 14 days and formed homogeneous mixtures at the 2 concentrations investigated.

- Achieved concentrations: Formulations were within acceptable limits of nominal concentrations.

TABLE 1 
Male animals: mean body weight (g) and standard deviation (S.D.) 
Week of study control 50 mg/kg bw /day 250 mg/kg bw/day 1000 mg/kg bw/day
Start Mean, S.D. 187.7, 8.67 193.8, 10.93 180.7, 14.35 187.5, 5.58
1 Mean, S.D.  238.9, 16.66 246.1, 13.43 229.2, 19.90 233.8, 8.08
2 Mean, S.D. 282.3, 29.91 281.5, 19.51 264.9, 23.32 266.2, 10.87
3 Mean, S.D. 311.1, 37.17 319.1, 24.94 295.0, 24.87 293.8, 14.74
4 Mean, S.D. 321.0, 38.23 332.4, 29.82 311.3, 26.42 298.5, 18.10
TABLE 2
Female animals: mean body weight (g) and standard deviation (S.D.) 
Week of study control 50 mg/kg bw /day 250 mg/kg bw/day 1000 mg/kg bw/day
Start Mean, S.D. 136.1, 5.74 137.4, 12.30 133.5, 7.98 138.0, 7.24
1 Mean, S.D. 163.5, 9.31 159.0, 14.12 150.4, 8.03 158.3, 7.66
2 Mean, S.D. 179.4, 11.11 176.6, 15.35 162.3, 9.83 168.6, 11.47
3 Mean, S.D. 198.2, 10.91 189.2, 15.61 174.7, 9.54 182.8, 15.17
4 Mean, S.D. 199.8, 14.0 1 193.1, 15.57 176.7, 12.11 185.8, 14.71
TABLE 3
Male animal group mean clinical chemistry
Occasion: Week 4 
Group  GOT (AST) Iu/L GPT(ALT) Iu/L ALK.PHOS Iu/L P.CHE Iu/L Na meq/L K meq/L Cl meq/L Ca mg/dL P mg/dL
1 Mean, S.D. 131, 42 46, 12 311, 36 61, 13 149, 2 4.0, 0.4 109, 1 10.3, 0.5 7.5, 0.5
2 Mean, S.D. 78, 59 44, 15 310, 91 68, 6 147, 2 4.1, 0.5 108, 1 10.2, 0.4 8.5, 0.4
3 Mean, S.D. 69, 54 43, 11 374, 50 66, 17 149, 3 4.0, 0.6 109, 1 10.7, 0.1 8.1, 1.1
4 Mean, S.D. 68, 32 44, 5 381, 70 55, 19 141, 0 3.9, 0.5 108, 1 10.4, 0.3 7.1, 0.7
TABLE 4
Female animal group mean clinical chemistry
Occasion: Week 4 
Group  GOT (AST) Iu/L GPT(ALT) Iu/L ALK.PHOS Iu/L P.CHE Iu/L Na meq/L K meq/L Cl meq/L Ca mg/dL P mg/dL
1 Mean, S.D. 87, 10 25, 4 183, 44 339, 134 148, 2 3.4, 0.3 111, 2 10.5, 0.3 6.3, 0.7
2 Mean, S.D. 106, 34 36, 6 235, 77 287, 108 147, 1 3.5, 0.5 111, 1 10.4, 0.2 6.1, 0.7
3 Mean, S.D. 99, 27 32, 6 266, 55 252, 42 147, 2 3.7, 0.2 108, 1 10.4, 0.1 7.1, 0.5
4 Mean, S.D. 102, 13 38, 11 252, 75 272, 93 147, 0 3.5, 0.4 109, 1 10.4, 0.2 6.8, 0.4
TABLE 3 cont.
Male animal group mean clinical chemistry - Occasion: Week 4 
Group  GLUCOSE mg/dL B.U.N. mg/dL T. BILI mg/dL CREAT mg/dL T. PROT g/dL ALBUMIN g/dL AG RATIO TOT. CHOL mg/dL
1 Mean, S.D. 90, 18  19, 3  0.2, 0.0  0.7, 0.1  6.3, 0.4 3.8, 0.1 1.5, 0.2 84, 15 
2 Mean, S.D. 100, 14  14, 1  0.2, 0.0  0.7, 0.1  6.2, 0.2 3.7, 0.1 1.5, 0.3 85, 13 
3 Mean, S.D. 92, 8  12, 1  0.2, 0.0  0.7, 0.1  6.3, 0.2 3.9, 0.1 1.6, 0.1 71, 13 
4 Mean, S.D. 104, 11  13, 2  0.2, 0.0  0.8, 0.0  6.3, 0.2 3.8, 0.1 1.5, 0.1 63, 4 
TABLE 4 cont.
Female animal group mean clinical chemistry
 - Occasion: Week 4 
Group  GLUCOSE mg/dL B.U.N. mg/dL T. BILI mg/dL CREAT mg/dL T. PROT g/dL ALBUMIN g/dL AG RATIO TOT. CHOL mg/dL
1 Mean, S.D. 91, 8  18, 2  0.2, 0.1  0.7, 0.1  6.3, 0.4 3.9, 0.1 1.6, 0.3 88, 22 
2 Mean, S.D. 99, 8  18, 3  0.2, 0.0  0.8, 0.1  6.2, 0.3 3.9, 0.1 1.7, 0.2 84, 10 
3 Mean, S.D. 109, 12  12, 1  0.1, 0.1  0.8, 0.0  6.2, 0.2 4.0, 0.3  1.8, 0.3 90, 12 
4 Mean, S.D. 106, 22  14, 3  0.2, 0.0  0.7, 0.1  6.2, 0.2 3.9, 0.2 1.7, 0.3 97, 25
TABLE 5
Male animal group mean organ weight (g) at terminal kill 
Group Bodyweight AL AR KL KR LI  GL  GR
1 Mean, S.D. 309.6, 39.6 0.023, 0.003 0.024, 0.004 0.948, 0.128 0.945, 0.129 7.992, 1.253  1.566, 0.163 1.547, 0.156
2 Mean, S.D. 319.1, 28.5 0.029, 0.006 0.025, 0.004 0.969, 0.084 1.001, 0.066 8.697, 0.810 1.578, 0.171 1.602, 0.187
3 Mean, S.D. 296.6, 26.0 0.026, 0.003 0.022, 0.005 0.955, 0.092 0.954, 0.064 8.015, 0.641 1.573, 0.071 1.585, 0.099
4 Mean, S.D. 284.8, 18.8 0.024, 0.006 0.024, 0.007 0.988, 0.076 0.961, 0.061 7.375, 0.546 1.600, 0.180 1.581, 0.205
TABLE 6
Female animal group mean organ weight (g) at terminal kill 
Group Bodyweight AL AR KL KR LI 
1 Mean, S.D. 187.3, 14.3 0.029, 0.003 0.026, 0.004 0.635, 0.093 0.661, 0.077 5.504, 0.461
2 Mean, S.D. 183.2, 17.4 0.031, 0.009 0.030, 0.005 0.647, 0.034 0.652, 0.054 5.569, 0.582
3 Mean, S.D. 167.7, 12.5 0.026, 0.005 0.024, 0.008 0.613, 0.063 0.630, 0.038 5.364, 0.335
4 Mean, S.D. 175.7, 13.1 0.032, 0.003 0.032, 0.003 0.616, 0.052 0.642, 0.053 5.475, 0.607 
TABLE 7
Male animal group mean organ/body weight (%)
Group Bodyweight AL AR KL KR LI  GL  GR
1 Mean, S.D. 309.6, 39.6 0.0074, 0.0007 0.0071, 0.0011 0.3062, 0.0186 0.3053, 0.0183 2.5745, 0.1553 0.5091, 0.0483 0.5027, 0.0452
2 Mean, S.D. 319.1, 28.5  0.0092, 0.0018 0.0080, 0.0014 0.3044, 0.0213 0.3148, 0.0241 2.7257, 0.0838 0.4955, 0.0461 0.5024, 0.0456
3 Mean, S.D. 296.6, 26.0 0.0087, 0.0010 0.0075, 0.0018 0.3221, 0.0161 0.3221, 0.0103 2.7048, 0.0859 0.5329, 0.0400 0.5368, 0.0464
4 Mean, S.D. 284.8, 18.8 0.0085, 0.0018 0.0083, 0.0022 0.3471, 0.0216 0.3383, 0.0252 2.5919, 0.1480 0.5645, 0.0844 0.5578, 0.0903
TABLE 8
Female animal group mean organ/body weight (%)
Group Bodyweight AL AR KL KR LI 
1 Mean, S.D. 187.3, 14.3 0.0154, 0.0016 0.0141, 0.0014 0.3379, 0.0299 0.3522, 0.0192 2.9386, 0.1127
2 Mean, S.D. 183.2, 17.4 0.0167, 0.0038 0.0162, 0.0026 0.3548, 0.0224 0.3578, 0.0374 3.0390, 0.1241
3 Mean, S.D. 167.7, 12.5 0.0156, 0.0032 0.0142, 0.0043 0.3669, 0.0408 0.3767, 0.0280 3.2170, 0.3723
4 Mean, S.D. 175.7, 13.1 0.0180, 0.0011 0.0184, 0.0028 0.3857, 0.0306 0.3887, 0.0193 3.1117, 0.1493

