Fructofuranosidase, β-

Endpoint summary

Description of key information

Sub-chronic Toxicity (OECD 408), oral, rat: NOAEL: 6400 mg/kg bw/day

Read-across from the source substance: Endoxylanase (CAS 9025 -57 -4)

Sub-chronic Toxicity (OECD 408), oral, rat: NOAEL: 1000 mg/kg bw/day

Read-across from the source substance: Alpha-amylase (CAS: 9000 -90 -2)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

refer to analogue justification provided in IUCLID section 13

Reason / purpose for cross-reference:

read-across source

Vehicle:

other: double distilled water

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

control and 400, 1600, 6400 mg/kg bw/day: An incidence of alopecia in a female at control group and incidence of hair thinning with hair regrowth in a female each at low and mid doses and in two females at high dose group were observed (incidental finding). For details please refer to table 6 in the " Any other information on results incl. tables" section.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

1600 mg/kg bw: in males and females higher food intake was observed in week 8, but was considered an incidental finding. In week 9, a lower food intake was observed in females, but considered an incidental finding.
6400 mg/kg bw/day: lower food intake was observed in week 9 in both sexes and in week 10 in males only. Since the food intake was not consistently affected the effect on food consumption was considered an incidental finding.
For details please refer to table 3 in the " Any other information on results incl. tables" section.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

400 mg/kg bw/day: a higher level of mean corpuscular volume (MCV) was observed in females, but considered incidental as the changes were isolated incidences.

1600 mg/kg bw/day: in males isolated incidences of higher level of MCH, platelets, prothrombin time, neutrophils, and lower level of lymphocytes were observed. Since there were no effects observed at high dose level, the observed effects were considered incidental. In males significantly higher level of MCHC was observed, but considered incidental as the corresponding changes were not observed in RBC and Hb. In females, an incidence of higher level of mean corpuscular volume and a higher level of haematocrit were observed, but considered incidental as the changes were isolated incidences.

6400 mg/kg bw/day: in males a significantly higher level of MCHC was observed, but considered incidental as the corresponding changes were not observed in RBC and Hb. In females, higher neutrophil percentage with lower lymphocyte percentage were observed compared to control, but considered incidental since they were within the range of biological variation.

For details please refer to table 4 in the " Any other information on results incl. tables" section.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

1600 mg/kg bw/day: the creatinine level was significantly higher in males. Although the increase was dose-correlated it was considered incidental as there were no corresponding histopathological changes in the kidneys.

6400 mg/kg bw/day: sodium, chloride and creatinine levels were significantly higher in males. Since the higher levels of chloride and sodium were within the normal range of variation, they were not considered to be treatment-related. Although the creatinine increase was dose-correlated it was considered incidental as there were no corresponding histopathological changes in the kidneys.

For details please refer to table 5 in the " Any other information on results incl. tables" section.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

1600 mg/kg bw/day: a significant decrease in the absolute and relative weight of ovaries was observed in females, but was not considered treatment-related as it was not observed at high dose and there were no corresponding gross and histopathological changes.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

control/1600 mg/kg bw/day:dilatation of pelvis in kidneys were considered incidental.

400 mg/kg bw/day: enlarged testes and heart were condisered incidental.

control, 400, 1600 and 6400 mg/kg bw/day: a few incidences of skin-alopecia were considered incidental.

6400 mg/kg bw/day: one male showed a white liver focus, this isolated finding was considered incidental.

For details please refer to table 6 in the " Any other information on results incl. tables" section.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

400 mg/kg bw/day: in males lower grip strength values were observed in forelimbs, but considered incidental as these changes were isolated incidences.

1600 and 6400 mg/kg bw/day: significantly higher incidence of landing foot splay was observed in males and females, but considered incidental as there was no dose-dependency and no change in gait observed in these animals. In mid dose males higher grip strength values were observed in hindlimbs, but considered incidental as these changes were isolated incidences. In females, significantly higher grip strength values were observed in forelimbs at mid and high dose and hindlimbs at high dose, but were also considered incidental findings since dose correlation was not evident.

For details please refer to table 7 in the " Any other information on results incl. tables" section.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

control, 400, 1600 and 6400 mgkg bw/day: perivascular lymphocytic infiltration in the lung (minimal to mild) was observed at all doses in females and in males at all doses with exception of the low dose. The incidence of perivascular lymphocytic infiltration in the lungs was not considered treatment-related as there was no intergroup statistical significance. Blood vessel mineralisation (minimal) was observed in males and females at all doses, except in males of the control group. Since the highest indidence was observed in females of the control group this finding was considered incidental.

400 and 6400 mg/kg bw/day: mineralisation in kidneys was observed in one male of the low dose group and one female of the high dose group.

control, 1600 and 6400 mg/kg bw: basophilic tubules (minimal) were observed in kidneys of males.

control and 6400 mg/kg bw/day: hyaline droplets and proteinaceous material were observed in tubular epithelium (minimal to moderate) of the kidneys in males.

The changes in kidneys and other tissues were considered incidental as the incidences were similar or higher in control.

For a detailed overview of all findings and details please refer to table 8 in the " Any other information on results incl. tables" section.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

6 400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed at this dose level

Critical effects observed:

no

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 August to 28 or 29 November 2001

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:

adopted: 21 September 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Version / remarks:

August 1998

Qualifier:

according to guideline

Guideline:

other: Commission Directive 87/302/EEC Part B:Methods for Determination of Toxicity: Subchronic Oral Toxicity Test: 90 day Repeated Oral Dose using Rodent species

Version / remarks:

18 November 1987

GLP compliance:

yes (incl. QA statement)

Remarks:

Ministry of Health Welfare and Sports, Inspectorate for Health Protection, Commodities and Veterinary Public Health, GLP Compliance Monitoring unit, The Netherlands

Limit test:

no

Species:

rat

Strain:

other: Hsd Cpb WU rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxicology Department, Rallis Research Center, Rallis India Limited, Bangalore, India
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 7 weeks
- Weight at study initiation: 208 ± 9.9 - 210 ± 9.7 (males), 153 ± 6.9 - 154 ± 7.3 (females)
- Housing: two rats per cage in in a sterilized suspended standard polypropylene rat cage (size: L 410 x W 282 x H 150 mm) with stainless steel mesh bottom and stainless steel top grill.
- Diet: rats/mice pellet food (Ssniff Spezialdiaten GmbH, Söest, Germany), ad libitum
- Water: deep borewell water passed through activated charcoal filter and exposed to UV rays in aquaguard on-line water filter-cum-purifier, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 -24
- Humidity (%): 30 -70
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24.08. 2001 To: 28./29.11.2001

