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

Carcinogenicity

Description of key information

Carcinogenicity Study (equivalent or similar to OECD 451), rat:

NOAEL (carcinogenicity) = 100 000 ppm (equivalent to 3650 and 4980 mg/kg bw/day in males and females, respectively)

Carcinogenicity Study (equivalent or similar to OECD 451), mouse

NOAEL (carcinogenicity) = 100 000 ppm (equivalent to 29950 and 18690 mg/kg bw/day in males and females, respectively)

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Jun 1980 - 21 Dec 1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

study was initiated before guideline was adopted

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
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 3 weeks
- Weight at study initiation: mean group weights ranged from 65 to 66 g in males and 59 to 62 g in females
- Fasting period before study: no
- Housing: group housing, 5/sex/cage, in suspended stainless stell cages, fitted with wire-mesh floors and fronts
- Diet: laboratory's stock diet for rats, modified by adding 10 % maize starch; this modification allowed the incorporation of test item and sucrose at the expense of starch, ad libitum
- Water: tap water, ad libitum
- Acclimation period: Not applicable as study animals were born at the facility. The rats were derived from parents which had been kept on the same diets already before mating and during the gestation and lactation period (in utero exposure, Sinkeldam, 1982). Weanling rats obtained from the second mating cycle (F 1b - rats) were used for this study.

DETAILS OF FOOD AND WATER QUALITY: Laboratory stock diet was analyzed regularly for nutrients and contaminants. Tap water was analyzed periodically for contaminants. Results of analyses made in the test period are presented in the report and gave no indication for a concern.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 1
- Humidity (%): 50 ± 10
- Air changes (per hr): approximatly 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 Jun 1980 To: 27 Dec 1982

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: weekly
- Mixing appropriate amounts with: laboratory's stock diet for rats, modified by adding 10 % maize starch; this modification allowed the incorporation of test item and sucrose at the expense of starch; the materials were thoroughly mixed into the carrier by means of a mechanical blender (Lödige)
- Storage temperature of food: not applicable as food was freshly prepared and filled into food hoppers

TEST ITEM INTAKE
- Test item intake was not specified in the report.
- Test item intake was calculated retrospectively based on mean body weights, food consumption and nominal dietary test item concentration at the end the study period. The dietary concentration of 2.5% resulted in test item intakes of approximately 960 and 1220 mg/kg bw/day in males and females, respectively. Dietary concentrations of 5% resulted in test item intakes of approximately 1630 and 2450 mg/kg bw/day in males and females, respectively. The dietary concentration of 10% resulted in test item intakes of approximately 3650 and 4980 mg/kg bw/day in males and females, respectively (Tables 2 and 3).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

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, 348, 504, 523, 614, 719, 797 and 881 the content of test item was determined by High Performance Liquid Chromatography (HPLC).

The stability and homogeneous distribution of test item in the diets were determined in the course of the multigeneration study (Sinkledam 1982, section 7.8.1) by analyzing five samples taken at five different places in the food container from one batch of each of the diets produced on December 6, 1979 after one day and after 14 days of storage at room temperature.

The results of the determinations show that the actual dietary levels of test item were close to the intended levels, indicating proper preparation of the diets (Table 1). 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 tan 5%. This is indicative of satisfactory homogeneous distribution of the test substance in the diets. The figures obtained after 14 days of storage at room temperature show that the test substance is stable under the conditions of storage.

Duration of treatment / exposure:

128 and 130 weeks for males and females, respectively

Frequency of treatment:

daily, 7 days/week

Post exposure period:

not applicable

Dose / conc.:

2.5 other: %

Remarks:

25 000 ppm (approx. 960 and 1220 mg/kg bw/day, for males and females, repectively, calculated from bw and food consumption data in the last study week)

Dose / conc.:

5 other: %

Remarks:

50 000 ppm (approx. 1630 and 2450 mg/kg bw/day, for males and females, repectively, calculated from bw and food consumption data in the last study week)

Dose / conc.:

10 other: %

Remarks:

100 000 ppm (approx. 3650 and 4980 mg/kg bw/day, for males and females, repectively, calculated from bw and food consumption data in the last study week)

No. of animals per sex per dose:

50 males and 50 females

Control animals:

yes

other:

Details on study design:

- Toxicokinetic data : not applicable
- 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: Yes
- Time schedule: every 2 weeks

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

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each cage 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: No

FOOD EFFICIENCY:
- Body weight gain in g/food consumption in g per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: daily during the first 4 weeks and daily during weeks 13, 25, 39, 51, 66, 77, 91, 103, 117 and 128 (females only)

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 26, 52, 78, 104 and 127; blood samples were collected from the tip of the tail of ten rats/sex/group
- Anaesthetic used for blood collection: No
- Animals fasted: No data
- How many animals: 10/sex/group
- Parameters examined: haemoglobin, packed cell volume, red blood cell count, thrombocyte count, white blood cell count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), differential white blood cell count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 26/27, 52/53, 78/79, 104/105, 127/128 (males) or 129 (females)
- Animals fasted: Yes (for determination of blood glucose only)
- How many animals:10/sex/group
- Parameters examined: glucose, total protein, protein electrophoresis, alkaline phosphatase (ALP), urea, albumin, glutamic-pyruvic transaminase (GPT), glutamic-oxalacetic transaminase (GOT), total bilirubin, cholesterol, creatinine, calcium (Ca), potassium (K), sodium (Na), urate
- other: determinations of vitamin B6 (pyridoxal phosphate; PLP) and oxalate were performed in samples of blood, treated with EDTA, and collected by orbits puncture from 5 animals/sex/group in week 108 of the study

URINALYSIS: Yes
- Time schedule for collection of urine: weeks 26, 52 (males) and 53 (females), 78, 104, 127 (males) and 129/131 (females)
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- How many animals: 10/sex/group
- Parameters examined: urine volume, densitiy, appearance, pH, protein, glucose, blood, ketones, urobilnogen, bilirubin, sediment (erythrocytes, leucocytes, epithelial cells, amorph materail, crystals, casts, bacteria, sperm cells, worm eggs)

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of the study (week 128 for males and week 130 for females) all surviving animals were killed by exsanguination from the abdominal aorta under ether anaesthesia and then examined grossly for pathological changes. The male animals were killed 2 weeks before the scheduled killing in week 130, because mortality in the sucrose control group had reached 80 %.

Animals that died spontaneously or were killed because they were moribund were also examined grossly. Organs of these rats were not weighed, but tissues were removed and preserved if autolysis was not too advanced.

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

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

Organs/tissues marked with an asterik were preserved without further examination.

The microscopic examination comprised:
a. Complete and detailed examination of all tissues not marked with an asterisk of all animals of the control groups and high dose group, and of liver and kidneys of all animals of the low and mid dose group
b. Examination of the spleen, thyroid, brain, testicles, adrenals, ovaries, uterus and pituitary of all animals of all groups for the presence of pre-neoplastic or neoplastic changes.
c. Histopathology of all grossly visible tumours or gross lesions suspected of being tumours of all animals.

Statistics:

The following statistical tests were applied:
Mortality: Fisher's exact probability test
Body weights: Analyses of variance (ANOVA) + Dunnett Multiple Comparison test
Food intake, food efficiency, water intake, organ weights: ANOVA + Dunnnett
Haematology, urine density and volume, blood chemistry: Mann/Whitney U-test (with and without a correction factor 4)
Histopathology (incl. tumor data): Fisher's exact probability test
Tumour data: Statistical test according to Peto et al. (1980)

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

After approximately 18 months, ageing symptoms developed in all groups. These symptoms included dyspnoea, emaciation, bloody discharge around nostrils and eyes, yellow and rough fur, focal alopecia, humpback position, malocclusion of incisors, pale eyes, blindness of one or both eyes, white and opaque cornea, cyanosis, weakness of muscles and focal dermatitis. These phenomena were either as frequent in controls as in test rats or their incidence was not clearly dose-related.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Mortality was similar amongst the control groups and the low dose group. Cumulative mortality data are presented in Table 4. Reduced mortality was noticeable in the mid dose group during the last half year of the study. The phenomenon occurred both in males and females and was found to be statistically significant at a number of stages. Mortality figures in high dose group were also lower than in controls during the last half year of the study in both sexes, but the differences with the controls never reached the level of statistical significance.
In males mortality reached 80% in sucrose control group after a feeding period of 127 weeks. That is the reason why for males the study was terminated in week 128, 2 weeks before the scheduled killing in week 130. The overall mortality at the end of the study amounted to 68% for males and 63% for females.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weights were slightly decreased in high dose males. The decreases occurred already at day 42, but did not become more marked in the course of the study. Only from day 42 to 181 they were statistically significant. The differences against the controls were generally not greater than 5%. The growth rate of the sucrose control group was very similar to that of the controls. The loss of weight, which was observed in each of the groups after 2 years (as a result of ageing) was most marked in the low dose group, resulting in some statistically significantly decreased values in this group.

Body weights of females did not show any obvious differences between the test item and both control groups during the major part of the study. However, by the end of the second year some statistically significant decreases in body weight occurred in mid and high dose groups. The greatest differences amounted to no more than 10% and were therefore not considered adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There were no outstanding differences in food intake among the different groups, either in males nor in females. High dose males showed a rendency to eat slightly more than controls. The differences were small and amounted to no more than 5% (during weeks 78 -104).
Achieved test item intake is presented in Tables 2 and 3.

Food efficiency:

no effects observed

Description (incidence and severity):

Food efficiency, calculated over the first four weeks, did not show any obvious differences amongst the various groups, in either sex. High dose males consistently showed slightly lower figures than did the controls, but the differences were not statistically significant.

