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Administrative data

Description of key information

NOAELs for the test item, very similar analoguous enzymes and the test item in mixture with another enzyme are ≥ 1000 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
not specified
Remarks:
Not reported in this summary report
Limit test:
no
Specific details on test material used for the study:
- Name as used in study report: glucanase: Enzyme ZS
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
distilled
Analytical verification of doses or concentrations:
not specified
Duration of treatment / exposure:
90 days
Frequency of treatment:
daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
The standard set of endpoints as mentioned in the guideline was monitored in all animals, but ophthalmoscopy and histopathology in the control and high dose groups only.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
The results showed higher values for red blood cell parameters in the two highest dose group females. The small changes observed in the haematology were not considered to be toxicologically relevant.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
The results showed lower creatinine in treated males. The small changes observed in serum biochemistry were not considered to be toxicologically relevant.
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
In functional tests, females in the highest group showed occasionally decreased hind limb grip strength, and all treated males a reduction in overall activity.
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no

No conclusions presented on the relevance of the functional findings.

Endpoint:
repeated dose toxicity: oral, other
Remarks:
short-term and long-term (up to subchronic) repeated dose toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Please refer to the read-across justification attached to section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOAEL
Remarks:
rat, 90day, gavage
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
quivalent to 83.7 mg TOS/kg bw/d
Sex:
male/female
Basis for effect level:
other: No treatment related adverse effects observed
Remarks on result:
other: Result read-across Penicilium funiculosum concentrated liquid
Dose descriptor:
LOAEL
Remarks:
rat, 5day, gavage
Effect level:
790 other: mg TOS/kg bw/d
Based on:
other: total organic solids
Sex:
female
Basis for effect level:
histopathology: non-neoplastic
Remarks on result:
other: Result read-across BD5088 alpha amylase
Dose descriptor:
NOAEL
Remarks:
rat, 14day, gavage
Effect level:
710 other: mg TOS/kg/d
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Remarks on result:
other: Result read-across BD5088 alpha amylase
Dose descriptor:
NOAEL
Remarks:
rat, 14day, gavage
Effect level:
2 200 other: mg TOS/kg/d
Based on:
test mat.
Sex:
female
Basis for effect level:
other: no treatment related effects observed
Remarks on result:
other: Result read-across BD5088 alpha amylase
Dose descriptor:
NOAEL
Remarks:
rat, 90day, gavage
Effect level:
890 other: mg TOS/kg/d
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse treatment related effects observed
Remarks on result:
other: Result read-across BD5088 alpha amylase
Dose descriptor:
NOAEL
Remarks:
rat, 14day, feed
Effect level:
360 other: mg TOS/kg/d
Based on:
test mat.
Sex:
male
Basis for effect level:
other: No adverse effects observed
Remarks on result:
other: Result read-across BD5088 alpha amylase
Dose descriptor:
NOAEL
Remarks:
rat, 14day, feed
Effect level:
310 other: mg TOS/kg/d
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No adverse effects observed
Remarks on result:
other: Result read-across BD5088 alpha amylase
Dose descriptor:
NOAEL
Remarks:
rat, 90day, feed
Effect level:
50 000 mg/kg diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment related adverse effects observed
Remarks on result:
other: Result read-across Amylofeed
Dose descriptor:
NOAEL
Remarks:
rat, 90day, gavage
Effect level:
>= 10 other: mL/kg/d
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects observed
Remarks on result:
other: Result read-across Ultraflo
Dose descriptor:
NOAEL
Remarks:
rat, 90day, feed
Effect level:
50 000 ppm
Based on:
test mat.
Remarks:
equivalent to 3371 mg/kg bw/d for males and 3836 mg/kg bw/d for females
Sex:
male/female
Basis for effect level:
other: No treatment related adverse effects observed
Remarks on result:
other: Result read-across Cellulase CP
Dose descriptor:
NOAEL
Remarks:
rat, 90day, gavage
Effect level:
>= 1 600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Remarks:
corresponding to 84.8 mg TOS/kg bw/day
Sex:
male/female
Basis for effect level:
other: No treatment related adverse effects observed
Remarks on result:
other: Result read-across beta-glucanase, celluclase and xylanase
Dose descriptor:
NOAEL
Remarks:
pig, 60day, feed
Effect level:
>= 300 000 other: U glucanase/kg feed and 220000 U xylanase/kg feed
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No treatment related adverse effects observed
Remarks on result:
other: Result read-across Rovabio
Dose descriptor:
NOAEL
Remarks:
rat, 90day, gavage
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment related effects of toxicological importance
Remarks on result:
other: Result read-across beta-glucanase and xylanase
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
food efficiency
gross pathology
haematology
mortality
organ weights and organ / body weight ratios
Remarks on result:
other: based on Read across VR002 Pyrolase HT Cellulase (CAS 9012-54-8)
Dose descriptor:
NOAEL
Effect level:
5 other: % (w/w)
Based on:
test mat.
Sex:
not specified
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
based on Read-across Endo-1,4-β-D-glucanase (CAS 9012-54-8)
Dose descriptor:
NOAEL
Effect level:
>= 2.8 other: g/kg bw/day
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
based on Read-across Endo-1,4-β-D-glucanase (CAS 9012-54-8)
Dose descriptor:
NOAEL
Effect level:
1 152.8 mg/kg diet
Based on:
test mat.
Sex:
male
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
based on Read-across Natugrain TS
Dose descriptor:
NOAEL
Effect level:
1 336.3 mg/kg diet
Based on:
test mat.
Sex:
female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
based on Read-across Natugrain TS
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Remarks:
Based on Read-across Hostazym
Key result
Critical effects observed:
no
Critical effects observed:
yes
Lowest effective dose / conc.:
790 other: mg TOS/kg bw/d
System:
gastrointestinal tract
Organ:
rectum
Treatment related:
not specified
Dose response relationship:
not specified
Relevant for humans:
not specified
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
There is a large number of studies of longer and shorter duration, either via oral gavage of feed with no treatment related effects at doses of 1000 mg/kg bw/day and higher.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

