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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Oct 2000 to 1 Feb 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other:

Remarks:

guideline study but no final report available (draft report available)

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Remarks:

reference to concurrent repeated dose toxicity study

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted in 1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted in 2016

Deviations:

yes

Remarks:

1000 instead of 4000 erythrocytes counted

Test guideline 3

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted in 1992

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

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

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

EC Number:

908-700-6

Cas Number:

64519-82-0

Molecular formula:

6-O-alpha-D-Glucopyranosyl-D-sorbitol: C12H24O11 1-O-alpha-D-Glucopyranosyl-D-mannitol: C12H24O11

IUPAC Name:

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

Test material form:

liquid

Test animals

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

Administration / exposure

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on 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

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily, 7 days/week

Post exposure period:

not applicable

Doses / concentrations

Dose / conc.:

10 other: %

Remarks:

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

No. of animals per sex per dose:

5 males and 5 females

Control animals:

yes

Positive control(s):

Mitomycin C (administered once in week 14, 24 h before collection of bone marrow cells)
- Route of administration: intraperitonreally
- Dose: 1.5 mg/kg bw
- Other: positive control rats were kept on purified control diet from the start of the 13-week treatment period

Examinations

Tissues and cell types examined:

femural bone marrow cells, numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 1000 erythrocytes/animal

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: The dietary level of 10% was selected as a high level which was previously found to be tolerable without obvious signs of toxicity.

TREATMENT AND SAMPLING TIMES:
test item animals and control animals: week 14, day 92/93/94
positive control animals: week 14, day 91

DETAILS OF SLIDE PREPARATION: At necropsy, bone marrow cells from one of the femurs was immediately collected into foetal calf serum and processed into glass drawn smear according to the method described by Schmidt (1976). Two bone marrow smears per animal were made and air­ dried, fixed in methanol and stained with May-Grünwald Giemsa.

METHOD OF ANALYSIS: Slides (one slide per animal) were read by moving from the beginning of the smear (label end) to the leading edge in horizontal lines taking care that areas selected for evaluation were evenly distributed over the whole smear.
The numbers of polychromatic and normochromatic erythrocytes (PE and NE, respectively) were recorded in a total of 1000 erythrocytes (E) per animal; if micronuclei were observed, these were recorded as micronucleated polychromatic erythrocytes (MPE) or micronucleated normochromatic erythrocytes (MNE). Once a total number of 1000 E (PE + NE) had been scored, an additional number of PE was scored for the presence of micronuclei until a total number of 2000 PE had been scored. Thus the incidence of MPE was recorded in a total of 2000 PE/animal and the number of MNE was recorded in the number of NE.

Evaluation criteria:

The study is considered valid if the positive controls give a statistically significant increase in the mean number of MPE/2000 PE and if the negative controls are within the historical range.

A response is considered positive if the mean number of MPE/2000 PE is statistically significant higher compared to the vehicle control.

A test substance is considered to cause chromosomal damage and/or damage to the mitotic apparatus if a clear dose-related increase in the mean number of MPE/2000 PE compared to the vehicle control is observed, and/or if a single positive dose is observed.

A test substance is considered negative in the micronucleus test if it produces no positive response at any dose level or time point.

The test substance or its metabolites are considered to have reached the general circulation and thereby the bone marrow, if the test substance reduces the mean number of PE/E, or causes systemic toxicity.

Both statistical significance and biological relevance will be considered in the evaluation.

Statistics:

Data on MPE and PE was subjected to a one way ANOVA with factor group (controls and test item groups) for each sex. If the ANOVA yielded a significant effect (p < 0.05), the ANOVA was followed by pooled error variance t-tests or if variances were inhomogenous, separate variance t-tests. These t-tests were applied to the negative control versus tets item treatments per sex. In addition, the positive control group and the negative control group for each sex, were compared using pooled error variance t-tests or if variances were inhomogenous, separate variance t-tests. All statistical tests were performed using BMDP statistical software (W.J. Dixon, BMDP Statistical Software Manual, University of California Press, Berkeley, 1992).

Results and discussion

Test results

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The incidences of MPE per 2000 PE of the positive control group differed significantly from the vehicle control group (p < 0.001) in both sexes, demonstrating the sensitivity of the system.

The incidences of PE per 1000 E of the positive control females differed significantly from the vehicle control group (p < 0.01) demonstrating the sensitivity of the system.

Any other information on results incl. tables

Table 1. Micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE)

	Group mean ± Standard Dev. numbers of micronucleated polychromatic erythrocytes (MPE) per 2000 polychromatic erythrocytes (PE)
	starch supplemented control diet	Test item 10%	Mitomycin C (1.5 mg/kg bw)
males	3.0 ± 1.7	1.4 ± 0.5	27.6 ± 4.0***
females	3.0 ± 1.2	3.2 ± 0.8	29.0 ± 6.6***

***p<0.001: two-sided asymptotic t-test, treated versus control

Table 2. Polychromatic erythrocytes (PE) per 1000 erythrocytes (PE)

	Group mean ± Standard Dev. numbers of polychromatic erythrocytes (PE) per 1000 erythrocytes (E)
	starch supplemented control diet	Test item 10%	Mitomycin C (1.5 mg/kg bw)
males	435.0 ± 136.7	387.0 ± 46.8	373.0 ± 26.0
females	465.2 ± 41.6	450.6 ± 39.0	284.8 ± 71.9**

**p<0.01: two-sided asymptotic t-test, treated versus control

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative