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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1989-11-17 to 1990-03-23
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1994
Report Date:
1994

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Test material form:
solid - liquid: aqueous solution

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Colworth Wistar
- Age at study initiation: 4-6 weeks
- Weight at study initiation (g): males: 132.9 - 138.3, females 101.7 to 104.5 (group mean values)
- Fasting period before study:
- Housing: singly housed in stainless steel or galvanised cages with stainless steel mesh floors awl doors, such that there was one animal per cage; 48 (four rows x six columns) cages were accommodated on each holding battery.
- Diet: ad libitum, MODAIN purified diet
- Water: e.g. ad libitum, potable tap water from the local water mains
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21+/-2
- Humidity (%): 55 +/-10
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12


Administration / exposure

Route of administration:
oral: feed
Vehicle:
other: MODAIN purified diet
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): most probably every other week (7 batches in the course of the 13 week study were prepared)
- Mixing appropriate amounts with (Type of food):
-- The formulation of the MODAIN purified diet (%) was Maize starch 65.0 %, Casein (spray dried) 20.0%, Solkafloc 5.0 %, Maize oil 5.0%, Modified AIN-76 minerals 3.5 %, AIN-76A vitamins 1.0 %, DL methionine 0.3 %, Choline bitartrate 0.2 %.
-- The test item was added to the diet at the expense of maize starch for the 0.40% and 1.00% dietary levels. The 1.00% diet was used to prepare the 0.10% dietary level by a 1:10 dilution with control MODAIN purified diet. Similarly the 0.40% diet was used to prepare the 0.04% dietary level by a 1:10 dilution with control MODAIN purified diet
- Storage temperature of food: cold
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Diets prepared for feeding at the beginning (Batch 1), middle (Batch 4) and end of the study (Batch 7) were sampled and analysed for test item to confirm dietary concentration, and for moisture, protein, fat, fibre and ash to confirm composition. Control MODAIN purified diet is routinely analysed for nutrients and contaminants. For confirmation of dietary concentration, the test item was extracted from the diet samples by sochlet extraction with a methanol: ammonia (100:1) mixture. The extracts were concentrated and analysed by HPLC on a 3µ Nucleosil C18 column using a mobile phase of methanol: water: ammonia (90:10:1) at a flow rate of 0.8 ml/min with detection by a light scattering detector. Quantitation was achieved by comparison of sample peak heights with those of external standards in the range 0.3 - 1.5 mg/ml.
Homogeneity and stability of the test substance within the diets were determined on a 5 kg batch of each diet containing (A) 1.00%, (B) 0.40% and (C) 0.10% test item (the 0.04% dietary level would be covered by the fact that it was prepared by a 1:10 dilution of the 0.40% dietary level with control MODAIN purified diet). Each batch was sampled at 5 positions in the mixing bowl as follows: A top centre, B middle centre, C bottom centre, D left centre and E right centre. Duplicate analyses were completed on each sample after it was prepared. The remainder of each batch of diet was divided into 2 parts, which were stored in an animal room (21 ± 2°C) or a cold room (4°C). Samples were taken for analysis after 7 and 14 days storage. Recovery tests were also carried out on diets spiked with the test item at levels covering the range of incorporation of the test item in the diet.
Duration of treatment / exposure:
mains study: 13 weeks
recovery period: further 32 days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0.04 other: %, nominal in diet
Dose / conc.:
0.1 other: %, nominal in diet
Dose / conc.:
0.4 other: %, nominal in diet
Dose / conc.:
1 other: %, nominal in diet
No. of animals per sex per dose:
main study groups: 12 males and 12 females per group
additional recovery groups: 5 males and 5 females (control and high dose group)
Control animals:
yes, plain diet

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked at least twice each day (once on Saturdays and Sundays) for signs of ill-health and reaction to treatment.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: All animals vere given a detailed examination twice a week.

BODY WEIGHT: Yes
- Time schedule for examinations: Each animal was weighed on the day that treatment commenced and then at weekly intervals for the duration 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
-- Food intake was recorded twice weekly for each animal throughout the study and weekly intakes were calculated.

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was recorded twice weekly for each animal throughout the study and weekly intakes were calculated.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at start and prior to the completion of the 13 week treatment
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the completion of the 13 weeks treatment feeding period and at the end of the recovery period
- Anaesthetic used for blood collection: Yes, Halothane anaesthesia
- Animals fasted: No
- How many animals: All surviving rats at the completion of the 13 weeks treatment feeding period and at the end of the recovery period.
- Parameters examined: Haemoglobin (Hb), Red cell count (RBC), Platelets (PLT), Reticulocytes (RET), White cell count (WBC), Lymphocytes (LYM), Neutrophils (NEU), Monocytes (MON), Eosinophils (EOS), Prothrombin time (PT), Activated partial thromboplastin time (APTT), Several indices are derived from these measurements: MCV - mean cell volume (mean of red blood cell volume histogram - femtolitres), HCT - haematocrit (red blood cell count times the MCV - litre/litre), MCH - mean cell haemoglobIn (haemoglobin divided by the red blood cell count picograms), MCHC - mean cell haemoglobin concentration (haemoglobin divided by the HCT grams/100 ml red cells), the following blood coagulation factors were measured: Prothrombin time, Activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the completion of the 13 weeks treatment feeding period and at the end of the recovery period
- Animals fasted: No
- How many animals: All surviving rats at the completion of the 13 weeks treatment feeding period and at the end of the recovery period.
- Parameters examined: in plasma: Sodium, Potassium, Chloride, Calcium, Inorganic phosphate, Magnesium, Aspartate transaminase, Alanine transaminase, Lactate dehydrogenase, 1-Hydroxybutyrate dehydrogenase, Creatine kinase, Alkaline phosphatase (pH 9.8), Pseudocholinesterase, 5-Nucleotidase, Triglyceride, Total cholesterol, Urea, Glucose, Creatinine; in serum: Total protein, Albumin; Serum electrophoresis separation yas performed on cellulose acetate plates followed by staining with Ponceau S and quantification of the fractions using the Profil Ecran scanning densitometer were performed for: Albumin Alpha-1-globulin, Alpha-2-globulin, Beta globulin, Gamma globulin, Albumin/globulin ratio

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected from a selection of the 13 week rats (eight male and eight female rats per group) and the recovery rats prior to treatment, after 32 and 88 days of treatment, and after 13 weeks treatment plus 32 days recovery time for recovery rats only.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No data
- Parameters examined analytically: Urine volume, Refractive index, Sodium, Potassium, Chloride, Calcium, Inorganic phosphate, Magnesium, Urea, Creatinine N-acetyl-beta-D-glucosaminidase, Gamma glutamyl transferase, Alanine aminopeptidase
- Parameters examined semi-quantitative using dip-stick strips: leukocytes, nitrite, glucose, protein, erythrocytes and haemoglobin

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All the animals which survived to the completion of the 13 weeks treatment feeding period and at the end of the recovery period received a detailed
necropsy. All macroscopic abnormalities were recorded as well as an assessment of the level of intra-abdominal fat deposition.
- Brain, heart, liver, kidneys, spleen, testes, caecum, empty caecum and adrenal glands were weighed prior to fixation. The ratio of organ weight to 100g of body weight at necropsy was calculated in each case and recorded as relative organ weight.
-- The following tissues were taken from each rat and preserved in 10% buffered formalin: Adrenal glands, Brain, Colon, Heart, Kidneys, Lungs, Mammary glands, Ovaries and fallopian tubes, Pituitary, Sciatic nerve, Sternum, Thyroid and parathyroid, Uterus, Aorta, Caecum, Duodenum, Ileum, Larynx Lymph nodes (cervical and mesenteric), Muscle, Prostate, Spinal cord, Stomach, Tongue, Bladder, Cervix, Head, Jejunum, Liver, Oesophagus, Pancreas, Rectum, Spleen, Thymus, Trachea
-- The following tissues were taken from each rat and preserved in Bouin's fixative: Epididymides, Seminal vesicles, Vagina, Femur and stifle joint, Skin, Sali vary glands, Testes
-- The eyes and harderian glands were taken from each rat and fixed in Davidson's fluid.

