Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

see document under "Attached justification"

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: guideline study but no final report available (audited draft report available)

Reason / purpose for cross-reference:

reference to other study

Remarks:

reference to concurrent micronucleus test

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted in 1998

Deviations:

yes

Remarks:

a concurrent micronucleus test was included in this study

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302/EEC, Official Journal of the European Union, no. L133

Version / remarks:

adopted in 1988

Principles of method if other than guideline:

A concurrent control group of 10 rats/sex was kept on basal diet supplemented with 10% pregelatinized wheat starch.

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspectorate for Health Protection, Commodities and Veterinary Public Health, Ministry of Health, Welfare and Sport, The Haque, The Netherlands

Limit test:

yes

Species:

rat

Strain:

Wistar

Remarks:

Crl:(WI)WU BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 135 - 191 g (males) and 109 - 135 g (females)
- Fasting period before study: no
- Housing: individually in suspended stainless steel cages (h x l x w = 18 x 32 x 18 cm), fitted with wire-mesh floor and front
- Diet: powdered diet: Rat & Mouse No. 3 Breeding Diet, RM3; SDS, Special Diets Services, Witham, England (ad libitum)
- Water: tap water for human consumption (ad libitum)
- Acclimation period: 13 days

DETAILS OF FOOD AND WATER QUALITY: Each batch of diet is analysed by the supplier for nutrients and contaminants (the CoAs of the used batches are attached to the report). Results of the routine physical, chemical and microbial examination of the drinking water as conducted by the supplier are made available to the CRO.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 50 - 70
- Air changes (per hr): approximately 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 Oct 2000 To: 26 Jan 2001

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

- DIET PREPARATION
- Rate of preparation of diet (frequency): approximately every three weeks (19 Oct 2000, 07 Nov 2000, 07 and 29 Dec 2000)
- Mixing appropriate amounts with: powdered diet (Rat & Mouse No.3 Breeding Diet, RM3; SDS, Special Diets Services, Witham, England)
- Storage temperature of food: in the freezer (≤ -18 °C) in plastic bags containing portions sufficient to cover the need for 3-4 days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses to determine the homogeneity and content of isomalt in the diet were conducted by means of a HPLC method, using the sum of the two main components (i.e. 6-O-glucopyranosyl-sorbitol and 1-O-glucopyranosyl-mannitol) for quantification. The stability of isomalt in the diet was not reconfirmed in the present study because in a previous study (TNO report V95.289, November 1995) levels of up to 10% of isomalt in rat diet were found to be stable upon storage for four days in the animal room or for four weeks in the freezer (≤ -18 °C).
The homogeneity and content of the test substance in the diet were checked by analysis in the first batch of diets prepared in the study (19 Oct 2000). For this purpose five samples of the diet containing the test item, taken at different locations in the feed container, were analysed. In addition, one sample of unsupplemented RM3 diet was analysed (of the same batch as used for the test diets). From the other batches of experimental diets samples were taken (one per diet) and stored in the freezer (≤ -18 °C). These samples were, however, not analysed.

Analytical result: Isomalt was homogeneously distributed and the content of isomalt was close to intended in the diets analysed.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

10 other: %

Remarks:

100 000 ppm (corresponding to ca. 7000 and 8400 g/kg bw/day in males and females, respectively)

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes

Details on study design:

- Dose selection rationale: The dietary level of 10% was selected as a high level which was previously found to be tolerable without obvious signs of toxicity.

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, on weekends and public holidays once daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 and once weekly therefter; on the day of scheduled necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight data: Yes

FOOD EFFICIENCY:
- Body weight gain in g/food consumption in g per unit time calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was measured during five-day periods in weeks 1, 6 and 12. Water consumption of females was also measured on days 92 and 93 (week 14). The results were expressed in g per animal per day.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at scheduled necropsy
- Anaesthetic used for blood collection: Yes (CO/O2)
- Animals fasted: No
- How many animals: all surviving
- Parameters examined: haemoglobin, packed cell volume, red blood cell count, reticulocytes, total white blood cell count, differential white blood cell count, prothrombin time, thrombocyte count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: fasting glucose: Day 87, all other parameters: at scheduled necropsy
- Animals fasted: No (except for determination of fasting glucose)
- How many animals: all surviving
- Parameters examined: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin, urea, creatinine, total bilirubin, total cholesterol, triglycerides, phospholipids, calcium, sodium, potassium, chloride and inorganic phosphate

URINALYSIS AND RENAL CONCENTRATION TEST: Yes
- Time schedule for collection of urine: Day 86/87
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (water for 24 hours, food for 16 hours)
- How many animals: all surviving
- Parameters examined: concentrating ability of the kidneys (urinary volume and density), appearance, dipstick measurements (pH, glucose, occult blood, ketones, protein, bilirubin, urobilinogen), microscopy of the sediment (red blood cells, white blood cells, epithelial cells, amorphous material, crystals, casts, bacteria, sperm cells, worm eggs)

NEUROBEHAVIOURAL SCREENING: Yes
- Time schedule for examinations: Days 84/85/86
- Dose groups that were examined: all
- Battery of functional tested: sensory activity / grip strength / motor activity / stimulus reactivity measurements / body temperature / landing foot splay

IMMUNOLOGY SCREENING: Yes
-The results of the routine measurements and examinations in this study from which primary indicators of immune toxicity can be derived were evaluated as an immunotoxicity screen. These routine tests included:
- haematology (total and differential white blood cell counts)
- clinical chemistry (total protein, albumin, albumin/globulin ratio, transaminases)
- body and organ weights (thymus, spleen)
- gross and microscopic examination of the lymphoid tissues (spleen, lymph nodes, thymus, Peyer's patches, bone marrow)
- microscopic examination of possible immune-related processes in non-lymphoid organs (e.g. mono-nuclear cell infiltrates in liver or kidneys)

OTHER:
The study was combined with a micronucleus test (refer to section 7.6.2). For this purpose, femural bone marrow (from one of the femurs) of five males and five females of each treatment group (the surviving animals with the lowest identification numbers) and of five rats/sex of an additional positive control group was used. The micronucleus test is presented in a separate report (refer to section 7.6.2, de Vogel, 2001).

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of the study, surviving rats were killed (on three successive working days) in such a sequence that the average time of killing was approximately the same for each group. The animals were killed by exsanguination from the abdominal aorta under CO/O2 anaesthesia. Subsequently, they were examined macroscopically for pathological changes.
A thorough necropsy was also conducted on the rat killed in extremis on Day 37 because of conditional decline.

ORGAN WEIGHTS: Yes
The following organs were weighed (paired organs together) as soon as possible after dissection to avoid drying, and the relative organ weights (g/kg body weight) were calculated based on the terminal body weight of the rats:
adrenals, brain, caecum (filled and empty), epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus and uterus

HISTOPATHOLOGY: Yes
Samples of the following tissues and organs of all animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde:
adrenals, aorta, axillary lymph nodes, brain (brain stem, cerebrum, cerebellum), caecum, colon, epididymides,*exorbital lachrymal glands, eyes, *femur with joint (1), GALT (gut associated lymphoid tissue, including Peyer's patches), *Harderian gland, heart, kidneys, liver, lungs, mammary gland (females), * mandibular (cervical) lymph nodes, mesenteric lymph nodes, *nasal turbinates, nerve-peripheral (sciatic), oesophagus, ovaries, *oviducts (= fallopian tubes), pancreas, parathyroid, parotid, salivary glands, pituitary, prostate, rectum, seminal vesicles with coagulating glands, skeletal muscle (thigh), skin, small intestine (duodenum, ileum, jejunum), spinal cord (at three levels), spleen, sternum with bone marrow, stomach (glandular and non-glandular), sublingual salivary glands, submaxillary salivary glands, testes, thymus, thyroid, *tongue, trachea/bronchi, urinary bladder, uterus (with cervix), vagina, *Zymbal's gland, all gross lesions

* tissues marked with an asterisk were preserved but not processed for histopathological examination, unless histopathological examination was conducted on the basis of the results of gross observations

(1) Femural bone marrow (from one of the femurs) of five males and five females of each treatment group of the study (i.e., the surviving animals with the lowest identification numbers) was used in a concurrent micronucleus test.

