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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
March 01, 2001 - October 18, 2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Due to the similarity between the two enzymes, same toxicological outcome is expected for pectate lyase.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
Adopted on 21 September 1998.
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Active enzyme protein of Pectin lyase (EC no. 232-894-5, CAS no.9033-35-6, EC name (1->4)-6-O-methyl-alpha-D-galacturonan lyase , Enzyme class no 4.2.2.10 )
Molecular formula:
n.a.
IUPAC Name:
Active enzyme protein of Pectin lyase (EC no. 232-894-5, CAS no.9033-35-6, EC name (1->4)-6-O-methyl-alpha-D-galacturonan lyase , Enzyme class no 4.2.2.10 )
Constituent 2
Reference substance name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available
IUPAC Name:
Protein as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 3
Reference substance name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Carbohydrates constituent of enzyme deriving from the fermentation or extraction process
Constituent 4
Reference substance name:
Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process
Constituent 5
Reference substance name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Molecular formula:
Not available. See remarks.
IUPAC Name:
Lipids as a constituent of enzyme deriving from the fermentation or extraction process
Test material form:
liquid
Details on test material:
- Lot/batch No.: PPJ 6991
- Expiration date of the lot/batch: 11 December 2010

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: SPF Sprague Dawley rats of the stock Mol:SPRD from M&B A/S, Ejby, Denmark
- Age (at delivery): 5-6 weeks
- Body Weight Range (at acclimatization): 83-100 g
- Housing: The rats were housed in transparent polycarbonate cages (macrolone type III, floor area: 810 cm2) with two animals in each cage, males and females separated. The cages were cleaned and the bedding changed at least twice per week.
- Diet (e.g. ad libitum): A complete pelleted rodent diet “Altromin 1324” was available ad libitum
- Water (e.g. ad libitum): Acidified (to pH 2.5) domestic quality drinking water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 3 °C
- Humidity (%): 55 ± 15%
- Air changes: 10 air changes per hour.
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hours light

IN-LIFE DATES: Animals arrived on 05 March 2001. Treatment started on 12 March 2001. In-life phase ended on 14 June 2001.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Rate of preparation of diet (frequency): Test material (pectin lyase) was prepared daily by diluting test material in tap water. Test material was kept frozen until use.
- Concentration in vehicle: 0, 10, 33 and 100% (v/v), corresponding to a dose of 0, 210, 693 and 2101 mg enzyme concentrate dry matter/kg body weight.
- Amount of vehicle (if gavage): constant volume 8 mL/kg body weight.
- Purity: Water for formulation
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose samples were analysed according to GLP. Before start of treatment and during weeks I and 13, triplicate (3) samples (10 ml each) from each dose formulation (groups 1-4) were collected pre-dose and sent to Novozymes A/S for analysis. The activity of test material was as expected (recovery range 98.3 - 111.1%).



Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Dose / conc.:
210 other: mg enzyme concentrate dry matter/kg bw/day
Dose / conc.:
693 other: mg enzyme concentrate dry matter/kg bw/day
Dose / conc.:
2 101 other: mg enzyme concentrate dry matter/kg bw/day
No. of animals per sex per dose:
10 female and 10 male rats
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The doses used in this study were selected on the basis of results from a 14-days dose range finding study performed on other similar enzyme preparations, where the highest dose (8 mL/kg/day) corresponding to 2101 mg enzyme concentrate dry matter/kg bw did not result in treatment related effects.
The lower doses were selected using an approximate ratio of 3.3 between doses.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
All visible signs of ill health and any behavioral changes were recorded daily. An additional morbidity/mortality check was performed in the afternoon.

DETAILED CLINICAL OBSERVATIONS: Yes, detailed clinical observations were performed outside the home cage once a week at similar times.

BODY WEIGHT: Yes
Animals were weighed on the day of arrival, on the first day of treatment (Day 1) and weekly thereafter and at necropsy.

FOOD CONSUMPTION: Yes
The consumption of food was recorded weekly for each cage

OPHTHALMOSCOPIC EXAMINATION: Yes. Before treatment started. Before termination of treatment, animals in group 1 and 4 were re-examined.