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

no effects observed

Description (incidence and severity):

Water intake was generally comparable in the various groups. A tendency towards a slightly higher water consumption was noticeable in the test item groups, especially in females. However, a clear dose-response relationship was not apparent. The water intake of the sucrose-fed animals was generally comparable to that of the controls. The highest figures for water intake were observed in all groups at the end of the study (week 117 and 128), probably as a result of ageing.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Several of the clinical chemistry blood values showed statistically significant increases or decreases in one or more test item groups as compared to the controls. In the high dose group such differences occurred for instance in blood urea nitrogen levels of males, cholesterol levels of both sexes, urate levels of males, total bilirubin content of females, GPT activity of males, and percentage β1-globulin of females. None of these differences, however, occurred at all stages during the study and for none of the parameters the differences with the controls tended to increase with increasing duration of the experimental period. The group fed sucrose showed differences similar to those in the high dose group in urea nitrogen, cholesterol and α-globulins. Based on these findings, no toxicological significance is attached to the differences between the high dose and the controls with respect to above parameters.

A few other parameters showed statistically significant differences between the controls in the mid and low dose group. Because of their absence in the high dose group these findings are considered fortuitous. A specific effect of sucrose on any of the parameters was not apparent.

The levels of pyridoxal phosphate showed large variation amongst the groups but did not suggest an effect of the test item or sucrose. Oxalate levels of the among groups were similar.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males fed test item or sucrose excreted greater volumes of more diluted urine than did the controls after half a year of feeding. A clear dose-relationship was, however, not observed. Moreover, the phenomenon did not occur at the other stages of the study, except for the group fed sucrose which showed the phenomenon also in study week 104.
High dose females showed a tendency to excrete a greater volume of more diluted urine at three out of five stages. No consistent changes in volume and density of the urine were observed in females fed sucrose.

The analyses of pooled urine samples and microscopic observations of the sediment showed no changes, which could be attributed to treatment.

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 relative weights of the caecum, both with the contents and after removal of the contents were clearly increased in both sexes of the high dose group.
Caecal enlargement is often observed in rats upon feeding carbohydrates or carbohydrate-like substances known to be slowly digested or poorly absorbed in the small intestine. Among such products are raw potato starch, chemically modified starches, pectins, alginates, xylitol and sorbitol. Unlike well digestible sugars and starches, substantial amounts of the above mentioned products may reach the large intestine where they induce an increased microbial fermentation. This gives rise to an increased production of volatile fatty acids, lowering of the pH, increased water retention in the intestinal contents and the faeces, increased excretion of faecal dry matter and distention and increased weight of the large intestine, particularly the caecum. The caecal enlargement is considered to be an adaptive rather than a pathological change. The test item has been reported to be incompletely digested both in the rat and man. Therefore, the caecal enlargement found in the present study is most likely due to the presence of non-digested test item in the large bowel, which escaped hydrolysis in the small intestine and is fermented in the large intestine by the gut flora.

An increased relative liver weight occurred in males of the low and high dose group. Because the difference to the controls showed no dose-related response and the liver weight was not increased in the mid dose group the increases observed were considered incidental findings.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At autopsy, no obvious treatment-related gross changes were observed. Caecal enlargement occurred to a slightly higher incidence in high dose males than in controls. Frequently observed and randomly distributed gross abnormalities included mammary gland nodules of various size, polyps in the uterus, tumour-like lesions in the pituitaries splenomegaly, discoloured and/or spotted adrenals, enlarged or discoloured kidneys with a granular appearance, unilateral testicular atrophy, livers with a pronounced lobular pattern, granular and/or pale extra orbital lachrymal glands (in males only), ovarian cysts, discoloured and/or spotted lungs, hydrothorax and enlarged hearts or hearts containing thrombi. Other gross lesions were seen in a small number of animals only. There was no indication that any of the grossly visible lesions was related to the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The kidneys of test item-fed females showed a dose-related decrease of calcareous deposits (mineralisation) in the intercortico-medullary layer, which was statistically significant in the high dose group. The number of animals with urothelial mineralisation localised in the renal pelvis (pelvic nephrocalcinosis) was significantly higher in high dose males and in mid and high dose females than in controls. In test item-fed females this phenomenon showed a distinct dose-response relationship. The difference was statistically significant both in high dose males and in mid and high dose females.

A dose-related increase in the incidence of hyperplasia of the local lining epithelium in the renal pelvis was observed in mid and high dose females. The difference to the control group was statistically significant in the high dose group. In males the incidence of this lesion was slightly, though not statistically significantly, higher in the high dose group than in the control group, but it was comparable with the sucrose control group.

A statistically significant decrease in the incidence of cortical hypertrophic cell foci in the adrenals in low and mid dose females was considered of no toxicological significance because the incidence of this phenomenon in sucrose-fed females was comparably low. Some other statistically significant differences in the liver (bile duct proliferation and focal hepatocellular vacuolation) were observed in the sucrose control group only.
No other significant differences in type or incidence of non-neoplastic changes, including hyperplastic changes, were observed between the groups.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The most common neoplastic lesions were phaeochromocytomas in the adrenals, fibroadenomas in the mammary glands, tumours in the pituitary, light cell solid adenomas in the thyroid and fibromatous polyps in the uterus.
The incidence of granulosa-theca cell tumours in the ovaries of low dose females (5/45; 11%) was statistically significantly higher than in controls (0/49: 0%). The incidence of this type of tumour was higher than the upper limit of the range of incidences found in control animals of seven historical studies (average 8/452, 1.7%; range 0/57, 0% to 2/50, 4%). However, there was no dose-response relationship (incidence at in mid and high dose: 1/47 and 1/49 animal each) and therefore the relatively high incidence of this tumour type in the low dose group is considered to be a fortuitous finding.

In the thyroids the incidence of small light-cell solid adenomas was statistically significantly increased in low and mid dose males, without showing a dose-response relationship. The total number of light-cell solid adenomas was comparable in all groups. Therefore, no toxicological significance is attached to this finding.

The tumours mentioned above and the other tumours noted are also common neoplasms in the strain of rats used. Although slight differences in the incidences occurred between the various groups, there was no indication that these differences were related to the feeding of the test item. A statistical analysis did not reveal any indication of a positive trend in tumour incidence.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

ca. 3 650 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no indication of increase tumour incidence up to and including the highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

ca. 4 980 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no indication of increase tumour incidence up to and including the highest dose tested

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

ca. 1 220 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

ca. 1 630 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Table 1. Analytical test item concentrations in the diet

	Nominal test item concentration (%)
Sampling date (study day)	0	2.5	5.0	10.0
	Analytical test item concentration (%)
-20	ND	3.0	5.7	10.2
69	ND	2.4	6.0	10.3
159	ND	1. 9	4.6	8.6
238*	ND	2.5	S.3	11.8
273	ND	1.8	4.0	8.4
348	ND	2.8	3.0	8.7
504	ND	2.4	4.5	8.8
523	ND	2.5	4.9	9.3
614	ND	2.8	5.0	9.9
719	ND	2.3	5.5	9.5
797	ND	2.4	5.2	9.8
881	ND	1.7	5.1	10.5
Mean	ND	2.4	4.9	9.7

ND = not detectable

* Samples taken from feeders in cages after the diets had been available to rats for one week

Table 2. Test item intake males

	Nominal concentration of test item in diet (%)
	0	2.5	5	10
Body weights (g, day 894)	506.5	472.2	489.1	485.2
Food consumption (g food/animal/week)	114.0	126.4	111.8	124.1
Food consumption (g food/animal/day)	16.3	18.1	16.0	17.7
Food consumption (g food/kg bw/day)	32.2	38.2	32.7	36.5
Test item intake (mg/kg bw/day)	0	956	1633	3654

Table 3. Test item intake females

	Nominal concentration of test item in diet (%)
	0	2.5	5	10
Body weights (g, day 914)	330.2	330.5	323.5	306.1
Food consumption (g food/animal/week)	118.8	112.5	111.1	106.6
Food consumption (g food/animal/day)	17.0	16.1	15.9	15.2
Food consumption (g food/kg bw/day)	51.4	48.6	49.1	49.8
Test item intake (mg/kg bw/day)	0	1216	2453	4975

Table 4. Cumulative mortality data (data taken from Smits-van Prooije publication)

	Cumulative mortality (no. of deaths)#
	Males				Females
Dietary concentrations (%)	0	2.5	5.0	10	0	2.5	5.0	10
Study week
12	0	0	0	0	0	0	0	0
24	0	0	1	0	0	1	0	0
36	0	0	1	0	0	1	0	0
48	0	0	1	0	0	2	0	0
60	0	1	1	1	1	2	0	0
72	1	1	3	1	3	2	1	l
84	2	3	4	3	4	9	4	4
96	II	7	8	8	8	10	6	7
108	21	18	14	12	17	16	11	15
120	31	32	20*	26	29	30	18*	20
126/130##	36	39	25*	31	36	35	24*	29

# initial group size: 50 animals/sex

## for males and females respectively

* p < 0.05, Fisher’s exact probability test

Table 5. Histopathological kidney findings (data taken from Smits-van Prooije publication)

	Incidence of lesions (no. of animals affected)
	Males				Females
Dietary concentrations (%)	0	2.5	5.0	10	0	2.5	5.0	10
No. of animals examined	50	47	49	47	48	47	48	49
Site and type of findings
Urothelial hyperplasia in the cortex:    slight	12	8	10	17	25	16	27	25
moderate	4	1	6	3	6	3	2	4
severe	1	0	0	5	0	0	0	2
total	17	9	16	25	31	19	29	31
Urothelial hyperplasia in the pelvis:    slight	8	12	10	16	11	12	19	21
moderate	4	3	5	8	7	1	8	10
severe	3	4	2	0	0	0	0	0
total	15	19	17	24	18	12	27	31*
Urothelial mineralization:                      very slight	5	3	6	11	5	15*	13	12
slight	7	2	5	13	9	13	19	23*
moderate	0	0	1	6	7	0	1	5
severe	0	0	0	1	0	0	0	0
total	12	5	12	31**	21	28	33*	40**
Corticomedullary mineralization:          very slight	1	0	0	1	8	7	14	3
slight	1	2	1	2	19	9	9	14
moderate	2	0	0	2	6	12	3	4
severe	4	2	1	5	33	28	26	21*
total	3	2	2	1	1	2	3	1
Cortical mineralization	8	8	4	12	14	10	12	18
Medullary mineralization	12	8	10	17	25	16	27	25

* p < 0.05, ** p < 0.01 Fisher’s exact probability test

Reference 2

Endpoint:

carcinogenicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

26 Jun 1980 - 21 Dec 1983

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 451 (Carcinogenicity Studies)

Version / remarks:

study was initiated before guideline was adopted

GLP compliance:

yes

Species:

mouse

Strain:

other:

Remarks:

Cpb: Swiss 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
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 3 weeks (weanlings)
- Weight at study initiation: 13.9 to 24.9 g (males), 12.1 to 20.2 g (females)
- Fasting period before study: no
- Housing: males: individual housing, females group housing (5/sex/cage); in macrolon boxes with wire screen covers
- Diet: laboratory's stock diet for rats, modified by adding 10 % maize starch; this modification allowed the incorporation of test item and sucrose at the expense of starch, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD QUALITY: Laboratory stock diet was analyzed regularly for nutrients and contaminants. Tap water was analysed periodically for contaminants. Results of analyses made in the test period are presented in the report and gave no indication for a concern.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 25
- Humidity (%): 30 - 75
- Air changes (per hr): 8 - 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: Jul 1980 To: Jul 1982

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on exposure:

DIET PREPARATION
- Rate of preparation of diet: every two months
- Mixing appropriate amounts with: laboratory's stock diet for rats, modified by adding 10 % maize starch; this modification allowed the incorporation of test item and sucrose at the expense of starch; the materials were thoroughly mixed into the carrier by means of a mechanical blender (Lödige); the mash was pelleted after adding 5% 1:1 water/molasses mixture. The diameter of the pellets was 10 mm.
- Storage temperature of food: at -20 °C until use

TEST ITEM INTAKE
- Test item intake was not specified in the report.
- Test item intake was calculated retrospectively based on mean body weights, food consumption and nominal dietary test item concentration at the end the study period. The dietary concentration of 2.5% resulted in test item intakes of approximately 6380 and 4050 mg/kg bw/day in males and females, respectively. Dietary concentrations of 5% resulted in test item intakes of approximately 14250 and 8890 mg/kg bw/day in males and females, respectively. The dietary concentration of 10% resulted in test item intakes of approximately 26950 and 18690 mg/kg bw/day in males and females, respectively (Tables 2 and 3).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

From each freshly prepared batch, a sample was taken and stored in a freezer at -20 °C. In a number of these samples (i.e., those prepared on 11 Jun 1980, 9 Oct 1980, 11 Dec 1980, 11 May 1981, 5 Nov 1981 and 20 Jan 1982) the content of test item was determined by High Performance Liquid Chromatography (HPLC).

From a batch of all test diets prepared on 11 Dec 1980, five samples taken at different places in the food container were analysed for the content and homogeneity of the test and control substance in the diets. Thereafter, the five samples of each diet were mixed, stored for two weeks at room temperature and analysed again.

The results of the analyses show that the actual levels of test item in the various diets were generally close to the intended levels (Table 1). Moreover, it appeared that in the diets stored for several months at -20 °C or for 2 weeks at about 22°C, no loss of test item occurred. In addition to the sucrose diet, all other diets contained 1-2% sucrose, mainly originating from the molasses and from the yellow maize.

Duration of treatment / exposure:

94 and 104 weeks for males and females, respectively

Frequency of treatment:

daily, 7 days/week

Post exposure period:

not applicable

Dose / conc.:

2.5 other: %

Remarks:

25 000 ppm (approx. 6380 and 4050 mg/kg bw/day, for males and females, repectively, calculated from bw and food consumption data in the last study week)

Dose / conc.:

5 other: %

Remarks:

50 000 ppm (approx. 14250 and 8890 mg/kg bw/day, for males and females, repectively, calculated from bw and food consumption data in the last study week)

Dose / conc.:

10 other: %

Remarks:

100 000 ppm (approx. 26950 and 18690 mg/kg bw/day, for males and females, repectively, calculated from bw and food consumption data in the last study week)

No. of animals per sex per dose:

50 males and 50 females

Control animals:

yes

other:

Details on study design:

- Toxicokinetic data : not applicable
- 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: Yes
- Time schedule: every 2 weeks

BODY WEIGHT: Yes
- Time schedule for examinations: before start of treatment, in week 1, 2, 3, 4, 6, 8, 10 and 12 and once every four weeks thereafter

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each cage 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: No

FOOD EFFICIENCY:
- Body weight gain in g/food consumption in g per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 13, 26, 54, 78 and 104; blood samples were collected by orbita punction
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: 10/sex/group
- Parameters examined: red blood cell count, white blood cell count, haemoglobin, packed cell volume, differential white blood cell count, thrombocyte count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC)

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of the study (week 94 for males and week 104 for females) all surviving animals were killed by decapitation under ether anaesthesia and then examined grossly for pathological changes.