HISTOPATHOLOGY: Yes
- Tissues were processed by conventional histological methods into paraffin wax and sections 4µm (nominal) in thickness were prepared and stained with haematoxylin and eosin for histological examination. Additional portions of liver were fixed in 10% formol saline for the preparation of cryostat sections that were subsequently stained with Oil Red 0 to demonstrate neutral fat.
- Full histological examination was confined to rats from the high dose group (1.00% test item) and control group.
Statistics:
- For body weights, food intakes, food efficiencies and water intakes, haematology, clinical chemistry and organ weights (including the ratio of organ weight to body weight at terminal kill), a statistical analysis was conducted using Student's t-test, F-test and Duncan's Multiple range test.
- A t-test versus control was used to show any significant differences between control group and any of the other treatment levels at the 5%, 1% and 0.1% probability levels. In conjunction with the F-ratio test in the analysis of variance, a Duncan's multiple range test at the 5% protection level was included to allow all pair-wise comparisons amongst the treatment levels to be carried out.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
- All rats showed no toxic symptoms, signs of distress or decrease in activity.
Mortality:
no mortality observed
Description (incidence):
All rats survived to the end of the 13 week feeding period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
- Statistically significant lower body weights and body weight gains were observed over the 13 weeks of treatment for only the female rats fed 0.10%
test item when compared with controls. These were both considered to be marginal effects and were not dose related.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
- No statistically significant differences in food intakes were observed over the 13 weeks of treatment for either male or female rats fed the test item when compared with controls.
- the dietary dose levels compared to the following mean daily ingestion levels based on product and based on the a.i. of 33.8 %
-- 0.04 %: 28 mg test item/kg body weight/day; 9.5 mg a.i./kg bw/day
-- 0.10 %: 71 mg test item/kg body weight/day; 24 mg a.i./kg bw/day
-- 0.40 %: 288 mg test item/kg body weight/day; 97 mg a.i./kg bw/day
-- 1.00 %: 731 mg test item/kg body weight/day; 247 mg a.i./kg bw/day
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
- Statistically significant lower food conversion efficiencies were observed over the 13 weeks of treatment for both male and female rats fed 1.00% test item and the female rats fed 0.10% test item. These statistically significant differences were not observed in the recovery rats in the 5 week recovery period.
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
- No statistically significant differences in water intakes were observed over the 13 weeks of treatment for either male or female rats fed the test item when compared with controls
Ophthalmological findings:
no effects observed
Description (incidence and severity):
- No treatment related ophthalmological effects were noted.
Haematological findings:
no effects observed
Description (incidence and severity):
- Some statistical significances were observed but these appeared randomly spread with no evidence of a dose-related response. It was considered that there were no direct effects that could be related to the feeding of the test item.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Main study groups:
- Rats fed the test item showed statistically significant differences from the control rats for the following plasma constituents. These observations largely reflect the reduced food conversion efticiency values recorded for these rats.
--I: Reduced plasma 5-nucleotidase for female rats fed 1.00% .
-- II:Reduced plasma triglyceride for male rats fed 1.00%, which was considered a marginal effect.
-- III: Reduced plasma urea for female rats fed 1.00%, which was considered a marginal effect.
Recovery groups:
These statistically significant differences were not observed in the recovery rats at Week 18.
Urinalysis findings:
no effects observed
Description (incidence and severity):
- Some statistically significant differences were observed but these appeared randomly spread with no evidence of a dose-related response. It was considered that there were no direct effects that could be related to the feeding of the test item.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Main study groups:
- The following statistically significant differences (P = 0.05) were observed, there was no histological correlate for these organ weight changes.
-- I: Reduced necropsy body weights for female rats fed 0.10% test item. This was considered a marginal effect. The reason for this reduced values is unclear as there was no evidence of a dose-related response.
-- II: Reduced heart and relative heart weights for female rats fed 0.10% test item. The reason for this reduced values is unclear as there was no evidence of a dose-related response.
-- III: Increased caecal and relative caecal weights for both male and female rats fed 1.00% test item.
-- IV: Increased empty caecal weights for male rats fed 1.00% test item, a pooled analysis of male and female rats fed 1.00% test item and
male rats fed 0.40% test item. The latter was considered to be a marginal effect.
-- V: Increased relative empty caecal weights for both male and female rats fed 1.00% test item, and for a pooled analysis of these male and female rats. Increased relative empty caecal weights were also observed for the female rats fed 0.10% test item (marginal effect), which was considered to be related to the reduced post mortem body weights observed for these rats (See I above).
-- VI: Reduced liver weights for female rats and for a pooled analysis of male and female rats fed 1.00% test item. Reduced liver weights were also observed for the female rats fed 0.10% test item (See (I) and (II) above)
-- VII: Reduced relative liver weights for both male and female rats fed 1.00% test item, and for a pooled analysis of these male and female rats.
The significant finding for the male rats alone was considered to be a marginal effect.
-- VIII: Increased testes weights for male rats fed 0.40% test item. This was considered to be a marginal effect.
Recovery groups:
- These statistically significant differences were not observed in the recovery rats at Week 18.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Main study groups:
- The abdominal fat depots were reduced in both male and female rats fed 1.00% test item when compared with the control animals. This feature corresponded to the decreased food efficiency values recorded for these rats.
- Enlargement of the caecum was noted at necropsy in 8/12 male rats and 7/12 female rats fed 1.00% test item, with some showing alteration of the colour of the contents from grey-green to green.
Recovery groups:
- An enlargement of the caecum was not observed in the recovery rats at Week 18 apart from a slight enlargement in one female recovery rat. It is considered that slight enlargement of the full caecum can occur as part of the background pathology of the Colworth Wistar rat and may account for this finding in one female rat fed 0.04% test item at Week 13 and one female recovery rat.

