D-cellobiose

Acute Toxicity: oral

Administrative data

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Remarks:

A 90-day repeated dose drinking water study in rats is available, in which no adverse effects were observed up to 6852 mg Cellobiose per kg body weight. The results of the study are used to assess the acute oral toxicity study.

Adequacy of study:

key study

Study period:

October 25, 2016, to October 13, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Remarks:

A 90-day repeated dose drinking water study in rats is available, in which no adverse effects were observed up to 6852 mg Cellobiose per kg body weight. The results of the study are used to assess the acute oral toxicity.

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 25, 2016, to October 13, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

version of September 21, 1998

Deviations:

no

GLP compliance:

yes

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Pfeifer & Langen GmbH & Co. KG, Batch no. CB 20.05.2016
- Purity, including information on contaminants, isomers, etc.: purity > 99%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: males: 51 days; females: 61 days
- Weight at study initiation: Males: 241.0 g - 285.5 g; Females: 206.8 g - 246.4 g
- Fasting period before study: no reported
- Housing: The animals were kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm.
- Diet (e.g. ad libitum): ssniff-R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: no reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: drinking water

Details on route of administration:

The oral route is the default route. In addition, the study was conducted for an application for authorisation of a novel food in accordance with Article 10 of Regulation (EU) 2015/2283.

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Remarks:

Samples of 10 mL of the test item-drinking water mixtures were taken (sterile) in test weeks 1 and 13 and stored at ≤-20°C until shipment for analysis. Germany

Details on analytical verification of doses or concentrations:

Samples were shipped to Analytisches Zentrum Biopharm GmbH Dr. Berthold Lausecker, Bitterfelder Str. 19, 12681 Berlin, Germany, and analysed for concentration, stability and homogeneity (GLP-compliant).

Duration of treatment / exposure:

90 days

Frequency of treatment:

Continuously (ad libitum) for 90 days

Dose / conc.:

25 mg/L drinking water

Remarks:

2.5%

Dose / conc.:

50 mg/L drinking water

Remarks:

5%

Dose / conc.:

100 mg/L drinking water

Remarks:

10%

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes, concurrent no treatment

Details on study design:

- Dose selection rationale: based on a 28-days dose-range finding study
- Post-exposure recovery period in satellite groups: 28-days with 20 rats
- Dose range finding studies: Yes

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations: Any signs of illness or reaction to treatment were recorded for each individual animal. Cage-side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity, and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before first exposure and then once per week
- Observations: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded at the time of group allocation, daily from the day of commencement of treatment up to and including test week 6 for dose adjustment, and thereafter weekly throughout the experimental period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examinations were performed on all animals before first dosing, at the end of the main study (test week 13) and at the end of the recovery period (test week 17)
- Dose groups that were examined:all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period and at the end of the recovery period
- Anaesthetic used for blood collection: light ether anaesthesia
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period and at the end of the recovery period
- Animals fasted: Yes (overnight)
- How many animals:all
- Parameters checked in table 2 were examined.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment period and at the end of the recovery period
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters examined: volime, pH, specific gravity, Protein, Glucose, Bilirubin, Urobilinogen, Ketones, Haemoglobin (HGB) (approx. values), Nitrite, microscopic examinations (Epithelial cells, Leucocytes, Erythrocytes, Organisms, further constituents (i.e. sperm, casts), Crystalluria)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: test weeks 13 and 17 (before any blood sampling for laboratory examinations)
- Dose groups that were examined:all
- Battery of functions tested: sensory activity / grip strength / motor activity (for details see Table 3 below)

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY:
On test day 92 (to complete the 24-hour exposure on test days 90/91) the main study animals were dissected following a randomisation scheme. Dissection of all animals allocated to the recovery period was performed on test day 120.
The animals were euthanized by carbon dioxide (CO2) inhalation, exsanguinated by cutting the aorta abdominalis, weighed, dissected and inspected macroscopically under the direction of a pathologist.
All superficial tissues were examined visually and by palpation and the cranial roof was removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection, all subcutaneous tissues were examined. The condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal, the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined. The lungs were removed and all pleural surfaces examined under suitable illumination. The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenal glands, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
The weights of the following organs of all animals were determined before fixation:
Adrenal gland (2)
Brain
Epididymis (2)
Heart
Kidney (2)
Liver Ovary (2)
Pancreas
Spleen
Testicle (2)
Thymus
prostate
seminal vesicles with coagulating glands
Uterus (incl. cervix)
Paired organs were weighed individually and identified as left or right. The relative organ weight [g/kg b.w.] was calculated.

HISTOPATHOLOGY: Yes (see table 4)

Optional endpoint(s):

Optional endpoints: No

Statistics:

Data for toxicology and pathology were captured, as far as possible; using the departmental computerized systems (Provantis® Integrated preclinical software, version 9.4.0, Instem LSS Ltd., Stone, Staffordshire ST15 0SD, United Kingdom). Raw data not fully compatible with the computerized systems were maintained on paper according to appropriate SOPs.
The test item groups 2 to 4 were compared to the control group 1. The following statistical methods were used:
- Multiple t-test based on DUNNETT, C. W.
- Exact test of R. A. FISHER (if applicable)
The following settings were used for the statistical evaluation of the parametricvalues captured by Provantis:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILKS test. In case of heterogeneity and/or non- normality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group was made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).

The following statistical methods were used for the data not captured with the Provantis system, i.e. Numerical functional tests: Body temperature / Hind leg splay / Grip strength / Spontaneous motility): STUDENT's t-test

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Treatment period (Table 1a):
The body weight, body weight gain and body weight at autopsy were not influenced in male animals treated with a concentration of 2.5% Cellobiose in drinking water and in female animals treated with concentrations of 2.5%, 5% or 10% Cellobiose in drinking water for 90 days compared to the control group.
The body weight of the male animals treated with a concentration of 5% Cellobiose in drinking water for 90 days was marginally reduced by up to 6% compared to the control group (statistically not significant at p ≤ 0.05) as of test day 15. The body weight gain from start (test day 1) to the end of test day 91 was 103% compared to 113% of the animals of the control group. Body weight at autopsy appeared not to be influenced. Female animals were not affected.
The body weight of the male animals treated with a concentration of 10% Cellobiose in drinking water for 90 days was slightly reduced by 5% to 7% compared to the control group (statistically significant at p ≤ 0.01 on test day 8 and at p ≤ 0.05 on test days 15, 22, 50, 57 and 64) as of test day 8. Body weight gain from start (test day 1) to the end of test day 91 was 100% compared to 113% of the animals of the control group. Body weight at autopsy was marginally reduced by 5% compared to the control group. Female animals were not affected.

Recovery period (Table 1b):
The body weight of male animals previously treated with a concentration of 10% Cellobiose in drinking water for 90 days was still marginally reduced by 5% com- pared to the control group (statistically not significant at p ≤ 0.05) during the 28-day treatment-free recovery period, however, body weight gain revealed a tendency towards normalisation.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Treatment period:
No test item-related influence was noted on relative food consumption of male animals treated with a concentration of 2.5% Cellobiose in drinking water for 90 days compared to the control group.
The relative food consumption of female rats treated with a concentration of 2.5% Cellobiose in drinking water and of male and female rats treated with concentrations of 5% or 10% Cellobiose in drinking water for 90 days was decreased in a dose-related way.
A marginal reduction (5% to 7% below the female control animals) was noted for the relative food consumption of low-dose-females (2.5% Cellobiose in drinking water) in test weeks 1 to 11 (statistically significant at p ≤ 0.05 or p ≤ 0.01 in test weeks 1 to 8).
A slightly decreased food intake (at maximum 10% or 13% below the control group) was noted for male and female animals treated with a concentration of 5% Cellobiose in drinking water during the 90-day treatment period (statistically significant at p ≤ 0.05 or p ≤ 0.01 except test weeks 9 and 13 (males) and test week 12 (females)).
Moderately decreased food consumption (at maximum 17% or 22% below the control group) was noted for male and female rats treated with a concentration of 10% Cellobiose in drinking water for 90 days (statistically significant at p ≤ 0.01).

Recovery period:
The relative food consumption of male and female rats previously treated with a concentration of 10% Cellobiose in drinking water for 90 days increased again to or slightly above the values consumed by the control group during the 28-day treatment-free recovery period.
Statistically significant differences in relative food consumption compared to the control (slight increase in test weeks 14 and 17) were not considered to be test item-related, as they were slight alterations in comparison to control animals is without any biological relevance and observed in females only.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Treatment period:
No test-item-related influence was noted on the relative and absolute drinking water consumption of the male and female animals treated with a concentration of 2.5% Cellobiose in drinking water and of the female animals treated with a concentration of 5% Cellobiose in drinking water for 90 days compared to the control group.
A marginal reduction (up to 19% below male control animals) was noted for the absolute drinking water consumption of mid-dose male rats (5% Cellobiose in drinking water) on few test days as of test week 2 (statistically significant at p ≤ 0.05 or p ≤ 0.01 on test days 8→9, 9→10, 14→15, 15→16, 18→19, 23→24, 31→32, 35→36, 38→39, 43→44 and 77→78). The weekly relative drinking water consumption of mid-dose males was not influenced.
Moderately decreased relative drinking water consumption (at maximum 16% or 26% below the control group) was noted for male and female rats treated with a concentration of 10% Cellobiose in drinking water for 90 days (statistically significant at p ≤ 0.01 or p ≤ 0.05 in test weeks 1 to 6 and 12 for males and in test weeks 1 to 8 and 10 for females). The daily absolute drinking water consumption was decreased at a maximum of 34% and 33% for male and female animals, respectively, compared to the control group (statistically significant at p ≤ 0.05 or p ≤ 0.01 on several test days).

Recovery period:
The drinking water consumption of male and female rats previously treated with a concentration of 10% Cellobiose in drinking water for 90 days increased again to or slightly above the values consumed by the control group during the 28-day treatment-free recovery period. Statistically significant increases in drinking water consumption compared to the control which were considered to be not test item-related, as they were slight alterations in comparison to control animals without any biological relevance and observed in females only.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No changes of the eyes and the optic region, i.e. adnexa oculi, conjunctiva, cornea, anterior chamber, lens, vitreous body and fundus were noted in the male and female rats of the main study treated with concentrations of 2.5%, 5% or 10% Cellobiose in drinking water for 90 days or of the recovery animals previously treated with a concentration of 10% Cellobiose in drinking water.
There was no indication of any impairment to auditory acuity.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test-item-related influence was observed for the haemoglobin content (HGB), the number of erythrocytes (RBC), leucocytes (WBC) and platelets (PLT), the percentage of reticulocytes (Reti), the haematocrit value (HCT), the relative and absolute count of neutrophilic granulocytes (Neut), lymphocytes (Lym), monocytes (Mono), eosinophilic granulocytes (Eos), large unstained cells (LUC) and basophilic granulocytes (Baso), the thromboplastin time (TPT), the activated partial thromboplastin time (aPTT), the mean corpuscular volume (MCV), the mean corpuscular haemoglobin (MCH), and the mean corpuscular haemoglobin concentration (MCHC).

Some statistically significant differences in haematological parameters compared to the control were considered to be not test item-related (see Table 2)

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test item-related influence was noted for the plasma levels of albumin and globulin, the albumin/globulin ratio, the plasma levels of bile acids, bilirubin, cholesterol, creatinine, glucose, protein (total), triglycerides, urea, calcium, chloride, potassium and sodium. Further, the plasma activity of alanine aminotransferase (ALAT), alkaline phosphatase (aP), aspartate aminotransferase (ASAT), lactate dehydrogenase (LDH) and gamma-glutamyl-transferase (Gamma-GT) was not influenced.

Statistically significant decreases in clinical chemistry parameters compared to the control were considered to be not test item-related (Table 3).

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

No test-item-related changes were noted for specific gravity and pH value of urine, and urine volume. The analyte concentrations of nitrite, protein, glucose, ketones, urobilinogen, bilirubin and haemoglobin were not influenced in male and female animals. No test-item-related changes were observed in urine colour and microscopically analysed urine sediments.

The statistical significance of the differences in urinary parameters compared to the control (increase in specific gravity in low dose group and decrease in urine volume in low dose group) were considered to be not test item-related, as they lacked a dose-response.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Treatment period / Recovery period
The neurological screening performed at the end of the treatment period in test week 13 did not reveal any test item-related influence in male and female rats treated with concentrations of 2.5%, 5% or 10% Cellobiose in drinking water, neither on any of the parameters examined during the functional observation tests nor on the fore- and hind limb grip strength or on the spontaneous motility. Furthermore, no test item-related influence was noted in rats previously treated with a concentration of 10% Cellobiose in drinking water at the end of the recovery period in test week 17.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No test-item-related influence was noted on the relative and absolute organ weights of male and female animals treated with concentrations of 2.5%, 5% or 10% Cellobiose in drinking water for 90 days compared to the control group, neither at the end of the treatment period on test day 92 nor at the end of the 28-day treatment-free recovery period on test day 120.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Treatment period:
No test-item-related influence was noted for male and female rats treated with concentrations of 2.5%, 5% or 10% Cellobiose in drinking water for 90 days at terminal sacrifice on test day 92.
Macroscopic changes were noted in the ovary (cystic), pancreas (pale / reddened), pituitary (enlarged), spleen (enlarged), stomach mucosa (reddened), thymus (reddened) and uterus (dilated) in individual animals of test-item-treated groups and the control group. These changes are considered to be incidental findings due to the low number of animals affected. Similar findings were also noted in control groups of subchronical / chronical rat studies at the same laboratory.

Recovery period:
No test-item-related pathological changes were observed in the organs of male and female rats previously treated with a concentration of 10% Cellobiose in drinking water for 90 days at the end of the 28-day recovery period.
Macroscopic changes were noted in the pituitary (enlarged) or uterus (dilated, filled with clear liquid) in 2 of 5 female animals of the previously high-dose group. These changes are considered to be incidental findings due to the low number of animals affected.

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Treatment period / Recovery period (restricted to group 1 (control) and group 4 (high dose)):
The histopathological examination was restricted to a variety of organs and tissues from the control and the high dose animals (groups 1 and 4).
Histological examination did not reveal any morphological changes in animals treated with a concentration of 10% Cellobiose in drinking water for 90 days which are considered to be related to the administration of the test item. No test- item-related changes were noted at the end of the 28-day recovery period.
A few minor microscopic changes were recorded for the organs and tissues exam- ined in this study. However, the type, incidence and severity of all microscopic findings observed did not indicate any relationship to treatment with the test item. All changes noted are regarded as spontaneous and to be within the normal back- ground pathology commonly seen in rats of this strain and age.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

>= 8 043 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Remarks on result:

other: All observed effects, i.e., marginally reduced body weight and food consumption, are considered to be secondary to a reduced drinking water intake at the highest dose level.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 6 852 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Remarks on result:

other: All observed effects, i.e., marginally reduced body weight and food consumption, are considered to be secondary to a reduced drinking water intake at the highest dose level.

Key result

Critical effects observed:

no

Table 1a: Bodyweight males (treatment period) (Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01)

					Day(s) Relative to Start Date
		-6	1	8	15	22	29	36	43	50	57	64	71	78	85	91
Group 1:	Mean	195.47	262.37	320.77	362.18	396.45	427.23	450.26	473.98	492.95	508.96	525.04	535.34	540.44	549.90	558.73
Control	SD	8.43	10.93	13.14	18.52	19.04	25.23	28.40	30.13	32.44	34.65	38.09	39.18	42.41	40.01	42.81
	N	15	15	15	15	15	15	15	15	15	15	15	15	15	15	15
Group 2:	Mean	195.94	262.54	322.50	367.02	403.12	436,74	459.24	481.73	497.07	509.50	525.36	535.09	536.95	545.00	555.59
2.5%	SD	7.54	10.86	11.93	14.68	19.74	26.35	26.92	30.80	27.88	35.15	41.00	41.96	44.37	50.20	53.53
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	%Diff	0.2	0.1	0.5	44621	44743	44594	2.0	1.6	0.8	0.1	0.1	0.0	-0.6	-0.9	-0.6
Group 3:	Mean	196.62	261.64	310.16	347.85	377.81	405.19	426.22	448.05	464.98	483.42	499.90	509.79	512.91	519.13	530.91
5%	SD	5.15	9.96	15.56	16.74	18.18	21.07	24.08	28.58	32.88	35.42	31.82	33.23	35.12	37.12	38.96
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
	%Diff	0.6	-0.3	-3.3	4.0	-4.7	-5.2	-5.3	-5.5	-5.7	-5.0	-4.8	-4.8	-5.1	-5.6	-5.0
Group 4:	Mean	197.13	263.30	303.37 **	343.16*	374.75*	404.65	425.33	447.37	460.37*	472.35*	486.29*	502.35	505.16	516.72	526.89
10%	SD	7.21	7.97	13.45	21.70	27.61	30.32	31.66	31.54	33.00	36.39	40.13	39.94	38.59	38.60	39.43
	N	15	15	15	15	15	15	15	15	15	15	15	15	15	15	15
	%Diff	0.8	0.4	-5.4	-5.3	-5.5	-5.3	-5.5	-5.6	-6.6	-7.2	-7.4	-6.2	-6.5	-6.0	-5.7