Applicant's summary and conclusion

Conclusions:
There was no effect of oral administration of test item in the diet at a dose level of 50 mg/kg/day for 28 days. At 250 and 1000 mg/kg/day there was a slight reduction in body weight gain and a slight increase in relative kidney weight. There was, however, no histopathological evidence of systemic toxicity.
Executive summary:

A study according to EU Method B.7 and OECD 407 (Repeated dose toxicity – oral) was carried out. The objectives of the study were to determine the palatability and oral toxicity of test item in the rat following administration for 28 days.

A total of 4 groups of 5 male and 5 female rats were offered test item admixed in the diet at dose levels of 0, 50, 250 and 1000 mg/kg bw/day. The dietary formulations were stable for 14 days and were homogeneous at the concentrations investigated. Week 1 formulations were accurately prepared.

There were no mortalities and no treatment-related observations. There were reductions in the body weight and body weight gain of female animals dosed at 250 mg/kg bw/day and male and female animals dosed at 1000 mg/kg bw/day. No treatment-related effects on food consumption were apparent. There were no effects of treatment on the hematological and clinical chemistry parameters measured. A slight increase in relative kidney weight in female animals dosed at 250 mg/kg bw/day and male and female animals dosed at 1000 mg/kg bw/day was the only apparent effect on organ weight. There was no gross or histopathologic evidence of toxicity in any of the tissues examined.

In conclusion, there was no effect of oral administration of test item in the diet at a dose level of 50 mg/kg/day for 28 days. At 250 and 1000 mg/kg/day there was a slight reduction in body weight gain and a slight increase in relative kidney weight. There was, however, no histopathologic evidence of systemic toxicity.