The tissues required for microscopic examination were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin. Histopathological examination (light microscopy) was performed on all organs and tissues listed above - except those marked with an asterisk- of all animals of the control group.

Statistics:

- Body weight: one-way analysis of covariance (covariate: body weight on day 0) followed by Dunnett's multiple comparison tests.
- 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 cheniistry values, volume and density of the urine, organ weights: one-way analysis of variance (Anova) followed by Dunnett's multiple comparison tests.
Independent from the results of Anova, the homogeneity of variances was tested by means of Bartlett's test. When the variances differed significantly (p < 0.01), Kruskal-Wallis nonparametric one-way analysis of variance followed by Mann-Whitney U-tests was used. If the results obtained with this test differed from those obtained with Anova+Dunnett tests, the results obtained with Kruskal-Wallis+Mann-Whitney U-tests were presented.
- Reticulocytes, relative differential white blood cell counts, urinary parameters except for volume and density: Kruskal-Wallis non-parametric Anova followed by Mann-Whitney U-tests.
- Histopathological changes: Fisher's exact probability test.

All analyses were two-sided. Group mean differences with an associated probability of less than 0.05 were considered to be statistically significant.

Statistical analyses were conducted by comparing the treatment group with the starch control group. Significant differences were indicated with *,** or ***.

Clinical signs:

no effects observed

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One male rat of the test item group was killed in extremis on Day 37 of the study. On the day of killing this rat showed dyspnoea, low body temperature and sluggishness, while nasal encrustation, growth retardation and decreased intake of food and water had been noticed earlier. Macroscopic and microscopic examination suggested that the primary cause of the conditional decline of this rat was ascites, but several other lesions were observed (red appearance of the lungs, prostate and seminal vesicles, oedematous and yellow mucosa of the stomach and enlarged, haemorhagic urinary bladder). Since none of the other animals in any treatment group showed a similar syndrome, the moribund condition of this rat was considered to be an incidental finding, not related to the administration of the test item.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Mean test item intakes are given in Table 1.

Food efficiency:

no effects observed

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

In week 1 and generally also in week 6 of the study, water intake was similar amongst the groups.
In weeks 12 and 14 (only recorded in females), water intake tended to be increased in both sexes of the test item group. The differences to the starch controls reached the level of statistical significance only on one occasion (day 78) for males. Mean water consumption values are presented in Tables 2 and 3.

The observed increase in water intake was not associated with histopathological alterations or any other relevant findings. They were therefore considered not adverse.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There were no treatment-related changes in clinical chemistry parameters.
Plasma potassium was slightly increased in males of the treatment group compared with starch controls, but all values were within the range of historical control data.

Urinalysis findings:

no effects observed

Description (incidence and severity):

The renal concentration test did not indicate impaired renal concentrating ability. Dipstick measurements and microscopy of the urinary sediment did not reveal any treatment-related changes.

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Description (incidence and severity):

The test item did not produce any primary indication of immune toxicity as evidenced by the absence of treatment-related changes in the relevant data from haematology, clinical chemistry, organ weights or pathology.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Compared to the starch control group, the absolute and relative weights of the filled and empty caecum were increased in the test item group in both sexes. These increases were generally statistically significant.
The caecal enlargement in the test item group was ascribed to incomplete absorption of isomalt and subsequent microbial fermentation in the large intestine, giving rise to an increased osmotic load attracting water. Such caecal enlargement is generally considered a physiological response of no toxicological significance.

- Compared to the starch control group, the relative weight of the liver was slightly increased in the test item group in both sexes.

- The relative weight of the kidneys was increased in females of the test item group.

The observed changes in organ weights were not accompanied by histopathological alterations or any other relevant findings. They were therefore considered not adverse.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Neurotoxicity screening:
The results of various routine measurements and examinations in this study (i.e., daily clinical observations, pathology, functional observation battery) did not indicate neurotoxic potential of the test items.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 7 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects noted up to and including 100 000 ppm

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 8 400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects noted up to and including 100 000 ppm

Key result

Critical effects observed:

no

Table 1. Mean test item intake

	Test item intake (mg/kg bw/day)
Males	6950
Females	8412

Table 2. Mean water consumption males (g/rat/day)

Time point	Starch control	Test item
Day 1	24.9	26.5
Day 2	26.4	27.2
Day 3	28.7	27.2
Day 4	26.2	26.5
Day 5	26.1	26.8
mean Week 1	26.5	26.8
Day 36	28.5	27.3
Day 37	31.6	29.6
Day 38	28.0	32.7
Day 39	28.0	31.4
Day 40	29.5	32.5
mean Week 6	29.1	30.6
Day 78	21.4	27.8*
Day 79	24.8	31.5
Day 80	25.8	29.9
Day 81	24.5	32.4
Day 82	24.4	28.6
mean Week 12	24.2	30.1

* p < 0.05 

Table 3. Mean water consumption females (g/rat/day)

Time point	Starch control	Test item
Day 1	18.9	20.9
Day 2	20.7	20.3
Day 3	21.4	20.8
Day 4	21.1	21.0
Day 5	21.0	21.1
mean Week 1	20.6	20.8
Day 36	21.6	23.0
Day 37	24.8	26.9
Day 38	23.4	25.0
Day 39	22.2	22.0
Day 40	22.3	24.3
mean Week 6	22.9	24.3
Day 78	19.7	20.6
Day 79	22.4	24.8
Day 80	22.4	27.5
Day 81	20.2	24.8
Day 82	22.3	27.1
mean Week 12	21.4	25.0
Day 92	20.3	23.4
Day 93	22.0	25.4
mean Week 14	21.1	24.4

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 409 (Repeated Dose 90-Day Oral Toxicity Study in Non-Rodents)

Version / remarks:

study was conducted before guideline was adopted; only a limited number of organs was preserved and histologically examined

GLP compliance:

no

Limit test:

yes

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 41 - 53 weeks
- Weight at study initiation: 7.1 - 11.8 kg
- Fasting period before study: no
- Housing: individually in cages; animals were given the possibility to excercise for at least 30 min/day
- Diet: Ssniff HH-meal, finely ground, from Sniff-Versuchstierdiäten, Soest, Germany (300 g/day)
- Water: tap water in dishes, drinking water quality (ad libitum)
- Acclimation period: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): approximately 22
- Humidity (%): approximately 50 (not regularly measured)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 Feb 1978 To: 09 May 1978

Route of administration:

oral: feed

Vehicle:

water

Details on oral exposure:

- DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: powdered, dry feed and the test item were mixed prior to administration with a handmixer with the same amount of tap-water (300 mL). To ensure homogeneity, the test item was put through a sieve prior to mixing throughout the whole trial. From the week 8 onwards sucrose was also sieved prior to mixing.