Animals that died spontaneously or were killed because they were moribund were also examined grossly. Organs of these rats were not weighed, but tissues were removed and preserved, as long as autolysis was not too advanced.

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

HISTOPATHOLOGY: Yes
Tissue samples of the following organs of all animals were preserved in a neutral, aqueous, phosphate-buffered, 4 % formaldehyde solution:
adrenals, aorta, bone and bone marrow, brain, caecum, colon, duodenum, eyes, heart, ileum, jejunum, kidneys, liver and gall bladder, lungs, lymph nodes (cervical and mesenteric), mammary gland, oesophagus, ovaries, pancreas, pituitary, prostate, salivary glands (parotid, sublingual and submaxillary), sciatic nerve, seminal vesicles, skeletal muscle, skin, spleen, stomach (glandular and non-glandular), testes, thyroid, trachea, urinary bladder, uterus, all gross lesions; all nodules, tissue masses and otherwise macroscopically abnormal tissues were preserved, along with samples of adjacent tissue where appropriate

All tissues to be examined microscopically were embedded in paraffin wax, sectioned at 5 µm and stained with haematoxylin and eosin.
Detailed microscopic examination was performed on the slides belonging to all animals of the control, high dose and sucrose control group. From all animals of the mid and low dose group the liver, spleen, brain, pitu itary, thyroid, lung and adrenals were examined microscopically.

All grossly observed tumours and lesions suspected of being tumours were examined in all groups.

Statistics:

The following statistical tests were applied:
Mortality: Fisher's exact probability test
Body weights: Analyses of variance (ANOVA) with the initial body weights as co-variables folloowed by Dunnett's Multiple Comparison test
Food intake: ANOVA and L.S.D. (Least Statistical Difference) test
Haematology: Mann/Whitney U-test followed by Dunnett's Multiple Comparison test
Histopathology (incl. tumor data): Fisher's exact probability test

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

There were no noticeable differences in appearance, behaviour or external abnormalities between the mice of the various test groups and those of the controls, that could be related to the feeding of the test item. In males, signs of inflammation of the preputial glands were noticed from day 76 onwards and occurred randomly distributed among the various groups, the controls included. The lesion was not considered to have caused the death of animals.
From about day 200 onwards an increasing number of male mice showed a swollen belly with firm palpable masses, which at autopsy were found to be related to a severe inflammation of the seminal vesicles. The enlarged seminal vesicles most probably caused obstruction of the urethra and, subsequent, dilatation or even rupture of the urinary bladder. This urogenital lesion caused the death of many male mice. This lesion was about equally distributed among test and control groups. There was no indication that the lesion was related to the test item.

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, non-treatment-related

Description (incidence):

From the cumulative mortality figures (Table 4) it appears that the test item did not increase death rate in either sex. Mortality was relatively high in males of all groups from day 280 onwards, but after 18 months (day 532) mortality in males of all groups, apart from the sucrose controls, was still below 50%. On day 657 mortality in males reached 84% in the control group and 82% in the sucrose controls. At this stage all male survivors were sacrificed (day 657 and 658). The death rate in females was quite common for the strain of mice used. All female survivors were sacrificed on day 728 and 729.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The females of the mid and high dose groups gained slightly less weight than did the controls from day 112 onwards, resulting in lower body weights during the remaining part of the experimental period. In the low dose group, growth rate was similar to that of the controls. Mean body weights in males were similar in all groups throughout the study.
The slight growth depression of the mid and high dose females, which was not associated with decreased food consumption, suggests a decreased utilization of these diets and is considered potentially adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption figures showed no consistent differences among the various groups.
Achieved test item intake is presented in Tables 2 and 3.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There was a statistically significant decrease in the number of white blood cells in low and high dose males on days 183 and 547. Since this phenomenon was not dose-related and did not occur on days 85 and 379 of the study it is probably an incidental finding. No marked or consistent changes occurred in any other of the parameters examined, either in males or in females.

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 absolute and relative weights of the filled and empty caecum were increased in high dose mice of both sexes. The differences versus the controls were statistically significant, except for the absolute empty caecum weight in males.
The weight of the other organs, either absolute or relative, did not suggest any treatment-related changes among the various groups.

The increase in the weight of the caecum was not accompanied by symptoms of diarrhoea. Treatment-related histopathological changes in the caecum have not been detected. These results confirm the earlier observations in a multigeneration study with rats (Sinkeldam, 1982, section 7.8.1-1). In that study caecal enlargement was found in F3b rats fed the test item at 10% at week 4 after weaning.