 

Table 1b: Bodyweight males (recovery period) (Anova & Dunnett: * = p ≤ 0.05; ** = p ≤ 0.01)

Sex: Male	Day(s) Relative to Start Date
		98	105	112	119
Group 1 :	Mean	568.92	585.30	593.18	595.30
Control	SD	47.56	54.03	54.75	53.21
	N	5	5	5	5
Group 4'	Mean	540.30	555.48	565.94	568.42
10%	SD	35.68	33.81	33.18	35.47
	N	5	5	5	5
	%Diff	-5.0	-5.1	-4.6	-4,5

 

Table 2: Statistically significant differences in haematological parameters not test-item related (A: the slight alteration in comparison to control animals is without any biological relevance; B: lacking dose dependence; C: only one gender affected)

Parameter	Increase (I)	Group/	Test day	Statistical significance	Reason
	Decrease (D)	Sex
Haemoglobin content (HGB)	D	3 f	92	p < 0.05	A, C
	D	4 f	92	p < 0.01	A, C
Erythrocytes /RBC)	D	3 f	92	p < 0.05	A
	D	4 m	92	p < 0.01	A
	D	4 f	92	p < 0.01	A
Platelets (PLT)	I	2 f	92	p < 0.01	B, C
	I	4 f	92	p < 0.05	A, C
Haematocrit value (HCT)	D	3 f	92	p < 0.05	A, C
	D	4 f	92	p < 0.01	A, C
Activated partial thromboplastin time (aPTT)	I	4 f	92	p < 0.01	A, C

 

Table 3: Statistically significant decreases in clinical chemistry parameters not test-item related (A: the slight alteration in comparison to control animals is without any biological relevance; B: lacking dose dependence; C: enzyme reduction without any toxological relevance ;D: only one gender affected)

Parameter	Group/	Test day	Statistical	Reason
	Sex		significance
Bilirubin	4 m	120	p < 0.01	A, C, D
Chloride	4 f	120	p < 0.05	B, D
Sodium	4 f	120	p < 0.05	A, D
Gamma-GT	4 m	120	p < 0.05	A, C, D

Conclusions:

In a GLP-compliant study according to OECD TG 408 in rats, cellobiose administered via drinking water did not induce any adverse effects. The experimental no-observed-adverse-effect level (NOAEL) was equal to or greater than 10% Cellobiose in drinking water (equivalent to ~ 6852 and ~ 8043 mg Cellobiose per kg body weight for males and females, respectively).

Executive summary:

In a GLP-compliant study according to OECD TG 408 in rats, cellobiose administered via drinking water did not induce any adverse effects. The experimental no-observed-adverse-effect level (NOAEL) was equal to or greater than 10% Cellobiose in drinking water (equivalent to ~ 6852 and ~ 8043 mg Cellobiose per kg body weight for males and females, respectively). All observed effects, i.e., marginally reduced body weight and food consumption, are considered to be secondary to a reduced drinking water intake at the highest dose level.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

version of September 21, 1998

Deviations:

no

GLP compliance:

yes

Test type:

other: 90-day drinking water study

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

D-cellobiose

EC Number:

208-436-5

EC Name:

D-cellobiose

Cas Number:

528-50-7

Molecular formula:

C12H22O11

IUPAC Name:

(2R,3S,4S,5R,6S)-2-(hydroxymethyl)-6-[(2R,3S,4R,5R,6R)- 4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxane-3,4,5-triol

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source (i.e. manufacturer or supplier) and lot/batch number of test material: Pfeifer & Langen GmbH
& Co. KG, Batch no. CB 20.05.2016
- Purity, including information on contaminants, isomers, etc.: purity > 99%
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL - Storage condition of test material: room temperature

Test animals

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: males: 51 days; females: 61 days
- Weight at study initiation: Males: 241.0 g - 285.5 g; Females: 206.8 g - 246.4 g
- Fasting period before study: no reported
- Housing: The animals were kept singly in MAKROLON cages (type III plus) with a basal surface of a pprox. 39 cm x 23 cm and a height of approx. 18 cm.
- Diet (e.g. ad libitum): ssniff-R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany) ad
libitum
- Water (e.g. ad libitum): ad libitum - Acclimation period: no reported
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