Route of administration:

oral: gavage

Vehicle:

other: double distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was dissolved in double distilled water and was prepared daily. The quantities of test substance was not weighed instead volume was measured based on specific gravity (specific gravity 1 06). Approximate quantities of 2000 mg (1.89 mL), 8000 mg (7.55 mL) and 32000 mg (30.19 mL) of test substance were separately mixed and volume of each was made up to 50 mL with double distilled water to get the test substance concentrations of 40, 160 and 640 mg.
- Dose volume: 10 mL/kg bw/day

VEHICLE
- Amount of vehicle: varied, depending on the body weights of the rats recorded during different intervals of treatment period

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test substance preparations were analysed for protein content by digesting the sample of the test item with concentrated sulphuric acid and total nitrogen content was estimated by Micro-Kjeldahl method on day 1, months 2 and 3 of the treatment period. The total protein content in the test item was calculated by multiplying total nitrogen content by a factor 6.25. The results showed mean concentrations of 34.40 ± 1.01, 135.50 ± 4.48 and 543.18 ± 17.33 mg of the test substance/mL as against the nominal concentrations of 40, 160 and 640 mg/mL.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Dose / conc.:

1 600 mg/kg bw/day (actual dose received)

Dose / conc.:

6 400 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Rationale for animal assignment: Grouping was done based on body weight stratification and distribution. The rats procured for the study were weighed and grouped in to body weight ranges (selected ranges: 191 - 200 g, 201 - 210 g, 211 - 220 g for males and 141 - 150 g, 151 - 160 g for females). These body weight stratified rats were distributed to all the study groups in equal numbers. The rats with extreme body weights were not used for the study. Grouping was done on last day of acclimatization.
- Fasting period before blood sampling for clinical biochemistry: overnight

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations: clinical signs of toxicity (once a day), pre-terminal deaths (twice a day)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to initiation of treatment and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: shortly before test substance administration and weekly thereafter

FOOD CONSUMPTION:
- 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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: one day before start of the treatment and at the end of the treatment period prior to sacrifice
- Dose groups that were examined: all dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: two days prior to sacrifice (blood smear: differential leucocyte count), at the end of the treatment period (blood collection)
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table 1 were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the treatment period (12/13th week of the treatment period)
- Dose groups that were examined: all dose groups
- Battery of functions tested: grip strength / motor activity / sensory activity / other: visual response, auditory response, proprioceptive response, righting reflex

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (liver, adrenals, kidneys, testes, ovaries, epididymides, uterus, thymus, spleen, brain, heart were collected and weighed)

HISTOPATHOLOGY: Yes (please refer to table 2 in the "Any other information on materials and methods incl. tables" section). All the tissues from control and high dose group rats and gross lesions from low and mid dose group rats were subjected to detailed histopathological examination. The lungs of animals in the low and mid dose groups were subjected to histopathological examination for evidence of infection to provide an assessment of the health status of the animals.

Statistics:

Body weights, cumulative net body weight gains, food consumption, laboratory investigations (haematology and clinical chemistry), organ weights and organ weight ratio data were compared by Bartlett's test for homogeneity of intra group variances. When the variances proved to be heterogeneous, the data were transformed using appropriate transformation. The data with homogeneous intra group variances were subjected to one-way analysis of variance (ANOVA - Snedecor and Cochran, 1987). Following ANOVA, When 'F' was found to be significant, Dunnett's pairwise comparison (Scheffe 1953) of means of treated groups with the mean of.the control group was done individually. Following a significant difference of a test group with the control group, the dose-response correlation was estimated by including the control and all the treated groups and tested by , t' test.
All analyses and comparisons are evaluated at 5% (P ≤ 0.05) level. Statistically significant differences (P ≤ 0.05) indicated by the a forementioned tests are designated by the superscripts as stated below:
+/- : Significantly higher(+)/lower(-) than the control group
d : Significant dose correlation

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

control and 400, 1600, 6400 mg/kg bw/day: An incidence of alopecia in a female at control group and incidence of hair thinning with hair regrowth in a female each at low and mid doses and in two females at high dose group were observed (incidental finding). For details please refer to table 6 in the " Any other information on results incl. tables" section.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

1600 mg/kg bw: in males and females higher food intake was observed in week 8, but was considered an incidental finding. In week 9, a lower food intake was observed in females, but considered an incidental finding.
6400 mg/kg bw/day: lower food intake was observed in week 9 in both sexes and in week 10 in males only. Since the food intake was not consistently affected the effect on food consumption was considered an incidental finding.
For details please refer to table 3 in the " Any other information on results incl. tables" section.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

400 mg/kg bw/day: a higher level of mean corpuscular volume (MCV) was observed in females, but considered incidental as the changes were isolated incidences.

1600 mg/kg bw/day: in males isolated incidences of higher level of MCH, platelets, prothrombin time, neutrophils, and lower level of lymphocytes were observed. Since there were no effects observed at high dose level, the observed effects were considered incidental. In males significantly higher level of MCHC was observed, but considered incidental as the corresponding changes were not observed in RBC and Hb. In females, an incidence of higher level of mean corpuscular volume and a higher level of haematocrit were observed, but considered incidental as the changes were isolated incidences.

6400 mg/kg bw/day: in males a significantly higher level of MCHC was observed, but considered incidental as the corresponding changes were not observed in RBC and Hb. In females, higher neutrophil percentage with lower lymphocyte percentage were observed compared to control, but considered incidental since they were within the range of biological variation.

For details please refer to table 4 in the " Any other information on results incl. tables" section.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

1600 mg/kg bw/day: the creatinine level was significantly higher in males. Although the increase was dose-correlated it was considered incidental as there were no corresponding histopathological changes in the kidneys.

6400 mg/kg bw/day: sodium, chloride and creatinine levels were significantly higher in males. Since the higher levels of chloride and sodium were within the normal range of variation, they were not considered to be treatment-related. Although the creatinine increase was dose-correlated it was considered incidental as there were no corresponding histopathological changes in the kidneys.