There is one, very briefly reported, 90-day oral gavage study in rats for the test substance itself. Therefore, read-across is also done to a substantial number of other repeated dose oral studies in rats (and one in pigs) for very similar analogous enzyme preparations.

 

In a study, reported to be done on ‘glucanase’ and assumed to be the test substance, rats were exposed via oral gavage to 50, 250 and 1000 mg/kg bw/day for 90 days in accordance to OECD TG 408 (Efsa, 2011). Some higher values for red blood cell parameters in the two highest dose groups of females and lower creatinine values in treated males were not considered toxicologically relevant. In functional tests, females in the highest group showed occasionally decreased hind limb grip strength, and all treated males a reduction in overall activity. The relevance of this finding is not discussed in the study report and no conclusions on NOAELs are reported.

 

In a Notification for BD5088 alpha-amylase enzyme preparation derived from Pseudomonas flourescens Biovar I, a number of repeated dose oral studies on rats is briefly reported (Innovase, 2003). Dose levels are described in units of total organic solids (TOS) and the preparation is reported to contain approximately 3%. A five-day oral gavage study resulted in a LOAEL of 790 mg TOS/kg bw/day, which is equivalent to approximately 26,300 mg preparation/kg bw/day. A 14-day oral gavage study and a 90-day oral gavage study resulted in NOAELs of above 710 mg TOS/kg bw/day and higher, which is equivalent to approximately 23,700 mg preparation/kg bw/day. Effects seen in the 5-day study included some microscopic alterations in the intestinal tract, most prominently in the rectum and some effects on nasal tissues / turbinates that were considered to be caused by reflux of the test material during gavage. In the 14-day study some effects on feed consumption, body weight and relative organ weight were seen as well as isolated observations of noisy respiration, that was probably caused by regurgitation of the test substance. Slight haematological effects were also seen. Effects in the 90-day study also included probable effects related to gavage, caused by reflux. Although some rats from all treatment groups had a degree of local respiratory tract inflammation, there were no adverse direct systemic effects considered to be related to the oral route of administration and therefore the NOAEL was concluded to be the top dose in the study (890 mg TOS/kg bw/day). To study the effects of the preparation without side-effects of gavage and reflux, a 14-day follow-up feed study was done. In this study, with 360 (males) and 301 (females) mg TOS/kg bw/day, no treatment-related adverse effects were observed.