The oral route is the default route. In addition, the study was conducted for an application for aut horisation of a novel food in accordance with Article 10 of Regulation (EU) 2015/2283

Doses:

25, 50 and 100 mg/L drinking water

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes

Details on study design:

Observations and examinations performed and frequency

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily
- Cage side observations: Any signs of illness or reaction to treatment were recorded for each
individual animal. Cage-side observations included skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity, and behaviour patterns. The onset, intensity and duration of any signs observed were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before first exposure and then once per week
- Observations: changes in skin, fur, eyes, mucous membranes, occurrence of secretions and
excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern). Changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-
mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each rat was recorded at the time of group all ocation, daily from the day of commencement of treatment up to and including test week 6 for dose ad justment, and thereafter weekly throughout the experimental period.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes - Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Examinations were performed on all animals before first dosing, at
the end of the main study (test week 13) and at the end of the recovery period (test week 17) - Dose groups that were examined:all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period and at the end of the reco very period
- Anaesthetic used for blood collection: light ether anaesthesia
- Animals fasted: Yes (overnight)
- How many animals: all
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period and at the end of the recov ery period
- Animals fasted: Yes (overnight)
- How many animals:all
- Parameters checked in table 2 were examined.

PLASMA/SERUM HORMONES/LIPIDS: No

URINALYSIS: Yes
- Time schedule for collection of urine: at the end of the treatment period and at the end of the recove ry period
- Metabolism cages used for collection of urine: Not specified
- Animals fasted: Yes
- Parameters examined: volime, pH, specific gravity, Protein, Glucose, Bilirubin, Urobilinogen,
Ketones, Haemoglobin (HGB) (approx. values), Nitrite, microscopic examinations (Epithelial cells, Leucocytes, Erythrocytes, Organisms, further constituents (i.e. sperm, casts), Crystalluria)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: test weeks 13 and 17 (before any blood sampling for laboratory
examinations)
- Dose groups that were examined:all
- Battery of functions tested: sensory activity / grip strength / motor activity (for details see Table 3
below)

IMMUNOLOGY: No

Statistics:

Data for toxicology and pathology were captured, as far as possible; using the departmental computer ized systems (Provantis® Integrated preclinical software, version 9.4.0, Instem LSS Ltd., Stone, Sta ffordshire ST15 0SD, United Kingdom). Raw data not fully compatible with the computerized systems
were maintained on paper according to appropriate SOPs.
The test item groups 2 to 4 were compared to the control group 1. The following statistical methods
were used:
- Multiple t-test based on DUNNETT, C. W.
- Exact test of R. A. FISHER (if applicable)
The following settings were used for the statistical evaluation of the parametricvalues captured by
Provantis:
Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and
SHAPIRO-WILKS test. In case of heterogeneity and/or non- normality of distribution, stepwise tr ansformation of the values into logarithmic or rank values was performed prior to ANOVA. If the
ANOVA yielded a significant effect (p ≤ 0.05), intergroup comparisons with the control group was made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
The following statistical methods were used for the data not captured with the Provantis system, i.e. Numerical functional tests: Body temperature / Hind leg splay / Grip strength / Spontaneous motility): STUDENT's t-test

Results and discussion

Effect levels

Key result

Sex:

male/female

Dose descriptor:

LD0

Effect level:

> 6 852 mg/kg bw

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Remarks:

The effect level is the no observed effect level from a 90-day repeated dose drinking water study in rats.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In a GLP-compliant 90-day repeated dose drinking water study in rats according to OECD TG 408, no adverse effects were observed up to 6852 mg Cellobiose per kg body weight. Based on this result, the acute oral toxicity will be higher than 6852 mg/kg bw.

Executive summary:

In a GLP-compliant study according to OECD TG 408 in rats, cellobiose administered via drinking water did not induce any adverse effects. The experimental no-observed-adverse-effect level (NOAEL) was equal to or greater than 10% Cellobiose in drinking water (equivalent to ~ 6852 and ~ 8043 mg Cellobiose per kg body weight for males and females, respectively). All observed effects, i.e., marginally reduced body weight and food consumption, are considered to be secondary to a reduced drinking water intake at the highest dose level.