For details please refer to table 5 in the " Any other information on results incl. tables" section.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

1600 mg/kg bw/day: a significant decrease in the absolute and relative weight of ovaries was observed in females, but was not considered treatment-related as it was not observed at high dose and there were no corresponding gross and histopathological changes.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

control/1600 mg/kg bw/day:dilatation of pelvis in kidneys were considered incidental.

400 mg/kg bw/day: enlarged testes and heart were condisered incidental.

control, 400, 1600 and 6400 mg/kg bw/day: a few incidences of skin-alopecia were considered incidental.

6400 mg/kg bw/day: one male showed a white liver focus, this isolated finding was considered incidental.

For details please refer to table 6 in the " Any other information on results incl. tables" section.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

400 mg/kg bw/day: in males lower grip strength values were observed in forelimbs, but considered incidental as these changes were isolated incidences.

1600 and 6400 mg/kg bw/day: significantly higher incidence of landing foot splay was observed in males and females, but considered incidental as there was no dose-dependency and no change in gait observed in these animals. In mid dose males higher grip strength values were observed in hindlimbs, but considered incidental as these changes were isolated incidences. In females, significantly higher grip strength values were observed in forelimbs at mid and high dose and hindlimbs at high dose, but were also considered incidental findings since dose correlation was not evident.

For details please refer to table 7 in the " Any other information on results incl. tables" section.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

control, 400, 1600 and 6400 mgkg bw/day: perivascular lymphocytic infiltration in the lung (minimal to mild) was observed at all doses in females and in males at all doses with exception of the low dose. The incidence of perivascular lymphocytic infiltration in the lungs was not considered treatment-related as there was no intergroup statistical significance. Blood vessel mineralisation (minimal) was observed in males and females at all doses, except in males of the control group. Since the highest indidence was observed in females of the control group this finding was considered incidental.

400 and 6400 mg/kg bw/day: mineralisation in kidneys was observed in one male of the low dose group and one female of the high dose group.

control, 1600 and 6400 mg/kg bw: basophilic tubules (minimal) were observed in kidneys of males.

control and 6400 mg/kg bw/day: hyaline droplets and proteinaceous material were observed in tubular epithelium (minimal to moderate) of the kidneys in males.

The changes in kidneys and other tissues were considered incidental as the incidences were similar or higher in control.

For a detailed overview of all findings and details please refer to table 8 in the " Any other information on results incl. tables" section.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

6 400 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effect observed at this dose level

Critical effects observed:

no

Table 3: Cagewise average food intake (g/rat/day)

Group	weeks
		1	2	3	4	5	6	7	8	9	10	11	12	13
males
control	mean	25.9	27.0	27.4	27.6	27.2	26.0	26.2	25.9	29.1	28.6	25.8	25.2	25.7
	SD	1.22	1.03	0.98	0.84	1.58	1.91	1.39	0.59	1.59	1.53	0.67	0.96	1.35
400	mean	26.4	27. 1	27.4	27.9	26.7	26.4	26.0	26.2	27.5	27.5	25.8	25.8	26.6
	SD	0.50	0.69	0.98	1.49	0.94	1.07	0.76	0.31	0.99	1.52	0.57	0.91	0.64
1600	mean	27.2	27.6	28.1	28.2	28.0	25.9	26. 9	27.6+	29.1	2 7.8	2 7.1	26.6	27.1
	SD	1.46	1.41	0.96	1.22	1.21	2.54	1.57	1.09	1.81	1.45	1.59	1.58	1.45
6400	mean	26.9	27.5	27.7	28.0	27.8	26.0	24.7	25.1	24.2-	25.9-	25.9	25.4	25.7
	SD	0.83	1.12	0.75	1.30	1.01	1.19	1.26	1.19	0.78	0.95	1.34	1.64	1.05
females
control	mean	17.9	18.0	18.3	19.2	19.7	19.9	18.8	17.8	22.3	20.6	17.4	17.6	17.6
	SD	0.71	0.63	1.00	0.87	0.90	1.57	1.21	1.25	1.34	1.38	0.98	1.69	0.79
400	mean	17.3	17.8	18.1	18.6	19.3	19.0	18.2	18.0	21.3	20.0	16.7	18.0	18.1
	SD	0.58	0.74	0.73	0.98	1.22	1.08	0.78	0.73	1.48	1.28	0.78	0.90	0.85
1600	mean	17.7	18.3	18.5	18.7	19.2	19.7	18.2	20.0+	18.1-	19.1	17.8	18.5	18.7
	SD	1.07	1.08	0.79	0.89	1.21	1.51	1.15	1.63	0.62	0.86	1.36	1.33	1.47
6400	mean	18.0	18.4	19.3	19.0	19.6	18.1	17.4	20.5	18.6-	19.4	16.6	17.4	17.9
	SD	0.60	0.56	0.78	0.68	0.57	0.78	0.41	0,45	1.02	0.82	0.75	0.36	0.93

SD Standard deviation

+ significantly higher than the control group

- significantly lower than the control group

 