HEMATOLOGY: Yes
- Time schedule for collection of blood: Week 14
- Anesthetic used for blood collection: Yes, CO2
- Animals fasted: Not specified
- How many animals: From all animals (80)
- Parameters checked:
Haemoglobin (Hb), Red blood cell count (RBC), Haematocrit (HT), Mean cell volume (MCV), Mean cell haemoglobin (MCH), Mean cell haemoglobin concentration (MCHC), White blood cell count (WBC), Differential leucocyte count (NEUTRO, LYMPHO, EOS, BASO, MONO), Platelet count (Plt)
Prothrombim time (Pt), Fibrinogen (Fib)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 14
- Anesthetic used for blood collection: Yes, CO2
- Animals fasted: No
- How many animals: From all animals
- Parameters checked:
Alanine aminotransferase (ALAT), Aspartate aminotransferase (ASAT), Alkaline phosphatase (ALKPH), Bilirubin (total) (BILI), Gamma-glutamyltransferase (GGT), Cholesterol (CHOL), Triglycerides (TRIG), Carbamide (UREA), Creatinine (CREAT), Glucose (GLUC) (whole blood), Sodium (Na), Potassium (K), Calcium (Ca), Magnesium (Mg), Inorganic phosphorus (P), Chloride (Cl), Protein (total), Protein electrophoresis (ALB, ALPHA1, APLHA2, BETA, GAMMA), Globulin
Albumin/Globulin (ALB/G) ratio

URINALYSIS: Yes
- Time schedule for collection of urine: In week 13
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes. only water was available.
Parameters checked:
Creatinine (CREAT), N-acetyl-β-D-glucosaminase (NAG), Gamma-glutamyltransferase (GGT), Specific gravity (SG), pH, Colour, Protein, Leucocytes (LEUC), Nitrite, Blood, Glucose, Ketones, Bilirubin (BILI), Urobilinogen (UROBIL)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Before termination of study
- Dose groups that were examined: All animals (80)
- Battery of functions tested: Reactivity to different types of stimuli (e.g. auditory, visual, tactile), Grip strength, Motor activity
Sacrifice and pathology:
At the end of the treatment period all animals were weighed, examined externally, anaesthetised with an i.p. injection of a barbiturate and killed by exsanguination. All animals were necropsied.
Gross pathology was performed and histopathology was performed on organs and tissue from the control group (Group 1) and the high dose group (Group 4).
Other examinations:
Weight of individual organs: yes
Statistics:
For all tests, the level of significance was defined as p<0.05.
The statistical analyses were made with SAS® procedures (version 8.1) described in "SAS/STAT® User's Guide, SAS OnlineDoc®, 1999, SAS Institute Inc., Cary, North Carolina 27513, USA and StatXact 4TM procedures described in StatXact 4 for Windows User Manual, 1998, Cytel Software Corporation, Cambridge, MA 02139, USA
Data were processed to give group mean values and standard deviations where appropriate.
Thereafter, each continuous variable was tested for homogeneity of variance with Barlett's test. If the variance was homogeneous, analysis of variance was carried out for the variable. If any significant differences were detected, possible intergroup differences were assessed with Dunnett's test. If the variance was heterogeneous, each variable was tested for normality by the Shapiro-Wilk method. In case of normal distribution, possible inter-group differences were identified with Student's t-test. Otherwise, the possible inter-group differences were assessed by Kruskal-Wallis's test. If any significant inter-group differences were detected, the subsequent identification of the groups was carried out with Wilcoxon Rank-Sum test. Ranked type of urinalysis data was analysed with Wilcoxon Rank-Sum test.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
During the daily observations, no clinical signs were seen that could be related to treatment with the test article. No significant differences were seen between groups in the open field test. No significant differences were seen between groups in the stimuli-induced clinical observations.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Animal no. 9 (group 1, male) was found dead on day 18 of the study. The necropsy showed oesophageal perforation. Microscopic findings consisted of moderate focal mucosal ulceration extending into the muscular layer and adventitia in oesophagus accompanied by moderate focal acute peri/epicarditis of heart, and slight focal acute pleuritis/subpleuritis of lungs.

