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

Endpoint summary

Description of key information

subchronic toxicity:

OECD 408, oral (feeding) rat, 90 days: NOAEL 100 000 ppm (equivalent to ca. 7000 mg/kg bw/day in males and ca. 8400 mg/kg bw/day in females)

Similar to OECD 409, oral (feeding) dog, 90 days: NOAEL 200 000 ppm (equivalent to ca. 6400 mg/kg bw/day in males and ca. 6000 mg/kg bw/day in females)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

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

Reference 2

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

 

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

7 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch 1 and 2) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, 8.6, of Regulation (EC) No. 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Reliable oral repeated dose toxicity studies with Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitola are available in the rat (90 days/1 year) and in the dog (90 days/1 year).

A reliable oral (dietary) repeated dose toxicity study (90-day) with Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol is available and was performed according to OECD 408 as a limit test (Jonker and Lina, 2001) and in compliance with GLP. In this study, the test item was administered in the feed at a level of 10% (100 000 ppm, dry matter) to groups of 10 rats/sex for 13 consecutive weeks. This level was equivalent to an overall test item intake of approximately 7000 and 8400 mg/kg bw/ day in males and females, respectively. A concurrent control group of 10 rats/sex was kept on basal diet supplemented with 10% pregelatinized wheat starch.

Observations and examinations of the animals included clinical signs, body weight, food/water consumption, haematology, clinical chemistry, urinalysis (including a urine concentration test), FOB, gross necropsy, organ weights and histopathology. The administration of the test item was tolerated without any adverse effects. One animal of the test item group was killed in extremis on day 37. The moribund condition of this rat was considered to be an incidental finding and not test item-related. Food consumption, food conversion efficiency and body weights did not reveal any test item-related changes. In the initial phase of the study, water intake was similar amongst the groups. At the end of the study water intake tended to be increased in the test item group in both sexes. However, the observed increases in water intake were not associated with histopathological alterations or any other relevant findings. They were therefore considered not adverse. Haematology and clinical biochemstry values showed no treatment-related changes. The urine concentration test did not indicate impaired renal concentrating ability. Dipstick measurements and microscopy of the urinary sediment did not reveal any treatment-related changes. Compared to starch controls, organ weights in the test item group showed the following changes: increased absolute and relative weights of the filled and empty caecum in both sexes, slightly increased relative liver weights in both sexes and increased relative kidney weights in females. The caecal enlargement was ascribed to incomplete absorption of the test item 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. The other observed variations in organ weights were not accompanied by histopathological alterations or any other relevant findings. They were therefore considered not adverse. Macroscopic examination of all rats at necropsy, and microscopic examination of a large number of organs and tissues did not reveal any treatment-related changes. Furthermore, there were no changes indicative of neurotoxic effects of the test item as assessed on the basis of the results of a FOB and various routine examinations in this study. The results of the routine tests evaluated as an immunotoxicity screen showed no potential for the test item to adversely affect the immune system.

It was concluded that a dietary level of 10% the test item was tolerated without obvious signs of toxicity. The NOAEL was therefore considered to be approximately 7000 and 8400 mg/kg bw/day in males and females, respectively.

 

A reliable oral (dietary) repeated dose toxicity study (90-day) in dogs with Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol is available and was performed equivalent or similar to OECD 409 (Hoffmann, 1978) before the introduction of GLP. In this study the test item was administered via the feed to 4 male and 4 female Beagle dogs/group at concentrations of 50 000, 100 000 and 200 000 ppm. In addition, the study included a reference group fed sucrose at 200 000 ppm. A concurrent control group of 4 dogs/sex was kept on basal diet. The resulting test item doses were equivalent to an overall intake of approximately 1600/1500, 3200/3000 and 6400/6000 mg/kg bw/day in males/females, respectively. The overall sucrose intake was 6200 (males) and 6000 (females) mg/kg bw/day.

Observations and examinations of the animals included clinical signs, body weight, food consumption, haematology, clinical chemistry, ophthalmoscopy, urinalysis, gross necropsy, organ weights and histopathology. Apart from moderately laxative effects at 200 000 ppm test item and to a lesser extent at 100 000 ppm the administration of the test substances was tolerated without any adverse effects at any test item group. The laxative effect observed after administration of 100 000 and 200 000 ppm 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.

It was concluded that a dietary level of up to 200 00 ppm Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol was tolerated without obvious signs of toxicity. The NOAEL was considered to be 6400 and 6000 mg/kg bw/day in males and females, respectively.

 

There are further studies available supporting the observed effects from the 90-day studies in rats and dogs. An additional 90-day rat study resulted in a NOAEL of 7290 and 9160 mg/kg bw/day for males and females, respectively. The NOAELs of one-year feeding studies in rats (Sinkeldam, 1981) and dogs (Hoffmann, 1981) were set at 3220 and 4320mg/kg bw/day in male and female animals, respectively and 2970 mg/kg bw/day for both genders.

Justification for classification or non-classification

Taken together, the available data on oral subchronic repeated dose toxicity of Reaction mass of Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol do not indicate adverse effects up to and including the highest doses tested which are well above the recommended limit values. Therefore, the available data did not identify any hazard for repeated dose toxicity, and are therefore conclusive but not sufficient for classification. Accordingly, Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol does not meet the classification criteria according to Regulation (EC) No 1272/2008.