Table 4: Summary of haematological results

males
		WBC g/L	RBC L/L	Hb g/L	Hct L/L	MCV fl	MCH pg	MCHC g/L	Plat g/L	P.T. S	Neut %	Lymp%	Eosi%	Mono%	Baso%
control	mean	5.7	8.76	162	0.432	49.4	18.5	375	957	18.4	14.6	82.2	2.4	0.8	0.0
	SD	1.76	0.31	3.88	0.016	1.46	0.44	9.25	139.14	1.34	5.50	5.71	2.27	1.03	0.0
400	mean	7.4	8.55	161	0.426	49.8	18.8	378	1002	19.5	1 6.0	78.3	4. 4	1.3	0.0
	SD	2.56	0. 39	6.53	0.021	1.61	0.34	11.89	99.08	2.08	5.01	6. l7	2.41	1.57	0.0
1600	mean	8.6	8.40	163	0.417	49.7	19.4+	391+	1081+	20.5+	26.3+	71.1-	2.1	0.5	0.0
	SD	2.64	0.39	8.09	0.022	1.02	0.49	11.73	71.41	1.85	9.93	10.54	1.91	0.53	0.0
6400	mean	7.2	8.76	167	0.428	48.9	19.0	390+	943	18.2	19.7	75.8	4.0	0.5	0.0
	SD	2.21	0.46	5.74	0.025	1.17	0.74	13.42	82.13	1.71	7.30	7.76	2.75	0.53	0.0
females
		WBC g/L	RBC L/L	Hb g/L	Hct L/L	MCV fl	MCH pg	MCHC g/L	Plat g/L	P.T. S	Neut %	Lymp%	Eosi%	Mono%	Baso%
control	mean	4.9	7.91	159	0.397	50.2	20.1	400	1034	15.9	11.3	85.8	1.8	1.1	0.0
	SD	1.03	0.29	4.57	0.014	1.11	0.65	9.60	82.49	1.60	6.04	7.13	1.62	1.45	0.0
400	mean	5.9	7.88	163	0.411	52.2+	20.7	397	974	15.0	18.2	78.8	1.7	1.3	0.0
	SD	1.45	0.31	5.10	0.018	1.64	0.80	12.83	116.11	1.03	8.72	8.72	1.34	0.67	0.0
1600	mean	5.9	7.95	164	0.415+	52.2+	20.6	395	1018	15.3	15.5	80.2	2.5	1.8	0.0
	SD	1.88	0.26	5.25	0.013	1.37	0.59	11.74	88.66	1.29	8.54	9.75	1.84	1.62	0.0
6400	mean	5.9	7.70	159	0.395	51.3	20.6	402	1006	16.1	22.3+	73.8-	2.7	1.2	0.0
	SD	1.58	0.24	4.26	0.014	1.39	0.55	8.14	89.83	1.09	8.03	8.95	1.83	1.48	0.0

SD Standard deviation

+ Significantly higher than the control group

- Significantly lower than the control group

 

Table 5: Summary of clinical chemistry results

	males
		Glu mmol/L	BUN mmol/L	Urea mmol/L	Tot. Pro g/L	AST U/L	ALT U/L	GGT U/L	Tot.Bil µmol/L	Creat µmol/L	Alb g/L	Pi mmol/l	Ca mmol/l	Chol mmol/l	Cl mmol/l	Na mEq/L	K mEq/L
control	mean	8.28	2.45	5.25	58.4	81	38	3	3.60	77	32.43	1.97	2.67	2.23	108	143.2	4.12
	SD	0.72	0.20	0.42	1.62	11.83	4.88	2.07	0.62	3.74	1.05	0.17	0.05	0.42	0.92	1.64	0.24
400	mean	8.79	2.73	5.85	59.7	88	36	4	5.19	82	33.22	2.13	2.72	2.29	109	143.9	4.10
	SD	0.90	0.33	0.70	3.00	35.36	4.58	5.09	2.46	7.07	1.97	0.18	0.06	0.46	1.51	1.84	0.36
1600	mean	8.52	2.49	5.32	58.3	81	34	3	4.70	85+	31.49	2.04	2.69	2.22	108	143.5	4.24
	SD	0.71	0.38	0.82	2.37	11.29	8.91	4.80	2.05	7.02	1.06	0.23	0.04	0.36	1.75	1.45	0.24
6400	mean	8.74	2.55	5.45	58.7	86	40	3	5.25	88d+	32.67	2.10	2.68	2.13	111+	145.5+	4.22
	SD	0.69	0.30	0.64	2.66	10.91	6.00	1.71	2.11	4.65	1.89	0.15	0.06	0.35	2.22	1.68	0.35
	females
		Glu mmol/L	BUN mmol/L	Urea mmol/L	Tot. Pro g/L	AST U/L		GGT U/L	Tot.Bil µmol/L	Creat µmol/L	Alb g/L	Pi mmol/l	Ca mmol/l	Chol mmol/l	Cl mmol/l	Na mEq/L	K mEq/L
control	mean	6.99	2.64	5.64	61.7	93	35	3	4.08	75	34.23	1.79	2.68	1.92	108	144.1	4.04
	SD	0.42	0.26	0.56	2.87	15.77	8.71	2.13	0.82	6.00	2.15	0.32	0.06	0.22	2.01	2.73	0.37
400	mean	7.12	3.01	6.45	63.0	100	34	4	4.59	80	34.36	1.81	2.68	1.91	108	146.1	3.95
	SD	0.79	0.80	1.72	3.08	18.06	6.84	4.03	1.99	8.14	1.90	0.34	0.08	0.20	2.42	2.43	0.39
1600	mean	7.08	2.77	5.92	64.3	112	36	5	4.54	79	35.65	1.67	2.76	2.19	109	145.6	4.10
	SD	1.06	0.32	0.68	3.06	32.9	5.14	2.67	1.65	10.55	2.09	0.29	0.08	0.33	1.89	2.96	0,30
6400	mean	7.15	2.95	6.31	63.8	101	39	3	3.66	83	34.69	1.82	2.71	2.05	109	145.5	3.98
	SD	0.56	0.56	1.20	2.21	14.70	7.95	0.74	0.44	4.20	2.35	0.22	0.06	0.38	2.04	3.40	0.46

SD Standard deviation

+ Significantly higher than the control group

- Significantly lower than the control group

d Dose-response

 

Table 6: Summary of necropsy findings

	males				females
Dose (mg/kg bw/day)	control	400	1600	64000	control	400	1600	64000
Number of animals examined	10	10	10	10	10	10	10	10
Number of animals showing gross pathology	1	2	1	1	1	3	2	2
Number of animals showing external pathology	0	0	0	0	1	1	1	2
Skin alopecia focal	0	0	0	0	1	0	0	0
Skin-hair thinning and regrowth focal	0	0	0	0	0	1	1	2
Number of animals showing visceral organ pathology	1	2	1	1	0	2	1	0
Liver-focus white	0	0	0	1	0	0	0	0
Kidney unilateral/bilateral pelvis dilated	1	0	1	0	0	2	1	0
Testes unilateral enlarged, abcess	0	1	0	0	0	0	0	0
Heart enlarged	0	1	0	0	0	0	0	0

 