The food not consumed within 24 hours to the time of the next feeding was weighed so that the total amount of test item or sucrose, could be individually determined. The time taken to consume the feed was also monitored.

- Storage temperature of food: at room temperature in sealed, plastic-lined, paper sacks

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of test item and sucrose for content and homogeneous distribution in the feed was carried out in week 10.

Analyses showed that the test item at the given concentrations was stable and distributed homogenously in the feed when stored for 14 days at room temperature.

Duration of treatment / exposure:

13 weeks (91 days)

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

5 other: %

Remarks:

50 000 ppm test item (corresponding to approximately 1500 and 1600 mg/kg bw/day for females and males, respectively)

Dose / conc.:

10 other: %

Remarks:

100 000 ppm test item (corresponding to approximately 3000 and 3200 mg/kg bw/day for females and males, respectively)

Dose / conc.:

20 other: %

Remarks:

200 000 ppm test item (corresponding to approximately 6000 and 6400 mg/kg bw/day for females and males, respectively)

No. of animals per sex per dose:

4 males and 4 females

Control animals:

yes

yes, plain diet

Details on study design:

- Dose selection rationale: not specified
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: every day at feeding time, exercise time and during cleaning procedures. External appearance was observed during weekly grooming of the coat and claws.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to dosing commencement as well as in weeks 4, 7 and 13
- The following parameters were examined: examination of reflexes, body temperatures, heart rates

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION: Yes (estimated)
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to dosing commencement as well as in weeks 4, 7 and 13
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to dosing commencement as well as in weeks 4, 7 and 13
- Anaesthetic used for blood collection: No
- Animals fasted: Yes, for 6 hours (250 mL water available)
- How many animals: all
- Parameters examined: haematocrit, haemoglobin, red and white cell count, platelet count, reticulocyte count, mean corpuscular haemoglobin content (MCH), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), differential blood count, thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to dosing commencement as well as in weeks 4, 7 and 13
- Animals fasted: Yes, for 6 hours (250 mL water available)
- How many animals: all
- Parameters examined: blood glucose, urea, creatinine, total protein, glutamic-pyruvic transaminase (GPT), glutamic-oxaloacetic transaminase (GOT), alkaline phosphatase (ALP), bilirubin, cholesterol, glutamate-lactate dehydrogenase (GLDH), sodium, potassium, calcium, triglycerides, oxalic acid, uric acid, serum protein electrophoresis

URINALYSIS: Yes
- Time schedule for collection of urine: prior to dosing commencement as well as in weeks 4, 7 and 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, for 6 hours (250 mL water available)
- Parameters examined: volume, specific gravity, pH, protein, sugar, blood, bilirubin, urobilinogen, ketone bodies, urinary sediment (semi-quantitative assessment of leucocytes, epithelial cells, erythrocytes, amorphous salts, triple phosphate and calcium oxalate)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: DETERMINATION OF α-GLUCOSIDASE ACTIVITY
- The activities of three α-glucosidases (maltase, sucrase and gluco-amylase) were determined in the mucosa of the duodenum

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- On conclusion of the study all dogs were sacrificed by exsanguination under Evipan(R) anaesthesia, and autopsied for pathological and anatomical changes.

ORGAN WEIGHTS: Yes
- The following organs were weighed: brain, heart, lungs, liver, kidneys, spleen, testes, prostate, ovaries, thyroid, adrenals, thymus and pancreas

HISTOPATHOLOGY: Yes
- The following organs were preserved: heart (left ventricular papillary muscle), lungs, liver, spleen, kidneys, parotis, pancreas, oesophagus, stomach (fundus and pylorus), duodenum, jejunum, ileum, colon, pituitary, thyroid, adrenals, testes, epididymides, prostate, ovaries, uterus, brain (cerebrum and cerebellum), eyes, optic nerve, sciatic nerve, thymus, mesenteric lymph nodes, urinary bladder, thoracic aorta, skeletal musculature (quadriceps muscle), bones (femoral bone) and bone marrow (sternum)

From all fixed organs and organ samples, 5 µm paraplast sections were prepared and stained with haematoxylin-eosin.

Additional paraplast sections of the kidneys were stained by the PAS reaction. Additional liver samples were fixed in ROSSMANN's fluid for demonstration of glycogen according to Best.

Statistics:

Any notable differences were examined for statistical significance using the Rank-Sum Test according to Wilcoxon.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

A consequence of treatment was a dose-dependent increase in the occurrence of diarrhoea or thin mushy faeces. This was a fairly constant feature in 7 of the 8 animals treated with the test item at 20%. In one animal of this group, this symptom was only occasionally observed, while the faeces were for the most part formed as with the animals teated at 10%. It was observed that up to six hours after administration of the food substance mixture most animals of all groups produced little or no faeces, while in the following 18 hours up to the next feeding, dose-dependent diarrhoea was observed. The animals treated at 10% had only occasional diarrhoea, while the faeces of most animals were loose or formed. Also with the animals treated at 20%, the faeces were not exclusively of thin mushy consistency, since formed parts were frequently found in addition to the fluid components.

In the animals treated at 20% sucrose and 5% test item faeces of normal consistency were seen during the whole of the study as was the case for the control animals.

The laxative effect observed after administration of 10 and 20% test item was evaluated as drug-related but not a toxic effect since there was no clinical evidence of impairment and the serum electrolytes were not changed.

In one control female an unintentional pregnancy was diagnosed in the 11th week of treatment, which on autopsy at the end of the study was found to have been in the 6th - 8th week.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

There was no notable difference between the mean body weights of the animals treated at 5, 10 and 20% test item and those of the untreated control animals. Mean body weights of animals treated at 20% sucrose were increased vs control animals and animals treated with test item at any dose.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

With few exceptions the offered amount of feed was completely consumed. Also with regard to the time during which the food was consumed, there were no differences between the groups which might have been attributable to treatment.
The resulting test item and sucrose intake is given in Table 1.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In some animals, particularly at the pre-examination, symptoms of conjunctivitis and/or superficial clouding of the cornea was observed as incidental finding and treated with Leukomycin(R) drops or with Scheroson F(R) drops.

Haematological findings:

no effects observed

Description (incidence and severity):

No haematological changes were noted throughout the study. Except for the pregnant control female which due to its pregnancy showed on final examination, an increased erythrocyte sedimenattion rate (ESR), increased platelet count and a decreased haematocrit, haemoglobin and erythrocyte count.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

There was a slight increase in alkaline phosphatase activity for the animals treated with 20% sucrose compared with the non-treated controls. The difference to the controls was statistically significant in week 4 week of treatment (p < 0.01). This slight increase was evaluated as a symptom of an increased load on the liver as a result of the high sugar content and a resulting enzyme induction.

Mean urea values of the animals in the treated groups were lower compared with the untreated control animals, at the time of both the intermediate and final examination. The differences were frequently found to be statistically significant compared with the control animals. The on average slight, but sometimes statistically significant reduction in plasma urea values was probably the result of a protein deficiency as there was no liver cell damage. In comparison with the non-treated control animals, the animals treated with 5 to 20% carbohydrate (test item or sucrose) in their standard feed received correspondingly less protein. This reduction in protein would account for the slightly reduced plasma urea values.

The creatinine values for the animals treated at 20% sucrose in the 13th week of treatment (p < 0.05) and in the animals treated at 20% test item in the 4th and 7th week of treatment (p < 0.01 or p < 0.05) were within the physiological range but statisitcally significantly increased compared with the non-treated control animals. There was no toxicological significance assigned to the increase in plasma creatinine, which was within the normal range. This was not caused by impaired renal function as the plasma urea values, urinalyses, macroscopical and microscopical organ findings and kidney weights showed no indication of kidney damage.