 

In a 90-day oral feed study with rats, doses of ‘Amylofeed’, which consists of mainly GBR 50010 (endo-1,3(4)-beta-glucanase), endo-1,4-beta-xylanase and alpha-amylase, no treatment-related adverse effects were observed up to the maximum dose (Efsa, 2013). Dose levels were 500, 5000 and 50,000 mg preparation/kg diet. Some transitory effects were observed in body weight gain and some changes in haematological parameters in treated groups, compared to controls, but these were within normal physiological range. There were also minor statistical differences in relative organ weights of no toxicological relevance. The NOAEL was concluded to be 50,000 mg/kg diet, which corresponds to approximately 3371 mg preparation/kg bw/day.

 

In a 90-day oral gavage study, according OECD TG 408, groups of ten male and ten female Wistar rats received Ultraflo at dosages of 1, 3.3 or 10.0 mL/kg/day (equivalent to 0.05, 0.17 or 0.51 g TOS/kg/day) (Novozymes, 2006). Ultraflo is a liquid enzyme concentrate. It is a multi-component enzyme preparation consisting of is a fungal xylanase (EC 3.2.1.8), an endo-cellulase, (EC 3.2.1.4, which is the target substance) and a beta-glucanase (EC 3.2.1.6). There were no treatment-related signs and no animals died. Weight gains, food intake and food conversion efficiencies were unaffected by treatment. There were no treatment-related ocular lesions. There were no haematological or biochemical findings related to treatment. Analysis of organ weights and the macroscopic examinations did not identify any treatment-related change. The histopathological examinations did not identify any histological alterations related to treatment. The NOAEL was concluded to be 10 mL/kg bw/day, equivalent to 510 mg TOS/kg/day.

 

In a Notification for a cellulase enzyme preparation derived from Penicillium funiculosum, two 90-day oral studies with rats are reported (Dupont, 2015). The notified product is very similar to the target substance or perhaps it is the target substance. It is reported to be endo-1,4-beta-glucanase with CAS-number 62213-14-3 and enzyme number 3.2.1.6. However, this CAS-number and enzyme number are generally used for endo-1,3-beta-glucanase. The first study is an oral gavage study, in accordance with OECD TG 408, with a cellulase preparation called “Penicillium funiculosum concentrated liquid”. Groups of 10 Sprague Dawley rats/sex each received daily 0, 7.5, 37.5 or 75 mg total protein/kg body weight in a constant aqueous volume of 5 mL/kg body weight via oral gavage. These doses corresponded to, respectively, 0, 50, 250 or 1000 mg of test material/kg/day in a constant volume of 5 mL/kg. There were no treatment-related deaths in this study. There were no biological or statistical differences between the control and treated groups with respect to clinical observations, feed consumption, body weight, body weight gain, haematology, clinical chemistry and ophthalmologic examinations. The relative brain weight in high dose group females was marginally increased but was of questionable toxicological significance in the absence of histopathologic findings. There were no treatment-related changes in histopathology. All morphological changes were those commonly observed in laboratory rats of the age and strain employed. The NOAEL is established at greater than 1000 mg of Penicillium funiculosum concentrated liquid /kg body weight/day. This is equivalent to 83.7 mg TOS/kg bw/day.

The second study is a feeding study with Cellulase CP from Penicillium funiculosum Strain 3094. Groups of 15 CD rats/sex each were provided with a diet containing 0, 2000, 10,000 or 50,000 ppm of Cellulase CP. These doses corresponded to, respectively, 0, 50, 250 or 1000 mg/kg/day in a constant volume of 5 mL/kg. There were no treatment-related deaths in this study. There were no biological or statistical differences between the control and treated groups with respect to clinical observations, feed consumption, body weight, body weight gain, haematology, clinical chemistry and ophthalmologic examinations. During week the study, some changes in haematological parameters were seen, but no differences were noted at study termination. There were slight increases in spleen weights noted in treated males. However, these values were still within the historical control values for CD rats and in the absence of similar effects in females, the findings were not considered as treatment-related. The blood glucose levels decreased in high dose females at weeks 6 and 12 and in high dose males at week 6 but all values were still within the historical control ranges for blood glucose in CD rats. No treatment-related changes in histopathology were found. All morphological changes were those commonly observed in laboratory rats of the age and strain employed. Under the conditions of this assay, it can be concluded that feeding of Cellulase CP in the diet for 90 continuous days did not result in systemic toxicity to rats. The NOAEL is established at 50,000 ppm (3371 mg/kg/day for males; 3836 mg/kg/day for females).