Table 7: Neurological examination results

Incidences - males
Dose(mg/kg bw/day)			control			400			1600			6400
Number of rats			10			10			10			10
1. Visual response
Normal (present)			10			10			10			10
Absent			0			0			0			0
2. Auditory response
Normal			10			10			10			10
No (absent)			0			0			0			0
Exaggerated			0			0			0			0
3. Gait
Normal			10			10			10			10
Abnormal			0			0			0			0
Ataxic			0			0			0			0
4. Landing foot splay(cm)
Mean±			6.5			7.0			7.8+			8.8+
SD			0.74			1.15			1.26			0.94
5. Righting reflex
on back
- Present	10					10			10			10
- Slow	0					0			0			0
- Absent	0					0			0			0
dropped
- Present	10					10			10			10
- Slow	0					0			0			0
- Absent	0					0			0			0
6. Motor activity (Score)
Mean ±			719			666			658			744
SD			122.26			92.56			191.67			201.92
7. Grip strength (g)
Forelimb
Mean±.		1054		984 -			1031			1089
SD		90.94		115.48			97.17			68.78
Hindlimb
Mean±		636				638			712+			670
SD		128.38				91 .43			79.40			140.45
Incidences - females
Dose(mg/kg bw/day)		control			400			1600			6400
Number of rats		10			10			10			10
1. Visual response
Normal (present)		10			10			10			10
Absent		0			0			0			0
2. Auditory response
Normal		10			10			10			10
No (absent)		0			0			0			0
Exaggerated		0			0			0			0
3. Gait
Normal		10			10			10			10
Abnormal		0			0			0			0
Ataxic		0			0			0			0
4. Landing foot splay (cm)
Mean±		6.1			6.1			7.8+			1.9+
SD		0.85			1.10			0.95			1.53
5. Righting reflex
on back
- Present		10			10			10			10
- Slow		0			0			0			0
- Absent		0			0			0			0
dropped
- Present		10			10			10			10
- Slow		0			0			0			0
- Absent		0			0			0			0
6. Motor activity (Score)
Mean ±		703			847			757			825
SD		200.42			107.87			149.62			139.75
7. Grip strength (g)
Forelimb
Mean ±		936		978			1067+			1034+
SD		127.15		135.58			89.40			96.54
Hindlimb
Mean±		575				611			619			664+
SD		85.55				98.63			97.57			102. 05

SD Standard deviation

+ Significantly higher than the control group

- Significantly lower than the control group

 

Table 8: Summary of selected Histopathological findings

	males				females
Dose (mg/kg bw/day)	control	400	1600	64000	control	400	1600	64000
Number of animals examined	10	10/-	10/-	10	10	10/-	10/-	10
Stomach- cystic glands	0	-	-	1	0	-	-	0
Colon- parasites	0	-	-	1	0	-	-	0
Pancreas						-	-
- Atrophy	1	-	-	0	0	-	-	0
- Adipocytes	1	-	-	0	0	-	-	0
- Increased cytoplasmic eosinopholia	0	-	-	0	1	-	-	0
Liver
- Haemorrhage	0	-	-	1	0	-	-	0
- Lymphocytic infiltration	1	-	-	0	0	-	-	0
- Necrosis	0	-	-	2	0	-	-	0
- Necrotic foci	0	-	-	1	0	-	-	0
Lungs
- Perivascular lymphocytic infiltration	1	0	1	3	1	1	4	3
- Granulocytic infiltration	1	0	0	0	0	0	0	0
- Mineralisation blood vessels	0	1	1	2	3	2	1	2
- Chronic pneumonia	1	0	0	1	0	0	0	0
- Chronic pneumonic foci	0	0	0	0	0	0	1	0
- Pneumonic foci	0	0	1	1	0	0	1	2
- Osseous metaplasia	0	1	0	0	0	0	0	0
Heart- dilatation	0	1	-	0	0	-	-	0
Kidneys
- Mineralisation	0	-	1	0	0	0	0	1
- Dilatation of pelvis	2	-	1	0	0	2	1	0
- Basophilic tubules	4	-	1	2	0	0	0	0
- Proteinaceous material in tubules	2	-	0	2	0	0	0	0
- Hyaline droplets-tubular epithelium	7	-	0	7	0	0	0	0
Testes
- Atrophy-seminiferous	0	1	-	0	/	/	/	/
- Spermatic granuloma	0	1	-	0	/	/	/	/
Epididymis- Lymphocytic infiltration	1	-	-	0	/	/	/	/
Ovaries- Follicular cyst(s)	/	/	/	/	0	-	-	1
Uterus- Dilatation	/	/	/	/	3	-	-	2
Thyroids
- Ultimobronchial cyst	1	-	-	1	0	-	-	0
- Ectopic thymus	1	-	-	1	0	-	-	1
- Accessory parathyroid	0	-	-	1	0	-	-	0
Parathyroids– Connective tissue proliferation	1	-	-	0	0	-	-	0
Pituitary
- Cysts(s)	1	-	-	1	0	-	-	0
- Dilated Rathke’s cleft	4	-	-	5	2	-	-	2
- Eosinopohilic vacuoles	0	-	-	1	0	-	-	0
Adrenals– Accessory adrenal	0	-	-	0	1	-	-	1
Skin
- Epidermal hyperplasia	0	-	-	1	1	0	0	0
- Necrotising dermatitis	0	-	-	0	0	0	0	1
- Parakeratosis	0	-	-	0	1	0	0	1
- Epithelial hyperplasia	1	-	-	0	0	0	0	0
Thymus
- Hemorrhage	2	-	-	0	2	-	-	1
- Epithelial hyperplasia	0	-	-	0	0	-	-	1
Mammary gland
- Hyperplasia	/	/	/	/	0	-	-	1
- Lymphocytic infiltration/	/	/	/	/	0	-	-	1
Rectum- Parasites	1	-	-	1	1	-	-	0

 

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

04 July - 04 October 2012

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:

adopted 21 September 1998

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 6 August 2001

GLP compliance:

yes (incl. QA statement)

Remarks:

National GLP Compliance Monitoring Authority, Department of Science and Technology, New Delhi, India

Species:

rat

Strain:

Wistar

Remarks:

HsdHanTM : WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Department of Safety Assessment, Advinus Therapeutics Limited, Bangalore, India
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: 177.728 ± 13.465 - 180.922 ± 12.343 (males), 139.653 ± 11.413 - 141.666 ± 9.259 (females)
- Housing: in groups of 2 per sex per cage in solid floor standard polysulfone cages (Size: L 425 x B 266 x H 175 mm), with stainless steel top grill and Steam sterilized clean corn cob bedding. Polycarbonate Rat huts were provided to the animals as an environmental enrichment objects in the cages.
- Diet: ssniff rats/mice pellet food - maintenance meal (ssniff Spezialdiäten GmbH., Soest, Germany), ad libitum
- Water: deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier (Eureka Forbes Ltd., Mumbai, India), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 24
- Humidity (%): 57 - 68
- Air changes (per hr): 13.5 - 13.7
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 4 July 2012 To: 04 October 2012

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Milli-Q

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Approximately 50 mL dose formulations from all dose groups including control group prepared on one day prior to initiation of treatment, month 2 and 3 were analysed for protein content by Micro-Kjeldahl method. The determination of protein content in enzyme samples was carried out at Analytical R&D of Advinus Therapeutics Ltd. as follows: The total nitrogen content in the sample was determined by digesting the sample with sulphuric acid and digestion mixture. A known volume of digested sample was distilled by using Micro-Kjeldahl distillation unit and the total nitrogen content was calculated. The protein content was calculated from the total nitrogen content in the sample by multiplying the total nitrogen content with a factor of 6.25. Results of analysis showed that the mean concentrations were within ± 10 % variation against the nominal concentrations of 8, 24 and 80 mg TOS/mL.

Duration of treatment / exposure:

90 days

Frequency of treatment:

daily

Dose / conc.:

100 mg/kg bw/day (nominal)

Remarks:

Dose is given in TOS/kg bw/day
This dose level corresponds to 1251.5 mg enzyme preparation /kg bw/day.

Dose / conc.:

300 mg/kg bw/day (nominal)

Remarks:

Dose is given in TOS/kg bw/day
This dose level corresponds to 3754.6 mg enzyme preparation /kg bw/day.

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Dose is given in TOS/kg bw/day
This dose level corresponds to 12515.6 mg enzyme preparation /kg bw/day.

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose selection was based on a repeated dose (14-day) oral toxicity study by gavage in Wistar rats (Study No. G8309, conducted at Advinus), in which the highest dose of 1000 mg TOS/kg bw/day was used. The results of the 14 - day study were taken into account when selecting the dose levels for this 90 - day study.
- Rationale for animal assignment: randomly, The body weight stratification method using ProvantisTM software. was used. Rats with extreme body weights were excluded from the study.
- Fasting period before blood sampling for clinical biochemistry: overnight

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations: clinical signs of toxicity (once a day), pre-terminal deaths (twice a day)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to initiation of treatment (Day 1) and weekly (± 2 days) thereafter
- Parameters checked: changes in skin and fur, eyes, mucous membrane, occurrence of secretions and excretions, autonomic activity, changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies and bizarre behaviour.

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of treatment before test substance administration and weekly thereafter except for week 13 wherein the body weights were recorded on Day 5 of the week. Fasting body weights were recorded prior to necropsy on Day 91.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
Cage wise feed consumption was monitored at weekly intervals except on week 13 wherein the food output was measured on day 5 of the week.

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: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Day 5 of acclimatization period and on Day 89 of treatment period
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment period on Day 91
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, fasted overnight (water allowed)
- How many animals: all animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period on Day 91
- Animals fasted: Yes, fasted overnight (water allowed)
- How many animals: all animals
- Parameters checked in table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: prior to sacrifice.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table 2 were examined. In addition, urine was also subjected to microscopic examination for sediments such as crystals, epithelial cells and casts.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the treatment period (13th week of the treatment period)
- Dose groups that were examined: all dose groups
- Battery of functions tested: grip strength / motor activity / landing hind limb foot splay/ sensory activity / other: physiological observations, body weight
- For further details please refer to table 3 in the "Any other information on materials and methods incl. tables" section.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 4)

HISTOPATHOLOGY: Yes (see table 4, for preserved organs of vehicle control and high dose group animals histopathology examination was carried out)

Statistics:

Data which were captured using ProvantisTM, including, body weights, food consumption, organ weights were analysed using built-in statistical tests. Derived data like net body weight gain and organ weight ratios were also analysed using above mentioned methods. For experimental data collected without use of ProvantisTM, the statistical analysis was carried out using the validated package in Excel and/or using licensed copies of SYSTAT Statistical package Ver.12.0. For all quantitative variables like functional observation parameters and laboratory investigations (haematology, coagulation and clinical chemistry) data were tested for normality and homogeneity of variances within the group before performing a one-factor ANOVA modeling by treatment groups. The non-optimal (non-normal or heteroschedastic) data were transformed and ANOVA was performed. Comparison of means between treatment groups and control group was done using Dunnett’s test when the overall treatment, ‘F’ test was found significant. All analyses and comparisons were evaluated at the 5 % (P < 0.05) level. Statistically significant differences (P<0.05), indicated by the aforementioned tests were designated by the superscripts throughout the report as stated below: +/-: Significantly higher/lower than the vehicle control group

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day: statistically significant lower net weight gain was observed in males during the period 43-50 days.

300 mg/kg bw/day: statistically significant lower net weight gain was observed in males during the period 57-64 days and in females during the period 85-90 days.

1000 mg/kg bw/day: statistically significant higher net weight gain was observed in females during the period 78-85 days. Statistically significant lower net weight gain was observed in females during the period 85-90 days.

Except these changes no significant changes were observed in the mean body weights and net weight gains at any of the tested doses in either sex. The observed statistically significant difference in the net weight gains is marginal and transient, hence considered as an incidental finding. For details please refer to table 5 in the " Any other information on results incl. tables" section.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day: statistically significant increase in RBC in males was not dose-correlated and thus considered toxicologically insignificant.

1000 mg/kg bw/day: statistically significant increase in MPV in males were either of lower magnitude or non- progressive and/or within the physiological range and hence considered toxicologically insignificant. Statistically significant increase in PT value in females was considered incidental, as the change was comparable to normal biological variation observed in rats. For details please refer to table 6 in the " Any other information on results incl. tables" section.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

300 and 1000 mg/kg bw/day: minimal decrease in splenic weights noted in mid dose females (12% and 13%; absolute to body weight and relative to brain weight) and in high dose females(13%; relative to brain weight) were considered to be biologically insignificant, as they were not associated with correlating microscopic findings at high dose level.
For details please refer to table 7 in the " Any other information on results incl. tables" section.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

300 mg/kg bw/day: one female with cysts in the kidney was observed.
1000 mg/kg bw/day: dilated unilateral ureter, hepato-diaphragmatic nodule in the liver, cystys in the kidney and dilated pelvis were observed in one female, respectively.
For details please refer to table 8 in the " Any other information on results incl. tables" section.

Neuropathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: statistically significant lower fore limb grip strength values in high dose males were observed when compared to concurrent vehicle group. In the absence of dose correlation and associated changes in any of the related parameters, this change along with statistical significance observed in the motor activity score of males and females were considered as incidental findings. The observed gross lesions noted across dose levels involving liver (hepatodiaphragmatic nodule), kidneys (dilated pelvis; cyst) and ureter (dialted) were confirmed microscopically. These findings were considered to be incidental changes of spontaneous nature. All the other tissues were examined grossly and no gross abnormalities were observed for all the animals.

For details please refer to table 9 in the " Any other information on results incl. tables" section.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no microscopic changes attributed to treatment in males and females. The few microscopic findings observed in all animals were considered as incidental background findings.
For details please refer to table 10 in the " Any other information on results incl. tables" section.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no advers effect observed at this dose level

Remarks on result:

other: corresponding to 12515.6 mg enzyme preparation /kg bw/day

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

6 400 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises an adequate and reliable study (Klimisch score 2 due to read-across), due to read across from reference substances with similar structure and intrinsic properties. Read-across is justified based on common defining feature of two critically located acidic residues, which make up the catalytic machinery responsible for the cleavage of glycosidic bonds. Regardless of their specific substrate-pattern all enzymes are proteins, i.e. polymers comprised from 20 naturally occurring amino acids, linked by peptide bonds. Depending on the specific sequence of the individual amino acids (primary structure) and the physical-chemical interactions between amino acid residues of the polymer and the surrounding (aqueous) medium, three-dimensional globular protein structures are formed which are decisive for their enzymatic activity and specificity. The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

There are no experimental data available on oral repeated dose toxicity of β-fructofuranosidase (CAS 9001-57-4). It must be noted however that β-fructofuranosidase is produced by a classical strain of Saccharomyces cerevisiae, which has a long history of safe use in food applications globally.Read-across from two appropriate analogue substances, endoxylanase (CAS 9025-57-4) and alpha-amylase was conducted in accordance with Regulation (EC) No 1907/2006, Annex XI, 1.5 in order to fulfil the standard information requirements defined in Regulation (EC) No 1907/2006, Annex VII and VIII, 8.1. Common functional groups and structural similarities of the source and target substances are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

 

In a reliable 90-Day repeated dose toxicity study, enzyme preparation containing xylanase activity (CAS 9025-57-4) was administered to rats orally by gavage. The study was performed according to OECD TG 408 and in compliance with GLP (Krishnappa, 2002). In this study, rats (10/sex/group) were administered the test substance at 400, 1600 and 6400 mg/kg bw/day daily. Controls received double distilled water, which also served as the vehicle for the test substance. The concentration of the test item in the suspensions was determined thrice, at the start of the treatment and in the 2nd and 3rd month of the treatment period. The differences between the nominal and actual concentrations were ± 5%.

 

Examinations included clinical signs, mortality, changes in body weights, food consumption, ophthalmological analysis and neurobehavioural parameters (functional observation battery: grip strength/motor activity/sensory activity/other: visual response, auditory response, proprioceptive response, righting reflex). Haematology and clinical chemistry analysis were performed at the sacrifice. Furthermore, all rats were subjected to detailed necropsy. Histopathological evaluation was carried out on all the tissues and organs collected from control and high dose group rats, gross lesions from low, and mid dose groups at the discretion of the pathologist.

 

There were no treatment-related clinical signs and no deaths observed during the study period. Single incidences of alopecia occurred in single females at all dose levels and in controls and were thus considered incidental. Body weight and body weight gain did not differ between treated and control animals at all doses tested. An increase in food consumption in week 9 in both sexes of the mid dose group and a lower food intake in females of the same dose group in week 9 was considered incidental. At the 6400 mg/kg bw/day dose, lower food intake was observed in week 9 in both sexes and week 10 in males, only. Since the food intake was not consistently affected the effect on food consumption was considered an incidental finding.

 

Ophthalmological examinations did not reveal any treatment-related findings. Also, all haematological findings were not considered-treatment related. In detail, at the low dose, a higher level of mean corpuscular volume (MCV) was observed in females, but considered incidental as the changes were isolated findings. At mid dose, isolated incidences of a higher level of mean corpuscular haemoglobin (MCH), platelets, prothrombin time, neutrophils, and lower level of lymphocytes were observed in males. Since there were no effects observed at the high dose level, the observed effects were considered incidental. In males, a significantly higher level of mean corpuscular haemoglobin concentration (MCHC) was observed, but considered incidental as the corresponding changes were not observed in red blood corpuscles (RBC) and haemoglobin (Hb). In females, a higher incidence of increased mean corpuscular volume and a higher level of haematocrit were observed, but also considered incidental as the changes were isolated incidences. At high dose, a significantly higher level of MCHC was observed in males, but considered incidental as the corresponding changes were not observed in RBC and Hb. In females, a higher neutrophil percentage with lower lymphocyte percentage was observed compared to control, but considered incidental since they were within the range of biological variation.

 

Also, clinical chemistry analysis did not reveal any treatment-related findings. At the mid dose, the creatinine level was significantly higher in males. At the high dose, sodium, chloride and creatinine levels were significantly higher in males. Since the higher levels of chloride and sodium were within the normal range of variation, these findings were not considered to be treatment-related. Although the creatinine increase was dose-correlated, it was considered incidental as there were no corresponding histopathological changes in the kidneys.

 

At the mid dose, a significant decrease in the absolute and relative weight of ovaries was observed but not considered treatment-related as it was not observed at high dose and there were no corresponding gross and histopathological changes. Gross pathology revealed: dilatation of pelvis in kidneys in the control and the mid dose group, enlarged testes and heart at the low dose, skin alopecia single animals of in all dose groups and one high dose male showed a white liver focus. All findings were considered incidental since they were isolated findings or there was no dose-correlation visible.