Apart from the pregnant animal, which - as a result of pregnancy - had reduced albumin and increased globulin in the 13th week of treatment, treatment with the test item or sucrose at concentrations up to 20% did not produce changes in serum protein.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was transient positive evidence of blood in some animals but this was not group-related. A second examination a few days after the 1st examination showed that the urine of those animals had no blood. Positive evidence of blood was obviously the result of minor accidental injury at the time the urine was collected.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No drug-related macroscopic organ changes were found. Individual animals in various groups had symptoms of parasitic invasion (ascarides in the small intestine, mild granulomatous pulmonary changes as a result of migrating ascarides larvae). One female in the sucrose group had a bean-sized cyst with corresponding cerebellar impression between the cerebellum and medulla oblongata.
One animal at 5% test item had a pituitary cyst the size of a pin head.

Six fetuses were noted in the uterus of the pregnant control female, her teats were swollen.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were occasionally cyst-like random changes in the pituitary, thyroid and adrenals.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

The activity of intestinal α-glucosidases (maltase, sucrase, glucoamylase) was not affected by treatment with the test item.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 6 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects noted up to and including 200 000 ppm

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 6 400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects noted up to and including 200 000 ppm

Key result

Critical effects observed:

no

Table 1. Mean intake of test item and sucrose

Group	Sex	Intake [mg/kg bw/day]
20% sucrose		Week 1	Week 7	Week 13
	male	6430	5980	5736
	female	6782	6213	5694
5% test item	male	1518	1471	1439
	female	1628	1610	1580
10% test item	male	3089	3046	3008
	female	3299	3196	3178
20% test item	male	6100	6028	5820
	female	6477	6711	6322

 

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 452 (Chronic Toxicity Studies)

Version / remarks:

study was conducted before guideline was adopted; only a limited number of organs was preserved and histologically examined

GLP compliance:

no

Limit test:

yes

Species:

dog

Strain:

Beagle

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: 18-29 weeks
- Weight at study initiation: 5.4-8.5 kg
- Fasting period before study: no
- Housing: single housing in cages (110 x 115 cm ground space); animals were given the possibility to excercise for at least 30 min/day except for weekends and public holidays
- Diet: "ssniff HH-diet for dogs, double grinding", from Sniff-Versuchstierdiäten, Soest, Germany (250 g/day from study start to week 7, 300 g/day from week 8 to 16, 330 g/day from week 17 to 26, 350 g/day from week 27 to 37 and 350 mg/day from week 38 onwards)
- Water: tap water in bowls (ad libitum)
- Acclimation period: not specified

DETAILS OF FOOD AND WATER QUALITY: not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): prevailing 22 (range 19-27.5)
- Humidity (%): prevailing 50 (up to 100% during cleaning procedures)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: Dec 1979 To: Dec 1980

Route of administration:

oral: feed

Vehicle:

water

Details on oral exposure:

- DIET PREPARATION
The test item was administered with the food at concentrations of 2.5, 5 or 10%.
For comparison, one group was dosed with 10% saccharose and another group received 10% maize starch. In order to obtain about the same caloric value in the food, the animals treated with 2.5 or 5% test item received 7.5 or 5% maize starch, respectively, to make up for the difference.

Preliminary mixtures were prepared for the groups with food containing 2.5 and 5% test item; premixes contained 25% test item and 75% maize starch, or 50% test item and 50% maize starch, respectively.
- Rate of preparation of diet (frequency): not specified
The food/ substance mixtures were prepared in a mixing granulator, type MGT (volume of mixing cylinder about 40 L). Thus were prepared food/ substance mixtures of 18 kg per group up to week 16 inclusive, 20 kg up to week 26 inclusive, 21 kg up to week 37 and 23.5 kg up to week 52 inclusive.
- Mixing appropriate amounts with: maize starch (preliminary mixtures) or powdered feed
- Storage temperature of food: not specified

Warm tap-water in the ratio of 1 : 1 was added to the feeding meal or to the food/substance mixture shortly before the feeding time. A homogeneous mash of the food/water mixture was prepared by means of a mechanical kneader in a kitchen appliance (model A 200) from the company Hobart, D-7600 Offenburg (kettle holding capacity 10 L). This food was generally offered to the animals in the morning, mainly between 9:00 and 9:30 a.m., so that in case of deferred food intake it remained at their disposal up to 22 h. The quantity of food left uneaten until the next feeding was weighed back, so that the quantity of food intake and thus the administered substance quantity had to be determined individually.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Prior to the study the preliminary mixtures were analysed for test item content and for stability exceeding 6 weeks (at least 55 days) at room temperature. At the same time, analytical examinations confirmed that the food/substance mixtures prepared from the preliminary mixtures contained the stated test item concentrations. These examinations also confirmed the homogeneous distribution of the aggregate mixture. The same applies to the concentration of saccharose in the saccharose comparison group.

The analytical control examinations were repeated at regular intervals during the entire experimental period.

A 24 h stability was confirmed for the test item in moist food, whereas saccharose remained stable for only 7 h. For this reason particular attention was paid to the saccharose group, for which the food intake had to be terminated within 7 h.

Duration of treatment / exposure:

1 year

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

2.5 other: %

Remarks:

25 000 ppm test item (corresponding to approximately 670 mg/kg bw/day)

Dose / conc.:

5 other: %

Remarks:

50 000 ppm test item (corresponding to approximately 1340 mg/kg bw/day)

Dose / conc.:

10 other: %

Remarks:

100 000 ppm test item (corresponding to approximately 2970 mg/kg bw/day)

No. of animals per sex per dose:

4 males and 4 females

Control animals:

yes

other: concurrent control animals received standard diet containing 10% saccharose

Details on study design:

- Dose selection rationale: not specified
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: every day at feeding time, care, exercise time and during cleaning procedures

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to dosing commencement as well as in weeks 4, 7,13, 26, 39 and 52
The following parameters were examined: control of reflexes, body temperatures, heart rates

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- 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 data: Yes

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: No

WATER CONSUMPTION: Yes (no details specified)

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to dosing commencement as well as in weeks 4, 7,13, 26, 39 and 52
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to dosing commencement as well as in weeks 4, 7,13, 26, 39 and 52
- Anaesthetic used for blood collection: No
- Animals fasted: Not specified
- How many animals: all
- Parameters examined: haematocrit, haemoglobin, red and white cell count, thromocyte count, reticulocyte count, mean corpuscular haemoglobin content (MCH), mean corpuscular volume (MCV), mean corposcular haemoglobin concentration (MCHC), differential blood count, thromboplastin time, partial thromboplastin time, sedimentation test

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to dosing commencement as well as in weeks 4, 7,13, 26, 39 and 52
- Animals fasted: Not specified
- How many animals: all
- Parameters examined: blood glucose, urea, creatinine, bilirubin, cholesterol, alkaline phosphatase (ALP), aspartate aminotranspherase (AST), alanine aminotransferase (ALT), glutamate dehydrogenase (GLDH), total albumin, sodium, potassium, calcium, triglycerides, uric acid, serum protein electrophoresis

URINALYSIS: Yes
- Time schedule for collection of urine: prior to dosing commencement as well as in weeks 4, 7,13, 26, 39 and 52
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes (while in metabolic cages, 6 h)
- Parameters examined: volume, specific weight, pH, albumin, glucose, blood, bilirubin, urobilinogen, ketone bodies, urinary sediment (semi-quantitative assessment)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: DETERMINATION OF α-GLUCOSIDASE ACTIVITY
- The activities of three α-glucosidases (maltase, saccharase and glucoamylase) were determined in the mucosa of the duodenum, jejunum and ileum

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
- At the end of the experimental period all dogs were killed by exsanguination under deep Evipan(R) anaesthesia. They were then dissected for macroscopic evaluation.