 

A 90-day oral gavage study with rats, in accordance to internationally accepted guidelines is reported for a preparation with as main enzymes beta-glucanase (endo-1,3-beta-glucanase), cellulase (the target substance: endo-1,4-beta-glucanase) and xylanase (endo-1,4-xylanase) (DSM, 2013). The dosages were at 100, 400 or 1600 mg/kg body weight/day. Treatment did not result in any treatment-related deaths. There were no general clinical signs associated with treatment, organ weights were not affected and ophthalmoscopic, macroscopic and microscopic examination did not reveal any abnormalities associated with the administration of the test material. The males receiving 1600 mg/kg bw/day had a slightly lower overall bodyweight gains than the Controls, and they ate 4 to 12 g food/animal/week less than the control, but these effects were not significant. When compared with the Controls, slightly longer prothrombin times (0.7 seconds longer) were recorded for both sexes at the highest dose with slightly longer activated partial thromboplastin times (3.6 seconds longer) also seen in the females. Changes in clotting times may be related to changes in hepatic metabolism. However, given that there are no macroscopic or microscopic treatment-related findings recorded in the livers of these animals and that the liver weights are unremarkable, these slight changes in haematology are of only minor importance. These effects resulted in no overt clinical manifestations such as premature death or anatomic abnormalities. Slightly low aspartate amino-transferase activities were observed in females at the highest dose. High levels of aspartate amino-transferase activity are markers of liver damage in rats, low activity levels are considered to be of no toxicological significance. The NOAEL for this study is therefore considered to be 1,600 mg/kg bw/day, corresponding to 84.8 mg TOS/kg bw/day.

 

In an oral gavage study, according to OECD TG 408 (90-days) with rats and a preparation named beta-glucanase BglS (main enzyme action: endo-1,3-beta-glucanase), no adverse effects were observed at all doses, up to 1000 mg TOS/kg bw/day, which is approximately equivalent to 9800 mg/kg bw/day preparation (Dupont, 2015). Groups of 10 CD rats per sex were treated with 0 (0.9% saline), 100, 300 or 1000 mg TOS/kg bw/day. The dosing volume was 5 mL/kg bw/day. No treatment-related deaths were noted during the 13-week period. There were no treatment-related changes in body weights, feed consumption and water intake. Haematology and clinical chemistry conducted after 13 weeks of treatment did not reveal any adverse effects. There were no biological or statistical differences between the control and treated groups with respect to clinical observation, feed consumption, water consumption, ophthalmologic examinations, body weights, and body weight gains. There were no treatment-related changes in haematology and clinical chemistry at the end of week 13. There were no differences in the functional observation battery, grip strength and locomotor activity assays between treated and control animals. At necropsy, there were no treatment related findings on organ weights, macroscopic findings and histopathologic examinations. All microscopic findings were considered to be within the background incidence of findings reported in this age and strain of laboratory animals. Under the conditions of this assay, the NOAEL is established at the highest dose tested, 1000 mg TOS/kg bw/day.

 

An oral feed tolerance study was performed in pregnant and lactating pigs (sows) with a preparation of endo-1,3(4)-beta-glucanase and endo-1,4-beta-xylanase produced from Penicillium funiculosum (Efsa, 2014). The study is a tolerance study in 40 pigs (sows), which were dosed with a preparation called Rovabio Excel AP, at dose levels of 3000/2200 U glucanase/xylanase/kg feed (2 times the recommended dose) or 300,000/220,000 U glucanase/xylanase/kg feed (200 times the recommended dose) for approximately 60 days (Efsa, 2014). Three sows had to be removed from the study, due to the need for antibiotic treatment, not related to the treatment with the test substance. Feed intake of the sows during lactation (average 5.1 kg/day), sows’ body weight at farrowing and at weaning, sow’s body weight changes (gestation and lactation) and the litter’s performance were not significantly different between treatments. Sows’ body condition score and back-fat loss during lactation was not modified by the treatments; however, the control group had initial values that were lower than those of the other two treatment groups. Haematological profile and biochemical blood parameters of the sows at weaning were, in general, not different between the treatments. The only modification found was in the calcium concentration, which was higher in the sows receiving 200 the recommended dose than in those receiving the control diet, but these values remained within the physiological range for sows. Based on these findings, a NOAEL can be set at 300,000/220,000 U glucanase/xylanase/kg feed, which roughly corresponds to 1700 mg preparation/kg bw/day.

 

An oral gavage study, according to OECD TG 408 (90-days) was done in rats with a preparation of endo-1,3-beta-glucanase and endo-1,4-beta-xylanase (DSM, 2013). 20 male and 20 female Wistar rats, dosed by oral gavage at dosages of 65, 250 and 1000 mg/kg body weight/day (volume-dosage of 10 mL/kg body weight/day) (DSM, 2013). No animals died during the treatment period. The appearance and behaviour of the animals was considered to be unaffected by treatment. In the last three weeks of treatment, salivation was seen, following dosing, in several animals receiving 1,000 mg/kg bw/day. Body weight gains, food consumption and food conversion efficiencies were unaffected by treatment. No treatment-related ophthalmoscopic abnormalities were detected. Haematological examination revealed slightly short prothrombin times in males receiving 1000 mg/kg bw/day when compared with the Controls. It is important to note that no macroscopic or microscopic liver abnormalities were noted, suggesting these findings are of minor significance. Changes in blood chemistry that were attributed to treatment comprised slightly low alanine aminotransferase activities and urea concentrations in females receiving 250 or 1,000 mg/kg bw/day with triglyceride concentrations also slightly high in females receiving the highest dosage. Slightly high bodyweight-relative kidney weights were recorded for males which had received 250 or 1000 mg/kg bw/day and the bodyweight-relative ovary weights were slightly high in females which had received 1000 mg/kg bw/day, when compared with the Controls. There were no macroscopic or microscopic findings which were considered to be related to treatment. It was concluded, that the NOAEL was 1000 mg/kg bw/day, corresponding with 199 mg TOS/kg bw/day.

 

An oral gavage study, according to GLP Guidelines, was performed in rats with VR002 Pyrolase HT Cellulase (DSM, 2013). 20 male and 20 female Wistar rats, dosed by oral gavage at concentrations of 250, 500 and 1000 mg/kg body weight/day. The dose volume was 10 mL/kg (BASFSE, 2014). The rats were administered once daily for a minimum of 14 consecutive days. Mortality and clinical observations were evaluated daily. Body weights and food consumption were recorded weekly. Blood for evaluation of hematology, coagulation and clinical chemistry was collected on Days 15. All surviving animals were sacrificed on Day 15. Selected tissues were harvested at necropsy and selected organs weighed. Histopathology was not performed. Tissues were saved for possible future histopathology. There were no early deaths during the study. There were no test article-related clinical signs of toxicity noted during the 14 day treatment period. There were no test article-related effects on body weights, body weight gains or food consumption noted during the study. There were no test article-related effects on hematology, coagulation or clinical chemistry parameters or erythrocyte morphology as determined on Day 15. There were no gross findings noted at necropsy. There were no test article-related changes in absolute or relative to body- or brain-weight ratios. Based on the findings in this study, the No Observed Effect Level (NOEL) following administration of 250, 500, or 1000 mg/kg VR002 once daily by oral gavage to Sprague-Dawley rats for 14 days was at least 1000 mg/kg.

 

A 14-Day Range Finding Study with the objective of providing data on the acceptability of the Cellulase (endo-1,4 -ß-D-glucanase) in rats upon administration via the diet for 14 consecutive days was performed (DYADIC, 2009). Clinical signs, growth, food consumption, food conversion efficiency, organ weights, and gross examination at necropsy were used as criteria for assessment of palatability and/or disclosing possible adverse effects. The results of the study showed that treatment with the cellulase enzyme test material via the diet up to a level of 5% (w/w) for a period of 14 days was well tolerated and did not result in any adverse effects.

 

A 13 -week oral toxicity study was performed with the Cellulase (endo-1,4 -ß-D-glucanase) (DYADIC, 2009). The oral toxicity of the cellulase test preparation was examined in a 13-week subchronic oral toxicity study with four groups of 20 male and 20 female rats. The test substance was incorporated in the feed at levels of 0% (control), 1%, 2% and 4%. Results of the study indicate that there were no treatment related clinical signs. The results of neurobehavioral observations and motor activity assessments did not indicate any neurotoxic potential of the test substance. Ophthalmoscopic examination did not reveal any treatment related changes. Growth, overall food intake and food conversion efficiency were not affected by treatment. Red blood cell variables, total white blood cell count and clotting potential were not affected by the treatment. There were no noticeable changes in clinical chemistry parameters as determined in 10 rats/sex/group at necropsy. Urinalysis, conducted in 10 rats/sex/group in week 13 of the study, did not reveal any treatment-related changes in renal concentrating ability, semi-quantitative urinary measurements or in microscopy of the urinary sediment. There were no significant changes in absolute or relative organ weights among the groups. Macroscopic examination at necropsy and microscopic examination did not reveal adverse histopathological changes. The no-observed-adverse-effect level (NOAEL) in this study was placed at the highest dose level applied, 4% in the diet (equivalent to 2.8 g/kg/body weight/day).

 

An additional 13-week oral toxicity study was performed with the Hostazym C (endo-1,4 -beta-glucanase) in Sprague–Dawley rats following administration by gavage, in accordance with OECD Guideline 408 (1983)(EFSA, 2013). Four treatment groups (10 per sex per group) were treated daily with 0, 100, 300 or 1000 mg/kg bw in glycerine/water (50:50 weight/volume). Clinical condition, food and water consumption and body weight were regularly monitored throughout the study. Haematological and clinical biochemical investigations were undertaken. At the end of the study all the animals were subjected to gross necropsy. Histopathological examination of the organs, as required by the mentioned OECD guideline, was conducted in rats in the control and high-dose groups.

An equivocal decrease in weight gain in female animals treated with 300 or 1000 mg/kg bw per day, compared with control rats, was observed over weeks 0–13, but statistically significant differences in group mean body weights in females were sporadic and the difference in total weight gain over the dosing period was not statistically significant. Moreover, no effect on body weight was seen in males. Therefore, this observation was considered to be unrelated to the administration of Hostazym C. In female animals receiving 100 mg or 1000 mg/kg bw per day, absolute adrenal weight was higher than in control rats. However, as no similar findings were seen in male animals and no histological differences were reported, this was considered unlikely to be a result of treatment with Hostazym C. It was concluded from the observations, laboratory investigations and terminal studies that administration of Hostazym C, by oral gavage, to Sprague–Dawley rats for 13 weeks at levels up to 1000 mg/kg bw per day produced no signs of toxicity attributable to administration of the test item.

 

A 90-day oral toxicity study was conducted with Natugrain TS (BASFSE, 2006), a preparation containing the test item in a mixture with xylanase. Groups of ten Wistar rats per sex per group were given daily 0, 1000, 4000 or 16000 mg/kg diet over a period of 3 months. The study was conducted in accordance with the current OECD guideline 408. Food consumption, water consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter. A functional observational battery (FOB) and measurement of motor activity was carried out after 88 resp. 90 days of treatment in males and 89 resp. 91 days of treatment in females. Clinicochemical, hematological examinations and urinalyses were performed towards the end of the administration period. Ophthalmological examinations were performed before and towards the end of the administration period. All animals were assessed by gross pathology, followed by histopathological examinations.

The oral administration of Natugrain TS - SD powder via the diet over a period of 3 months caused no substance-related adverse effects. Therefore, the no observed adverse effect level (NOAEL) under the conditions of this study was16000 ppm in male (about 1152.8 mg/kg body weight/day) and female (about 1336.3 mg/kg body weight/day) Wistar rats.

Justification for classification or non-classification

Based on the available information classification for repeated dose toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008.