Animal No 62 (group 4) was found dead on test day 19. The necropsy showed oesophageal perforation. Microscopic findings consisted of marked diffuse acute inflammation in the adventitia of oesophagus, accompanied by slight focal acute peri/epicarditis of heart, and pleuritis/subpleuritis of lungs. These acute inflammatory changes in the organs of the thorax cavity are consistent with oesophageal perforation. Mucosal damage of oesophagus would also be an expected microscopic finding, however perforation site was not observed microscopically in this case.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no clear effects of treatment on body weights.
The very slightly lower weight gain for the group 3 males from day 22 onwards was due almost entirely to one rat (No 49) that gained consistently less weight during this time (it is not possible to comment on this individual's food consumption because the animals were housed in pairs). As there was no effect in the group 4 animals, this one case is considered to be fortuitous.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no clear effects of treatment on food consumption.
There were some slight differences between treated groups and controls and, on isolated occasions, these were statistically significant (males of groups 2 and 4 in week 4; males of group 3 in week 11, females of group 2 and 4 in week 13). In the males, group 3 was associated with lower values from week 2 onwards; this was largely attributable to cage 23, though its occupants (Nos 45 and 46) gained weight normally. In the females, all treated groups tended to have slightly lower values than controls, though there was no indication of any dose-response relationship. The overall conclusion is that these minor differences are fortuitous.
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
not specified
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no findings that could be related to treatment.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no significant differences between treated and control groups in any of the haematological parameters investigated, except for a significantly higher mean cell volume of group 3 females erythrocytes. This finding was considered incidental as no dose dependency was present and as there were no significant findings in the males.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no findings that could be related to treatment with the test article.
The following significant observations were considered incidental as no dose dependency was present and as no significant findings were made in the males: 1) the alanine aminotransferase activity of females of group 4 was significantly lower than that of the control group; 2) the aspartate amino-transferase activity of fema_les of group 3 was significantly higher than that of the control group; 3) the albumin concentration of females of group 3 was significantly higher than that of the control group.
Urinalysis findings:
no effects observed
Description (incidence and severity):
There were no significant differences between treated and control groups in any of the urine parameters investigated.
Marked amounts of crystals and bacteria were seen in some of the urine samples from males and females from all groups. This is a normal finding in urine samples from rats.
Behaviour (functional findings):
no effects observed
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no significant differences in any organ weights (absolute or relative) between the treated and the control groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes were observed in the necropsy. The macroscopic changes recorded in some organs are considered typical/incidental findings for rats of this strain and age.
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOEL
Effect level:
>= 2 101 mg/kg bw/day (nominal)
Based on:
other: mg enzyme concentrate dry matter/kg bw
Sex:
male/female
Basis for effect level:
other: There were no adverse effects observed at the highest does tested.
Remarks on result:
other:
Remarks:
There were no adverse effects observed at the highest does tested.

Target system / organ toxicity

Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Oral administration (gavage) of pectin lyase to Sprague-Dawley rats at doses of 210, 693 and 2101 mg enzyme concentrate dry matter/kg/day for a period of 13 weeks was generally well tolerated. No test item-related deaths occurred and no changes in daily or weekly observations as well as functional observational battery (performed during week 13) including grip strength and locomotor activity were observed. Furthermore, no test item-related effects on body weights or food consumption, no ophthalmoscopic findings, no changes in clinical laboratory investigations of toxicological relevance as well as no macroscopic and microscopic findings were noted.
Based on the results of this study, the no observed adverse effect level (NOAEL) for the test item pectin lyase was set at or above 2101 mg enzyme concentrate dry matter/kg/day.
Executive summary:

The objective of the study was to assess the toxicity of pectin lyase, batch PPJ 6991 administered daily by oral gavage to rats for 13 weeks. The study was conducted in accordance with the OECD Guideline 408, adopted on 21 September 1998. The rat was selected as the test model because of its suitability in this type of study. Oral treatment was chosen to comply with the intended route of exposure in humans. The selected doses were based on the results from a 14-day dose range finding study. Eighty SPF Sprague Dawley rats (40 males and 40 females) of the stock Mol:SPRD were used in this study. The animals were allocated to four groups (10 males and 10 females each) and treated once daily by gavage for 91 days with tap water (control, group I), 10% pectin lyase PPJ 6991 (group 2), 33% pectin lyase PPJ 6991 (group 3) or 100% pectin lyase PPJ 6991 (group 4). This corresponded to 0, 210, 693 and 2101 mg enzyme concentrate dry matter/kg b.wt., respectively. The dose formulations for group 2 and 3 were prepared by diluting the 100% solution with tap water to achieve· 10% and 33%, respectively. The dose volume was 8 ml/kg bwt. Clinical signs were recorded daily. Detailed clinical observations were performed once weekly. During week 11 of the study, the animals were examined for sensory reactivity, grip strength and motor activity. Body weight and food consumption were recorded weekly.


Ophthalmoscopy was performed on all animals before start of treatment, and on the animals of groups I and 4 during week 12 of the study. Before termination of treatment, blood samples were taken for haematology and clinical chemistry, and urine was collected for urinalysis. The animals were killed and subjected to a macroscopic necropsy. Specified organs/tissues were weighed and specified organs/tissues were sampled for microscopic examination. The clinical observations revealed no treatment-related effects. No differences were seen between treated and control animals in clinical signs, body weight, body weight gain, food consumption, ophthalmoscopy, haematology, clinical chemistry, urinalysis or organ weight. There were no macroscopic or microscopic findings that could be related to treatment with the test article.


In conclusion, daily oral (gavage) treatment with pectin lyase, batch PPJ 6991 in concentrations up to 2101 mg enzyme concentrate dry matter/kg bw/day for 13 weeks resulted in no treatment-related effects in rats. The no-observed adverse effect level (NOAEL) of pectin lyase, batch PPJ 6991 was at or above 2101 mg enzyme concentrate dry matter/kg bw/day.