ORGAN WEIGHTS: YES
- The following organs were weighed: heart, lung, liver, kidneys, spleen, testes, ovaries, thyroid gland, adrenals, thymus, prostate, brain and pancreas

HISTOPATHOLOGY: Yes
- The following organs were preserved in Bouin's liquid or in 10 or 4% formaldehyde solution: heart, lung, liver, speen, kidneys, pancreas, parotis, pituitary, thyroid, adrenals, testes, epididymides, prostate, ovaries, uterus, esophagus, stomach, intestines, mesenteric lymph node, thymus, gall bladder, urinary bladder, brain, eye, optic nerve, sciatic nerve, aorta, skeletal muscle, bones and marrow

The bones were decalcified in EDTA. Paraplast cuttings of about 5 µm thickness were prepared of all the fixed organs and organ samples and were coloured with H&E. Additional Paraplast cuttings of kidneys were stained by means of the PAS reagent. Slices of frozen liver, fixed in formol, were stained with Oil Red O in order to trace lipids.

Statistics:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Diarrhoea (pappy to liquid faeces) has dose-dependently been more frequently observed, in all animals treated with the test item. Animals treated at 2.5% rarely showed diarrhoea, but more often pappy faeces, whereas the faeces of high dose animals were generally of a liquid to watery nature. Animals dosed at 10% saccharose showed only a slightly increased in the frequency of watery to liquid faeces in comparison with the maize starch control animals.

For one animal at 2.5% and for two animals at 5%, sanguinolent glairs in their faeces have been reported once for each animal during the treatment period. This was probably due to a parasitic origin in the intestinal tract, but it bears no relation with the administered test item.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No test item-related effects on food consumption were noted. Also with regard to the time during which the food was consumed, there were no differences between the groups which might have been attributable to treatment. Animals in the 10% saccharose group had pratically emptied their bowl within 7 h (maximum defined stability of the food/substance mixture).

The resulting mean test item intake amounted to 8 170, 16 070 and 33 000 mg/animal/day, respectively. The mean saccharose intake was 33 000 mg/animal/day. Based on mean body weights at the end of the study period these intakes correspond to daily test item intakes of approximately 670, 1340 and 2970 mg/kg bw/day. The mean saccharose intake was 2845 mg/kg bw/day.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Very few macroscopic findings were noted at necropsy. These findings did not give an indication of test item-related effects.

For one animal at 2.5% test item, the ileum presented a medium-graded blotched mucosa of the size of 10 pence. For another animal in this group extensive hypoplasia was noted for one epididymis. For one animal at 10% test item, the region of the left metacarpal ligament presented a bald, blotched skin, of the size of 10 pence, with a formation of scurf. For one animal in the saccharose group a medium-graded blotched mucosa of the size of a penny was noted.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

In the different groups, certain animals presented some low-graded unspecific sequels of inflammation in different organs :
In kidneys such a process has been observed as ascending round cell infiltrations of the papilla. In the lungs were found small parabronchial infiltrations with poly-morphonuclear leucocytes.

For the dog with the skin finding at metacarpus (10% test item) the histological diagnosis revealed an unspecific purulent dermatitis.

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Activity of the three α-glucosidases (maltase, saccharase and glucoamylase) in duodenum, jejunum and ileum showed the characteristic enzyme pattern for each intestinal section. These patterns were not affected by treatment with the test item or with saccharose.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 2 970 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects noted up to and including 100 000 ppm

Key result

Critical effects observed:

no

Cross-reference 4

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: A one-year feeding study with the test item in rats was carried out. The study was part of a life-span oral toxicity and carcinogenicity study with the same test item and was primarily intended to obtain interim information on pathological changes, if any, after a one-year feeding period.
- Short description of test conditions: The test substance was fed to groups of 10 male and 10 female rats at levels of 0 (control), 2.5, 5 and 10% in the diet. An additional control group was fed sucrose at a level of 10% in the diet. The rats were derived from parents which had been kept on the same diets already prior to mating, and subsequently during the gestation and lactation period.
- Parameters analysed / observed: Observations were made of general appearance and growth and food and water intake were measured. After 52 weeks all rats were killed and examined grossly. Seven different organs were weighed. Tissue samples of a wide range of organs were examined microscopically.

GLP compliance:

yes

Species:

rat

Strain:

Wistar

Remarks:

Cpb:WU; Wistar random

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Central Institute for the Breeding of Laboratory Animals TNO, Zeist, The Netherlands
- Other: The rats used for the study were pre-treated in utero with the test or control substance. Parental animals were fed the test and control diets for 12 weeks before mating, during pregnancy and during lactation. Nine weeks after the first mating the animals were mated again to produce the F1b litters. When these F1b litters had reached weaning age, 10 male and 10 female pups were selected from each group from as many litters as possible and with a birth-date range as narrow as possible. The selected animals were kept on the same diets as their parents had received.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: not specified
- Weight at study initiation: not specified
- Fasting period before study: no
- Housing: five per sex per cage, in suspended stainless steel cages, fitted with wire mesh floors and front
- Diet: TNO-CIVO stock diet for rats, modified by adding 10% maize starch (ad libitum)
- Water: tap water (ad libitum)
- Acclimation period: not specified

DETAILS OF FOOD AND WATER QUALITY: The diet has been regularly analysed by the supplier for nutrients and contaminants. Tap water has been analysed periodically for contaminants. Results of analyses made in the test period are presented in the report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 40-60
- Air changes (per hr): not specified (well-ventilated)
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 Jun 1980 To: 26 Jun 1981

Route of administration:

oral: feed

Details on route of administration:

The animals were fed ad libitum from weighed feeders which were filled once a week with the diets.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

- DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with CIVO stock diet for rats, modified by adding 10% maize starch. The materials were thoroughly mixed into the carrier by means of a mechanical blender (Lodige) at levels of 0 (control), 2.5, 5 or 10% (test item) and 10% (sucrose).
- Storage temperature of food: room temperature

TEST ITEM INTAKE
- Test item intake was not specified in the report.
- Test item intake was calculated retrospectively based on mean body weights and food consumption on study day 364. The calculation of test item intake is detailed in Tables 2 and 3.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and homogeneous distribution of the test item and sucrose in the diets was determined by analyzing five samples taken at five different locations in the food container from one batch of each of the diets after one day and after 14 days of storage at room temperature. From each freshly prepared batch a sample was taken and stored in a freezer at -20 °C. In samples of the batches produced on days -20, 69, 159, 238, 273 and 348 the content of test item and sucrose was determined by High Pressure Liquid Chromatography (HPLC).

The results of the analyses show that the actual dietary levels of test item and sucrose were close to the intended levels, indicating proper preparation of the diets. The analysis of five samples taken from each diet immediately after preparation showed a coefficient of variation (= standard deviation divided by the mean value) which is generally less than 5%. This is indicative of satisfactory homogeneous distribution of the test- and control item in the diets. The figures obtained after 14 days of storage at room temperature show that test item and sucrose are stable under the conditions of storage.