Enlargement of various parts of the intestinal tract is a well-known pbenomenon in rodents when their food contains poorly digestible substances, such as dietary fiber, raw potato starch, modified starches, lactose, pectins, alginates, etc. Unlike well digestable sugars and starches, substantial amounts of the above mentioned products reach the large intestine where they induce an increased microbial fermentation. This gives rise to an increased production of volatile fatty acids, lowering of the pH, increased excretion of faecal dry matter and distention and increased weight of the large intestine, particularly the caecum. The caecal enlargement is considered to be an adaptive rather than a pathological change. In the present study with mice, the enlargement is most likely due to the presence of non-digested test item in the large bowel, which passes the small intestines unhydrolysed and is fermented in the large intestines by the gut flora. The type of intestinal enlargement mentioned above is generally not considered to be of toxicological significance.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At autopsy, no obvious treatment-related gross lesions were observed. A high incidence of urogenital abnormalities, such as inflammation of preputial glands and/or seminal vesicles, accompanied by distended or even ruptured urinary bladders, was noticed in males of all groups. The inflamed preputial glands contained necropurulent material from which the bacterial strain Staphylococcus aureus could be isolated. Incubation of necropurulent material from the affected seminal vesicles was negative for bacteriae. Probably a viral or mycoplasm infection was responsible for this abnormality.
Similar urogenital lesions have been found in male animals belonging to other carcinogenicity studies performed in this laboratry with the same strain of mice.
Other frequently occurring non-neoplastic gross alterations were splenomegaly, ovarian cysts, hydrometra and pale discoloration of the adrenals, kidneys, liver and pituitary. All other non-neoplastic gross changes were seen in a few animals only.

In both sexes, there were no noticeable differences between the test groups and the controls in the incidence of lesions suspected of being tumour.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All non- and pre-neoplastic lesions observed were about equally distributed among the various groups, or they occurred in one or a few animals only.
At microscopy, many preputial glands, seminal vesicles and coagulating glands showed severe inflammatory reactions, which were in accordance with the observations at autopsy. A high incidence of adenitis of the preputial glands occurred in males of all groups. The affected glands showed cystic dilatation and infiltration of polymorphonuclear and/or mononuclear inflammatory cells. Sometimes abcesses were present. In the seminal vesicles and in the coagulating glands granulomatous vesiculitis and adenitis were observed. Occasionally, the heavily distended glands contained necropurulent exudate. Inflammatory cells were scattered throughout the stroma. Abcesses were regularly seen.
Dilatation of the urinary bladder, sometimes accompanied by submucosal haemorrhages was observed in a few male rats.
The incidence of the lesions in preputial glands, seminal vesicles and coagulating glands was very high in males of all groups, but there was no evidence that these lesions were related to treatment with the test item.
The other non-neoplastic and pre-neoplastic lesions noted are common findings in aged mice of the strain used. Therefore, these abnormalities are not ascribed to the ingestion of the test compound.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Neoplastic lesions were most frequently observed in the liver (neoplastic nodules and hepatocellular carcinomas), in the lung (alveologenic carcinomas), and in the haematopoeietic system (lymphomas). Although the incidence of these neoplastic lesions varied considerably in the different groups, there was no evidence that any of the neoplastic alterations was related to treatment with the test ietm.

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

ca. 26 950 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no indication of increase tumour incidence up to and including the highest concentration tested

Key result

Dose descriptor:

NOAEL

Remarks:

carcinogenicity

Effect level:

ca. 18 690 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no indication of increase tumour incidence up to and including the highest concentration tested

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

ca. 26 950 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects noted up to and including the highest concentration tested

Key result

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

ca. 4 050 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

Key result

Critical effects observed:

no

Table 1. Analytical test item concentrations in the diet

	Nominal test item concentration (%)
Sampling date	0	2.5	5.0	10.0
	Actual test item concentration (%)
20 JUN 1980	ND	3.3	5.4	9.9
09 OCT 1980	ND	3.4	5.5	9.5
11 DEC 1980*	ND	2.4 (0.08)	4.5 (0.01)	9.4 (0.14)
11 DEC 1980**	ND	2.4	4.6	9.3
11 MAY 1981	ND	2.4	4.8	9.6
05 NOC 1981	ND	2.3	5.0	10.2
20 JAN 1982	ND	2.7	4.3	9.7

ND = not detectable (detection limit 0.1%)

* mean + SEM (in brackets) of five samples taken at different places in the food container

** prepared 11 Dec 1980, stored at -20 °C until 04 Feb 1981 and at room tempertateure for two weeks

Table 2. Test item intake males

	Nominal concentration of test item in diet (%)
	0	2.5	5	10
Body weights (g, day 644)	46.4	48.8	45.8	45.8
Food consumption (g food/animal/week)	83.3	87.2	91.4	86.4
Food consumption (g food/animal/day)	11.9	12.5	13.1	12.3
Food consumption (g food/kg bw/day)	256.5	255.3	285.1	269.5
Test item intake (mg/kg bw/day)	0	6382	14255	26949