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Main study groups:
- There was no evidence of an adverse effect of treatment in any of the tissues examined including inspection of seminal vesicles, prostate, epididymides, testes, mammary glands, ovaries and fallopian tubes, uterus, cervix and vagina.
.
-- Caecum: There was no evidence of an effect of treatment on the morphology of the caecum.
-- Liver: There were no findings to explain decreased liver weight in the rats fed 1.00% test item.
-- Incidental findings: A variety of incidental findings was recorded in rats from all groups with no evidence of an effect of treatment on their character, incidence or distribution. The findings are consistent with the normal spectrum of spontaneous lesions encountered in young laboratory rats.
Recovery groups:
- Since there was no evidence of an adverse effect of treatment in any of the tissues examined, no histological evaluation was undertaken for the recovery rats.
Histopathological findings: neoplastic:
not examined
Details on results:
HOMOGENEITY AND STABILITY OF TEST MATERIAL IN DIET
- Analyses of diet obtained from various sites during mixing and preparation indicate that the test item at dietary levels of 0.10%. 0.40% or 1.00% was distributed uniformly in the diets and was stable in diet when stored for up to 2 weeks in either the cold room or the animal room. It is considered
that the 0.04% dietary level is covered by the fact that it was prepared by a 1:10 dilution of the 0.40% dietary level with
control MODAIN purified diet.

PROTEIN AND PROTEINE ELECTROPHORESIS
Main study groups:
- Statistically significant lower serum total protein levels were observed for both male and female rats fed 1.00%. These observations largely reflect the reduced food conversion efficiency values recorded for these rats.
Recovery groups:
These statistically significant differences were not observed in the recovery rats at Week 18.

Effect levels

open allclose all
Key result
Dose descriptor:
NOEL
Remarks:
effects relevant to humans
Effect level:
247 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: see "remarks"
Dose descriptor:
LOEL
Effect level:
97 mg/kg bw/day (actual dose received)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Based on transient effects on food conversion efficiencies and organ weights of caecum and liver, judged as not relevant to humans in view of an potential serious health risk due to missing histopathological correlate and proved reversibility.