Results of the determinations of test item and sucrose in the diets during the course of the experiment generally showed some deviation from the intended levels, but this is rather due to the analytical procedure applied than to mixing of an incorrect amount of test item or sucrose into the diets. In the diets without any sucrose added an average amount of about 1% sucrose was found. The origin of this amount of sucrose is not known.

Duration of treatment / exposure:

1 year

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

2.5 other: %

Remarks:

25 000 ppm (corresponding to ca. 790 and 1070 mg/kg bw/day in males and females, resp.; calculated from mean body weights and food consumption on day 364)

Dose / conc.:

5 other: %

Remarks:

50 000 ppm (corresponding to ca. 1500 and 2240 mg/kg bw/day in males and females, resp.; calculated from mean body weights and food consumption on day 364)

Dose / conc.:

10 other: %

Remarks:

100 000 ppm (corresponding to ca. 3220 and 4320 mg/kg bw/day in males and females, resp.; calculated from mean body weights and food consumption on day 364)

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes

other:

Details on study design:

- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable

Positive control:

not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: general condition and behaviour of the animals were checked frequently
- Cage side observations: special attention was paid to the consistency of the faeces to establish the occurrence of diarrhoea, if any

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during the first 26 weeks, every two weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/week: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in g/g food consumed: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily during the first 4 weeks and and daily during weeks 13/14, 25, 39 and 21

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
In week 53 the rats were killed by exsanguination from the abdominal aorta under ether anaesthesia and then examined grossly for pathological changes. Males were killed at day 364; females at day 365.

ORGAN WEIGHTS: YES
The following organs were weighed: brain, caecum (filled and empty), heart, kidneys, liver, testes

HISTOPATHOLOGY: Yes
Tissue samples of the following organs of all animals were preserved in a neutral, aqueous, phosphate-buffered, 4% formaldehyde solution:
adrenals, aorta, brain, caecum, colon, duodenum, eyes, heart, ileum, jejunum, kidneys, liver, lungs, lymph nodes (cervical and mesenteric), mammary glands, oesophagus, ovaries, pancreas, pituitary, prostate,salivary glands (parotid, sublingual and submaxillary), sciatic nerve, seminal vesicle, skeletal muscle, skin, spleen, sternum with bone marrow, stomach (glandular and non-glandular), testes, thyroid, trachea, urinary bladder, uterus

All tissues and organs listed for microscopic examination were embedded in Paraplast®. Sections of 5 µm thickness were cut and stained with haematoxylin and eosin. In addition kidney sections were stained with periodic acid-Schiff and sections of frozen formalin-fixed liver with Oil-red-O. Detailed microscopic examinations were carried out on tissues and organs mentioned above of all rats of the control group, the 10% test item group and the 10% sucrose group and on liver and kidneys of all rats of the 2.5% and 5% test item groups. In addition the spleen, thyroid, brain, adrenals and pituitary of all rats of the low- and mid-dose test item groups were examined for the presence of preneoplastic or neoplastic changes.

Statistics:

Body weights were subjected to analysis of co-variance, with the initial body weights as co-variables, followed by Dunnett's Multiple Comparison test. Food intake and food efficiency figures were subjected to analysis of variance followed by the LSD test. Relative organ weights were subjected to analysis of variance followed by Dunnett's Multiple Comparison test. Absolute group mean differences that exceeded the significant differences are marked as statistically significantly different only when the F probability, obtained by analysis of variance is equal to or less than 5%.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Statistically significant differences did not occur between the groups fed test item or sucrose and the controls. However, in males body weights were generally slightly higher than in controls. This difference was associated with a lower initial body weight in the control group. In females a similar phenomenon occurred: i.e. relatively high body weights in the sucrose group, associated with a high initial weight. The initial body weights of the test item-fed groups were similar. The body weights remained similar during the whole experimental period.
The growth rate of the sucrose-fed group was generally comparable with that of the group fed 10% test item. Females fed 10% sucrose, however, became slightly heavier than those of the 10% test item group during the second half of the study.

Body weight gain over the whole one-year period is expressed as a percentage of the initial body weights is presented in Table 1. These figures show that neither the test item nor sucrose had any consistent effect on the growth rate of rats at the dose levels tested.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Although the food intake values of the groups fed the test item or sucrose was generally slightly higher than those of controls, both in males and females, statistically significant differences amongst the groups occurred only occasionally in males. From the grand means it appears that in the group fed 10% test item the males consumed 9.6% more than the controls did, while in females the difference was 3.9%.

Food efficiency:

no effects observed

Description (incidence and severity):

Food efficiency figures were very similar in the various groups, both in males and in females. In males fed 10% test item, food efficiency was slightly lower than in controls during the first five weeks. Thereafter, the figures were comparable or even higher than in controls.

Water consumption and compound intake (if drinking water study):

no effects observed

Description (incidence and severity):

No statistically significant differences in water consumption were noted throughout the study. Water intake values in males fed 10% of the test item were generally slightly higher than those of males of other groups during the major part of the study. In females, however, an opposite tendency was observed: in the 10% test item group and also in the other test groups water intake values were generally lower than in controls.

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

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):

The only statistically significant change in relative organ weights which could be ascribed to the feeding of the test item consisted of an increased weight of the filled and empty caecum in males of the high dose group.
Caecal enlargement is known to be induced by a variety of substances which are not fully digestible, such as dietary fiber, raw potato starch, modified starches, lactose, pectins, alginates etc. It is considered to be an adaptive rather than a pathological change. In the present study, the enlargement is most likely due to the presence of non-digested test item in the large bowel, which passed the small intestines unchanged and is fermented in the large intestines by the gut flora.

The decrease in relative brain weight which occurred in females fed 10% sucrose, is ascribed to the relatively high body weights in this group and the well-known inverse relationship between body weight and the weight of this organ.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Gross examination at autopsy did not reveal any abnormality attributable to the feeding of the test item, except for an enlarged/swollen caecum in two males of the high dose test item group. The other gross lesions observed consisted of e.g. discoloured kidneys and hydrometra. All other gross lesions occurred in one or a few animals only.
Caecal enlargement is known to be induced by a variety of substances which are not fully digestible, such as dietary fiber, raw potato starch, modified starches, lactose, pectins, alginates etc. It is considered to be an adaptive rather than a pathological change. In the present study, the enlargement is most likely due to the presence in the large bowel of non-digested test item, which passed the small intestines unchanged and is fermented in the large intestines by the gut flora.

Although the incidence of gross lesions varied among different groups, in no case a consistent or relevant dose-related increase was seen. Moreover, the observed abnormalities were age-associated and common findings in the strain of rats used. Therefore, these findings are not ascribed to the ingestion of the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The observed non-neoplastic histopathological changes are common findings in the strain of rats used. Moreover, the incidences of the observed lesions are comparable with those found in other 12-month studies with rats of the same strain performed at the laboratory. Therefore, these lesions were not ascribed to the treatment.

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

The observed neoplastic lesions consisted of fibromatous polyps in the uterus (three females of the high dose group and one female of the sucrose group); adenomas in the pituitary (two females of the mid dose group); a ganglioneuroma in the adrenal of one female of the low dose group and a brain tumour (most probably a meningioma) in one female of the control group. Furthermore, a phaeochromocytoma was found in an adrenal of one male of the mid dose group.

The observed neoplastic histopathological changes are common findings in the strain of rats used. Moreover, the incidences of the observed lesions are comparable with those found in other 12-month studies with rats of the same strain performed at the laboratory. Therefore, these lesions were not ascribed to the treatment.