Table 3. Test item intake females

	Nominal concentration of test item in diet (%)
	0	2.5	5	10
Body weights (g, day 728)	45.2	50.3	42.5	42.2
Food consumption (g food/animal/week)	51.9	57	52.9	55.2
Food consumption (g food/animal/day)	7.4	8.1	7.6	7.9
Food consumption (g food/kg bw/day)	164.0	161.9	177.8	186.9
Test item intake (mg/kg bw/day)	0	4047	8891	18687

Table 4. Cumulative mortality data (data taken from Smits-van Prooije publication)

	Cumulative mortality (no. of deaths)#
	Males				Females
Dietary concentrations (%)	0	2.5	5.0	10	0	2.5	5.0	10
Study week
12	0	0	0	0	0	0	0	0
24	1	0	0	1	0	0	1	0
36	2	3	3	1	2	0	0	1
48	8	6	7	6	2	1	2	1
60	16	11	13	13	4	3	4	1
72	25	23	23	21	4	3	4	1
84	28	28	28	24	13	10	11	7
94/96##	28	28	28	24	24	16	20	21
104					30	19	25	28

# initial group size: 50 animals

## for males and females respectively

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

3 650 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

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

Justification for classification or non-classification

The available data on the carcinogenic potential 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 are conclusive but not sufficient for classification according to Regulation (EC) No 1272/2008.

Additional information

Two reliable dietary lifetime carcinogenicity studies are available with Reaction mass of 1-O-α-D-glucopyranosyl-D-mannitol and 6-O-α-D-glucopyranosyl-D-glucitol (CAS 64519-82-0) in the rat and mouse.

A reliable oral (dietary) carcinogenicity 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 451 (Sinkeldam, 1983) and in compliance with GLP in Wistar rats. Groups of 50 animals of each sex were fed 0, 2.5, 5 or 10% test item in the diet for nearly 2.5 years. These dietary concentrations resulted in approximate exposures of 960/1220, 1630/2450 and 3650/4980 mg/kg bw/day in males/females in the order of ascending dietary concentrations, respectively. Control groups received either basal diet with 10% maize starch or basal diet with 10% sucrose. Test item and sucrose were included in the diet at the expense of maize starch. Study animals were obtained from a multigeneration study (Sinkeldam, 1982), during which their parental animals were already treated at the same dose levels of the test item. Accordingly, administration of the test item was already started in utero. Treatment with the test item did not affect the appearance or behaviour, nor did it cause diarrhoea. Mortality rate was unaffected. Body weights of high dose rats were generally slightly lower than those of controls. Periodic examinations for haematological criteria, clinical chemistry of the blood, urine composition and kidney function did not reveal any changes of toxicological significance. Caecal enlargement was observed in rats of the high dose group, but the microscopic structure of the caecal wall was unaffected. The caecal enlargement in all test item groups 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. An increased number of treated male and female rats showed hyperplasia of the urothelium in the renal pelvis accompanied by mineralization, whereas the number of females showing corticomedullary mineralization was decreased in the treated groups. The incidence, type or location of neoplasia provided no evidence of a carcinogenic potential of the test item. Feeding 10% sucrose did not induce significant differences compared with the controls fed 10% maize starch.

It was concluded that the NOAEL for carcinogenicity in this study was 10% (corresponding to approx. 3650 and 4980 mg/kg bw/day in males and females, respectively). Based on the noted kidney findings the NOAEL for general toxicity was considered to be 1630 and 1220 mg/kg bw/day in males and females, respectively.

A second reliable oral (dietary) carcinogenicity study 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 451 (Dreef-van der Meulen, 1983) and in compliance with GLP in Cpb: Swiss random mice. Groups of 50 animals of each sex were fed 0, 2.5, 5 or 10% test item in the diet for nearly 2.5 years. These dietary concentrations resulted in approximate exposures of 6380/4050, 14250/8890 and 26950/18690 mg/kg bw/day in males/females in the order of ascending dietary concentrations, respectively. Control groups received either basal diet with 10% maize starch or basal diet with 10% sucrose. Test item and sucrose were included in the diet at the expense of maize starch. Treatment with the test item did not affect the appearance or behaviour, nor did it cause diarrhoea. Mortality rate was unaffected. Body weights of mid and high dose females were slightly lower than those of controls from study day 112 onwards. Periodic examinations for haematological parameters did not reveal any changes of toxicological significance. Absolute and relative weights of the filled and empty caecum were increased in high dose mice of both sexes. The increase in caecal weight was not accompanied by symptoms of diarrhoea. Treatment-related histopathological changes in the caecum have not been detected. 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. Histopathology did not reveal any test item-related non-neoplastic findings. Further, neither the incidence, type nor location of neoplasia provided evidence of a carcinogenic potential of the test item. Feeding 10% sucrose did not induce significant differences compared with the controls fed 10% maize starch.

It was concluded that the NOAEL for carcinogenicity in this study was 10% (corresponding to approx. 26950 and 18690 mg/kg bw/day in male and female mice, respectively). In the absence of any adverse effects in males the NOAEL for general toxicity was considered to be 10% (26950 mg/kg bw/day), while for females, based on reduced body weights the NOAEL was considered to be 2.5% (4050 mg/kg bw/day).