Target system / organ toxicity

Critical effects observed:
no

Any other information on results incl. tables

In deviation to human anatomy, the small intestine of rats is unremarkable, but rats possess a prominent caecum. Enlargement of the rat caecum with or without subsequent effects on caecum weight, food conversion and nutritional status is a common and frequently response to feeding poorly-absorbable or osmotically-active substances, such as xylitol, sorbitol,sucralose or natural sugars like d-ribose, general changes in nutritional diet composition or application of compounds with effects on the caecal microflora. In the absence of histopathological changes, the rat caecum alterations are taken as physiological adaptive responses and considered to be of no toxicological significance (for instance, see: Leegwater, D. C., DE Groot, A. P. & VAN Kalmthout-Kuiper, M. Fd. Cosmet. Toxicol. (1974) The aetiology of caecal enlargement in the rat. 12: 687-697 or CHMP Assessment Report for Doribax, EMEA (European Medicines Agency), Doc.Ref.: EMEA/309032/2008 (2008) or A Report on Sucralose from the Food Sanitation Council, The Japan Food Chemical Research Foundation, Food Sanitation Council Notice, No.5 January 6, (1999) or the Options of the Scientific Committee on Cosmetic Products and Non-Food Products intended for Consumers, SCCNFP/0514/01 and SCCNFP/0539/01 (2002)).

Applicant's summary and conclusion

Conclusions:
Up to and including the highest tested dose of of 1% in feed (corresponding to 731 mg product/ kg bw/d and 247 mg a.i./kg bw/day), Coco AAPB was tolerated with no evidence of any systemic toxicity relevant in view of an potential serious health risk for humans in this 90 day rat feeding study with 38 day recovery.
Executive summary:

In a subchronic toxicity study according OECD guideline 408, Coco AAPB (a.i. 33.8 %) was administered to 12 male and 12 female Colworth Wistar rats per dose via food at dose levels of 0.00%, 0.04%, 0.10%, 0.40% and 1.00% (corresponding to 0, 28, 71, 288 and 731 mg product/kg bw (9.5, 24, 97 and 247 mg a.i./kg bw/day)) for 90 days. Additional animals in satellite groups (control and high dose, 5 males and 5 females, each) were kept for further 32 days without treatment to detect recovery from, or persistence of, toxic effects. Concentrations in diet formulations were analytically verified and substance intake was calculated from recorded food consumption.

 

The substance was tolerated without any systemic effects relevant in view of an potential serious health risk for humans. Up to and including the highest dose tested of 1% in feed (corresponding to 731 mg product/ kg bw/d and 247 mg a.i./kg bw/day), there were no dose related effects on mortality, clinical signs, body weight, food consumption, water consumption, haematology, urinalysis and histopathology including inspection of seminal vesicles, prostate, epididymides, testes, mammary glands, ovaries and fallopian tubes, uterus, cervix and vagina.

 

The only treatment related effects ascertainable at termination of treatment but not after the recovery period were reduced food conversion efficiencies and organ weight changes in the caecum and liver. The enlarged caecum observed at necropsy in some male and female rats fed 0.40% and 1.00% Coco AAPB was reflected in the statistically significant increases recorded in the absolute and relative, full and empty caecal weights for both male and female rats fed 1.00% Coco AAPB. These animals fed 1.00% Coco AAPB also showed reduced absolute and relative liver weights, reduced abdominal fat depots corresponding to the reduced food efficiency and several possibly associated plasma and serum biochemical changes. There was no histopathological correlate for the organ weight changes in caecum and liver. All alterations were completely reversible in the recovery group after 32 days without treatment.

 

Enlargement of the rat caecum with or without subsequent effects on caecum weight, food conversion and nutritional status is a common and frequently response to feeding poorly-absorbable or osmotically-active substances, such as xylitol, sorbitol, sucralose or natural sugars like d-ribose, general changes in nutritional diet composition or application of compounds with effects on the caecal microflora. In the absence of histopathological changes, the rat caecum alterations are taken as physiological adaptive responses and considered to be of no toxicological significance. An increase in liver weight without any histopathological correlate is commonly not considered to reflect an adverse effect but should be considered as an adaptive metabolic response which in known to be reversible. As also in this study, the increase in liver weight was without any histopathological correlate and has been proved to be reversible, the liver weight alteration is not considered to be an adverse effect relevant in view of an potential serious health risk for humans.

 

Therefore, the NOEL derived from this study relevant in view of a potential serious health risk for humans is the highest tested dose of 1% in feed (corresponding to 731 mg product/ kg bw/d and 247 mg a.i./kg bw/day).

 

The LOEL is 0.4% in feed (corresponding to 288 mg product/ kg bw/d and 97 mg a.i./kg bw/day), based on transient effects on food conversion efficiencies and organ weights of caecum and liver, judged as not relevant to humans in view of a potentially serious health risk due to missing histopathological correlate and proved reversibility.