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 3 220 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects noted up to and including 100 000 ppm

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 4 320 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effects noted up to and including 100 000 ppm

Key result

Critical effects observed:

no

Table 1. Mean body weight gain over study period [%]

Group	Males	Females
Control	756	380
2.5% test item	698	381
5.0% test item	742	388
10% test item	757	378
10% sucrose	726	404

 

Table 2. Test item intake males

	Nominal concentration of test item in diet (%)
	0	2.5	5.0	10
Body weights (g, day 364)	560.1	565.7	583.5	583.9
Food consumption (g food/animal/week)	127.7	125.5	125.7	131.5
Food consumption (g food/animal/day)	18.2	17.9	18.0	18.8
Food consumption (g food/kg bw/day)	32.6	31.7	30.8	32.2
Test item intake (mg/kg bw/day)	0	792	1539	3217

Table 3. Test item intake females

	Nominal concentration of test item in diet (%)
	0	2.5	5.0	10
Body weights (g, day 364)	302.6	303.3	300.8	299.1
Food consumption (g food/animal/week)	87.9	90.8	94.2	90.4
Food consumption (g food/animal/day)	12.6	13.0	13.5	12.9
Food consumption (g food/kg bw/day)	41.5	42.8	44.7	43.2
Test item intake (mg/kg bw/day)	0	1069	2237	4318

Cross-reference 5

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

study was conducted before guideline was adopted; no ophthalmoscopic examinations were performed; histopathology was only performed on organs from 5 males and 5 females/group

GLP compliance:

no

Limit test:

yes

Species:

rat

Strain:

Wistar

Remarks:

W.74

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Females nulliparous and non-pregnant: not specified
- Age at study initiation: approximately 5 - 6 weeks
- Mean weight at study initiation: males 85 g, females 80 g
- Fasting period before study: no
- Housing: individually in Makrolon cages (type II) on dust-free wooden granules
- Diet: Altromin R powder feed (ad libitum)
- Water: tap water (ad libitum)
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): not specified
- Air changes (per hr): not specified
- Photoperiod (hrs dark / hrs light): not specified

IN-LIFE DATES: From: Feb 1978 To: Mar 1978

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On three occasions during the study achieved test item concentration in the diet were analytically determined. The test substance was extracted from the dietary admix with water for 30 min and chromotographically analysed. Results on achieved concentraions are presented in Table 1.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily, 7 days/week

Dose / conc.:

3.3 other: %

Remarks:

33 000 ppm (corresponding to ca. 2240 and 3230 mg/kg bw/day in males and females, respectively)

Dose / conc.:

10 other: %

Remarks:

100 000 ppm (corresponding to ca.7290 and 9160 mg/kg bw/day in males and females, respectively)

Dose / conc.:

30 other: %

Remarks:

300 000 ppm (corresponding to ca. 23 650 and 29 730 mg/kg bw/day in males and females, respectively)

No. of animals per sex per dose:

15 males and 15 females

Control animals:

yes

yes, plain diet

Details on study design:

- Dose selection rationale: not specified
- Rationale for selecting satellite groups: not applicable
- Post-exposure recovery period in satellite groups: not applicable

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

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: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 5 and 12
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Not specified
- How many animals: 5 males and 5 females/group
- Parameters examined: red and white cell count, platelet count, reticulocyte count, haemoglobin, haematocrit, differential blood count, mean corpuscular haemoglobin content (MCH), mean corpuscular volume (MCV), thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 5 and 12
- Animals fasted: not specified
- How many animals: 5 males and 5 females/group
- Parameters examined: alkaline phosphatase (ALP), glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), creatinine, urea, blood glucose, cholesterol, bilirubin, total serum protein, uric acid, oxalate

URINALYSIS: Yes
- Time schedule for collection of urine: weeks 5 and 12
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Not specified
- Parameters examined: semiquantitave determination of glucose, blood, protein and pH, ketobodies, bilirubin, urobilinogen, urinary sediment, protein (quantitative)

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

OTHER: DETERMINATION OF α-GLUCOSIDASE ACTIVITY
- The activities of three α-glucosidases (maltase, sucrase and gluco-amylase) were determined in the mucosa of the small intestine (jejunum) of 2 males and 2 females/group.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes

ORGAN WEIGHTS: Yes
- The following organs were weighed: thyroid, thymus, heart, lungs, liver, spleen, kidneys, adrenals, testes and ovaries

HISTOPATHOLOGY: Yes
- The following organs from 5 males and 5 females/group were preserved in Bouin's fluid (transferred after approximately 30 hours into
70% alcohol): heart, lungs, liver, spleen, kidneys, pituitary, thyroid, adrenals, testes, epididymes, prostate, seminal vesicle or ovaries and uterus, salivary glands, pancreas, oesophagus, stomach, intestines, lymph nodes, thymus, urinary bladder, brain, eyes, aorta, trachea, skeletal muscles (quadriceps muscle), bones (femoral bone) and bone marrow (sternum).

additional liver samples were fixed in ROSSMAN'S fluid for demonstration of glycogen and fixed in formolin-calcium for evidence of fat.

Organ material from the remaining animals was fixed in 10% formaldehyde solution.

The bones were decalcified with ethylene dinitrilo­tetraacetic acid tetra-sodium salt (EDTA).
From the organs or organ samples fixed in Bouin's fluid, 5 µm paraplast sections were prepared and stained with haematoxylin-eosin.
Additional paraplast sections of the kidneys were stained using the PAS reaction. Frozen sections of liver samples of approximately 15 µm fixed in formalin were stained with Oil Red O to demonstrate fat.

Paraplast sections of the liver samples fixed according to ROSSMANN were stained for the demonstration of glycogen according to BEST.

Statistics:

The following were calculated: arithmetic group mean values, standard deviations, upper and lower confidence limits at the confidence level of 1-α = 95% and 1-α = 99%.

Comparison of values for the trial groups with the control groups was carried out using the Mann, Withney and Wilcoxon significance test (U-Test) at the significance level α = 5% and α = 1%.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mild diarrhoea occurred in about one third of the animals at 10% test item, mainly in the first two weeks of the experiment. All the animals treated at 30% test item had severe diarrhoea during the first two weeks of the experiment. As a result of diarrhoea the region around the anus and root of the tail was sore in some of the animals. As the trial progressed the intensity of the diarrhoea decreased and from about the 5th week of the experiment faeces were normal in the majority of animals.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight development in males and females at 3.3% test item and also in females at 10% were similar to the control. For males at 10% slightly lower body weights were noted during the first 8 weeks (- 6%). At 30% males body weights were significantly lower than those of the control. For females at 30% lower weights were noted mainly during the first 6 weeks.

In week one male and female animals at 30% sucrose were significantly lighter than the control, however, the male rats were significantly heavier than the control animals from the 9th week of the experiment.

The reduced body weights at 30% item might at least in part have been attributable to the noted diarrhoea.
No toxicological relevance was attached to the temporary significant reduction in body weight occurring only in e male rats at 10% test item, since the difference from the control group was small (max. 6%) and the increases in body weight from about the middle of the study were equivalent to those of the control group. Also in the case of the significantly lower bodyweights of the male and female rats of the sucrose group which occurred temporarily (after 1 week of study), these depressions in growth rate were certainly a consequence of the "unphysiological" food composition, where in the case of the test item there was the additional laxative effect. The regression of these symptoms (depression of growth and diarrhoea) as the experiment progressed is regarded as adaptation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Male and female rats given up to 10% test item consumed approximately the same amount of powder feed as the control animals. The male animals in the 30% test item group and also the male and female animals in the sucrose group had a slightly (< 20%) reduced feed intake.