 

Neurobehavioural examinations did not reveal any effects on motor activity, sensory activity, visual response, auditory response and righting reflex. At the low dose, lower grip strength values were observed in forelimbs in males, but considered incidental as these changes were isolated findings. At mid and high dose, a significantly higher incidence of landing foot splay was observed in males and females, but also considered incidental as there was no dose-dependency and no change in gait observed in these animals. In mid dose males, higher grip strength values were observed in hindlimbs but again considered incidental as these changes were isolated findings. In females, significantly higher grip strength values were observed in forelimbs at the mid and the high dose and hindlimbs at the high dose, but these were considered incidental findings since dose-correlation was not evident.

 

The frequently observed microscopic changes were perivascular lymphocytic infiltration (minimal to mild) and mineralisation of blood vessels (minimal) in the lungs. Both findings were observed in the control animals and at all dose levels. The incidence of perivascular lymphocytic infiltration in the lungs was not considered treatment-related as there was no intergroup statistical significance. The frequently observed findings in the kidneys were basophilic tubules (minimal) in control, mid and high dose groups, hyaline droplets in control and high dose groups and tubular epithelium (minimal to moderate) in control and high dose groups. The changes in kidneys were considered incidental, as the incidences were similar or higher in control. All other findings in any other organ or tissue were considered incidental since the incidences were similar or higher in the control group.

 

Taken together, the results of this study indicated that oral administration of enzyme preparation containing xylanase activity to Wistar rats at concentrations of 400, 1600 and 6400 mg/kg/day did not have any treatment-related effects on the above described parameters. As no changes of toxicological significance were noted in rats at any dose tested, the highest dose, 6400 mg/kg/day, was considered to be the no observed adverse effect level (NOAEL).

 

 

A further reliable 90-Day repeated dose toxicity study, enzyme preparation of bacillus subtilis containing alpha-amylase activity was administered to rats. The study was conducted according to OECD TG 408 and in compliance with GLP (Satish, 2013). In this study, rats (10/sex/group) were administered the test substance orally by gavage at 100, 300 and 1000 mg/kg bw/day. Controls received Milli-Q water, which also served as the vehicle for the test substance. The concentration of the test item in the suspensions was determined thrice, at the start of the treatment and in the 2nd and 3rd month of the treatment period. The differences between the nominal and actual concentrations were within ± 10% variation against the nominal concentrations of 8, 24 and 80 mg TOS/mL.

 

Examinations included clinical signs, mortality, changes in body weights and weight gains, food consumption, physical abnormalities, eye abnormalities and functional neurobehavioural parameters (functional observation battery: grip strength, motor activity, landing hind limb foot splay, sensory activity, physiological observations and body weight). Haematology, clinical chemistry analysis and urinalysis were performed at the sacrifice. Furthermore, all rats were subjected to detailed necropsy. Histopathological evaluation was carried out on all the tissues and organs collected from control and high dose group rats.

 

There were no treatment-related clinical signs and no deaths observed during the study period.

Single effects on body weight gain lower weight gain in males at the low dose (43-50days) and the mid dose (57-64 days), in females of the mid dose (85-90 days), higher weight gain in females (78-95 days) of the high dose and lower weight gain in males of the high dose (85-90 days) were observed. Since the observed statistically significant difference in the net weight gains were marginal and transient, they were considered as an incidental finding. Effects on food intake were not observed and also ophthalmological examinations did not reveal any treatment-related findings.

 

All observed haematological findings were not considered-treatment related. In detail, at the low dose, the statistically significant increase in RBC was not dose-correlated and thus considered toxicologically insignificant. At the high dose, the statistically significant increase in MPV in males were either of lower magnitude or non- progressive and/or within the physiological range and hence considered toxicologically insignificant. In the high dose females, the statistically significant increase in PT value was deemed to be incidental, as the change was comparable to the normal biological variation observed in rats.

 

Clinical chemistry analysis and urinalysis did not reveal any treatment-related findings.

 

At the mid dose, a minimal decrease in splenic weights was noted in females (12% absolute to body weight and 13% relative to brain weight) and in high dose females (13% relative to brain weight). As these observations were not associated with correlating microscopic findings at the high dose level, they were considered biologically insignificant. There were no effects on organ weights or organ body weight/brain weight ratio observed in any other organs.

 

Neurobehavioural examinations did not reveal any treatment related significant changes at any of the tested dose levels. Statistically significant lower forelimb grip strength values in high dose males were observed when compared to concurrent vehicle group. However in the absence of dose-correlation and associated changes in any of the related parameters, this change along with statistical significance observed in the motor activity score of males and females were considered as incidental findings.

 

There was no test item related gross changes observed in male and female rats. The observed gross lesions noted across dose levels involving liver (hepatodiaphragmatic nodule), kidneys (dilated pelvis; cyst) and ureter (dilated) were considered to be incidental changes of spontaneous nature. Histopathology did not reveal any microscopic changes attributed to treatment in males and females in any dose group. The few microscopic findings observed in all animals were considered as incidental background findings.

 

Taken together, the results of this study indicated that oral administration of enzyme preparation from Bacillus Subtilis containing alpha-amylase to rats at concentrations of 100, 300 and 1000 mg/kg/day did not have any treatment-related effects on the above described parameters. As no changes of toxicological significance were noted in rats at any dose tested, the highest dose, 1000 mg/kg/day, was considered to be the no observed adverse effect level (NOAEL).

Conclusion on repeated dose toxicity

The read-across approach is detailed in the analogue justification. The target and source substances are considered unlikely to differ in their repeated dose toxicity potential via the oral route. Since no adverse effects were observed in the study with the source substance endoxylanase (CAS 9025-57-4) a NOAEL of 6400 mg/kg bw/day was determined, the highest dose tested. Also in the study with the source substance enzyme preparation of Bacilis subtilis containg alpha amylase, no adverse effects were observed up to the highest dose tested and a NOAEL of 1000 mg/kg bw/day was determined. The target substance β-fructofuranosidase (CAS 9001-57-4) is not expected to differ in its repeated dose oral toxicity potential. CLP: not classified

 

Justification for classification or non-classification

Based on the available data, the registration substance is considered not to meet the classification criteria according to Regulation (EC) No. 1272/2008 (CLP).