Mean food consumption and amounts of test item ingested are shown in Table 2.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

effects observed, treatment-related

Description (incidence and severity):

Water intake for male and female rats in the low and mid dose group was not affected. In high dose animals water consumption was increased (10 to 20% in males and females, resp.), while animals treated with sucrose consumed less water than the control.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No differences between test item-treated and control animals were noted after 5 or 12 weeks of treatmnet.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

After 5 weeks of treatment male animals treated at 30% test item had a significantly (p < 0.05) higher bilirubin plasma content as well as significantly (p < 0.05 and p < 0.01) lower urea and blood sugar, while male animals of the sucrose group had significantly (p < 0.05 and p < 0.01) lower GOT and GPT and urea values.

Female animals at 30% test item had significantly (p < 0.05) higher alkaline phosphatase (ALP) activities as well as significantly (p < 0.05 and p < 0.01) lower protein and urea values. In the animals of the sucrose group the latter was also significantly (p < 0.05) lower than in the control females.

Blood sugar values in all female treatment groups was significantly but not strictly dose-dependently higher than in the control animals, with females fed with sucrose exhibiting the mean highest blood sugar content.

After 12 weeks of treatment, ALP activities, as well as GOT and GPT were not affected in males treated at up to 10% test item. However, at 30%, ALP activity as well as bilirubin content were significantly higher, while the urea content was significantly lower (p < 0.01) than in the control animals. In all test item groups the oxalic acid content was significantly (p < 0.05 and p < 0.01) increased, although this was not dose-dependent.

In female rats of all test item groups the bilirubin content was significantly (p < 0.01) and dose­dependently increased, while the urea content at 30% test item was significantly (p < 0.05) decreased.

The male animals of the sucrose group had significantly (p < 0.05) lower ALP, GOT, GPT activities and urea contents as well as significantly higher (p < 0.05 and p < 0.01) plasma creatinine and oxalic acid contents in comparison to the control group.In female rats at 30% sucrose, the ALP activity and bilirubin content was significantly (p < 0.01 and p < 0.05) lower, while the creatinine content was significantly (p < 0.01) higher in comparison with the control group.

In conclusion, the clinical-chemistry parameters up to and including 10% test item gave no indication of treatment-related organ damage. The values for these groups which deviated significantly from those of the controls (blood sugar in females after 5 weeks, oxalic acid in males, bilirubin in females each after 12 weeks) are not regarded as an expression of damage, due to - in
part - absence of dose dependency (blood sugar, oxalic acid) and because corresponding results in the other sex or other time points of determination were not obtained. In addition, the values lie within the biological deviation found for untreated rats of this strain.

For the same reason no toxicological significance is attached to the results obtained after 30% test item for the parameters blood sugar (significantly lowered in males and increased in females, in each case after 5 weeks), ALP (significantly increased in females after 5 weeks and in males after 12 weeks) as well as for plasma protein (significantly lowered in females after 5 weeks). Also there was no correlation between the histopathological findings for the animals of this group and the results cited above.

The significantly raised bilirubin values of the male rats after 30% test item at both investigation times and those of the female rats at the end of the study were in the case of individual animals (particularly females at the end of the experiment) either at the limit or beyond the norm for rats of this age. In the absence of any correlation, the toxicological significance of this finding is unclear. At 30% test item for both male and female rats at both examination times, the urea level was significantly lowered and was accompanied by significantly lower kidney weights. The urea values for the male rats treated with sucrose (at both examination times) and the female rats (after 5 weeks) were significantly lower than those of the controls. Since low urea levels are not regarded as evidence of renal damage and urinalyses, creatinine determinations and histopathological investigations gave no indication of possible renal pathology, these findings are not regarded as toxicologically relevant. The decreased urea concentrations might well have resulted from the high carbohydrate and lowered protein content in the diet of both these dose groups.

Urinalysis findings:

no effects observed

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):

Absolute kidney weights in males and females at 30% test item were significantly (p < 0.01) reduced.

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

Activity of the three intestinal α-glucosidases (maltase, sucrase and glucoamylase) was not affected by the test item or sucrose.

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 7 290 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 9 160 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

Table 1. Actually achieved test item concentrations in the feed

	Nominal concentration (ppm)
	33 000	100 000	300 000
Analysis Date	Actual concentration (ppm)*
11Apr1978	33 660	109 000	279 000
28Apr1978	30 000	87 000	279 000
29May1978	30 200	89 000	288 000
Mean	31 300	95 000	282  000

Table 2. Mean feed and test item intake

Test item concentration in the diet (%)	Mean food intake (g/animal/day)	Mean test item intake (mg/kg bw/day)*
Males
0	19.50	0
3.3	19.47	2239
10	18.88	7291
30	17.34	23649
30 (sucrose)	17.82	18374
Females
0	15.26	0
3.3	16.75	3233
10	15.79	9164
30	15.16	29728
30 (sucrose)	12.80	23147

* Based on animal weights in the middle of the study

Cross-reference 6

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

exposure-related observations in humans: other data

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Endpoint addressed:

acute toxicity: oral

other: metabolic effects

Qualifier:

no guideline followed

Principles of method if other than guideline:

Acute doses of 50 g test item were given to 8 metabolically healthy volunteers and compared against 50 g fructose or saccharose to 12 each Type II diabetics. Measurements were conducted over a period of 3 hours in respect of capillary blood glucose, serum insulin, free fatty acids in serum, as well as blood lactate and pyruvate. Clinical signs were observed for 12 hours following treatment.

GLP compliance:

no

Ethical approval:

not specified

Details on study design:

The study was conducted on 8 metabolically healthy female volunteers plus 24 type II diabetics. Volunteers and patients were accepted for participation in the study on a volunteer basis only after being informed in detail regarding the procedures of the study. All the diabetics were under treatment with oral antidiabetics (sulfonylurea preparations) and diet. In each case the medication was stopped on the evening before the loading test. he metabolically healthy volunteers received the test item loading only once. The 24 patients were subdivided into two groups of 12 each and received either test item and frutose or test item and sucrose as part of two controlled cross-over studies.

The average age of the volunteers in years was 32.7 ± 4.8, and the body weight according to Broca was 85 ± 12%.

Blood samples were taken of capillary blood to determine the blood glucose, and samples of venous blood were taken through a polyethylene cannula inserted into a cubital vein to determine the serum insulin, serum free fatty acids, blood lactate and blood pyruvate levels. Blood samples were taken after fasting as well as 10, 15, 20, 30, 45, 60, 90, 120,150 and 180 minutes after beginning the oral loading.

Exposure assessment:

not specified

Details on exposure:

TYPE OF EXPOSURE: oral
The loading tests were performed with 50 g test item, 50 g fructose or 50 g sucrose in accordance with the standard conditions of an oral glucose tolerance test. The test items were dissolved in 400 mL water and were to be drunk by the test person within 5 minutes.

Results:

In metabolically healthy volunteers, neither blood glucose nor serum insulin or lactate showed any significant change in total level over the observation period of 180 min. The same also applies to the free fatty acids and pyruvate. Only the significant curvature in the blood glucose and insulin curves indicated a certain interim, although minimal, loading. One volunteer complained of meteorism and flatulence within the first 12 h after loading, and another also complained of mild diarrhea.

In the two groups with diabetics there were no increases in blood sugar or serum insulin levels after consuming isomalt, in contrast with observations after sucrose or fructose administration. Gastrointestinal symptoms (meteorism, flatulence, and mild diarrhoea) occurred in 2 - 4 persons in each of the 2 groups.