Registration Dossier

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29-08-2016 to 19-10-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report Date:
2017

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Version / remarks:
Revised 3 October 2008.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
- Lot/batch No.: PPF41399
- Expiration date of the lot/batch: 31-05-2026
- Storage condition of test material: Frosen

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Crl:CD(SD) rat.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS Limited
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 41-47 days
- Weight at study initiation: 193 to 256 g for males; 150 to 184 g for females
- Fasting period before study: None
- Housing: 5 animals of the same sex per cage
- Diet: Teklad 2014C Diet ad libitum (removed overnight before blood sampling for hematology or blood chemistry).
- Water: Ad libitum. Potable water from the public supply via polycarbonate bottles with sipper tubes.
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature : 20-24°C
- Humidity : 40-70 % RH
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 22 August 2016 To: 20 Spetember 2016

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Remarks:
reverse osmosis water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test items (0, 10 and 100% active enzyme) were provided (deep-frozen) in a number of containers, which were further divided at Envigo into daily aliquots as required. The daily aliquots of formulation were thawed overnight in a refrigerator (2 to 8°C) if being used for dose administration on the following day, or were thawed at ambient temperature (15 to 25°C) if used for dose administration on the same day. For either case, formulations were allowed to equilibrate at room temperature before dosing and were administered within 24 hours of the commencement of thawing procedures..
VEHICLE
- Concentration in vehicle: 67, 217, 435, 870, 3420, and 6841 KMTU/kg bw/day.
- Amount of vehicle (if gavage): constant volume 20 mL/kg body weight.
- Purity: Reverse osmosis water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dose samples were analysed according to GLP.
The purpose of this phase of the study was to determine whether the enzyme activity (KMTU/g) in the dose solutions prepared week 1, corresponded to the expected activity.
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
67 other: KMTU/kg/day
Dose / conc.:
217 other: KMTU/kg/day
Dose / conc.:
435 other: KMTU/kg/day
Dose / conc.:
870 other: KMTU/kg/day
Dose / conc.:
3 420 other: KMTU/kg/day
Dose / conc.:
6 841 other: KMTU/kg/day
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The highest dose (100% active enzyme) was the maximum practical dose and represented administration of the enzyme, as received, at a volume dosage of 20 mL/kg body weight. The lower doses were formulated from the highest dose.

Positive control:
Not included

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment.

BODY WEIGHT: Yes
- Time schedule for examinations: The weight of each animal was recorded one week before treatment commenced (Day -7), on the day that treatment commenced (Day 1), weekly throughout the study (Days 8, 15, 22 and 28) and before necropsy (Days 29 or 30).

FOOD CONSUMPTION:
- The weight of food supplied to each cage, that remaining and an estimate of any spilled was recorded for the week before treatment started and for each week throughout the study. In Week 4 food consumption was only recorded over 6 days because of the placement of animals in metabolism cages for urine collection on the final day of the treatment period.

WATER CONSUMPTION:
- Time schedule for examinations: During Weeks -1, 1 and 3 of treatment, water consumption was recorded by weight (over a three day period on each occasion) for each cage of animals, using water bottles fitted with sipper tubes.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food. Day 29 for males and day 30 for females.
- Anaesthetic used for blood collection: Yes (isoflurane anaesthesia)
- Animals fasted: Yes, overnight
- How many animals: From all animals
- Parameters checked:
Haematocrit (Hct)
Haemoglobin (Hb)
Erythrocyte count (RBC)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Red cell distribution width (RDW)
Total leucocyte count (WBC)
Platelet count (Plt)

Differential WBC count:
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Blood samples were collected after overnight withdrawal of food. Day 29 for males and day 30 for females.
- Animals fasted: Yes, overnight
- How many animals: From all animals
- Parameters checked:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Total bilirubin (Bili)
Bile Acids (Bi Ac)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Total protein (Total Prot)
Albumin (Alb)

URINALYSIS: Yes
- Time schedule for collection of urine: Urine samples were collected overnight at the following occasions: day 29 for males and day 30 for females.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked:
Clarity and Color (App) - by visual assessment
Volume (Vol) - using a measuring cylinder
pH - using a pH meter
Specific gravity (SG) - by direct refractometry using a SG meter
Ketones (Keto)
Bile pigments (Bili)
Urobilinogen (Urob)
Blood pigments (UBld)
Protein (T-Prot)
Glucose (T-Gluc)
Epithelial cells (Epi)
Leucocytes (WBC)
Erythrocytes (RBC)
Casts
Other abnormal components (A)

NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations: Sensory reactivity and grip strength assessments were performed (before the 1st daily dose) on all animals during Week 4 of treatment.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / : Yes
Sacrifice and pathology:
All animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative. The retained tissues were checked before disposal of the carcass.
Other examinations:
Bone Marrow
Organ Weights
Histology
Statistics:
The main tests used were Dunnett’s test, Barlett’s test, Williams’ test, and Fisher’s exact tests. Significant differences between the groups compared were expressed at the 5% (p<0.05) or 1% (p<0.01) level.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Description (incidence and severity):
Chin rubbing after dosing was observed at all dose levels and in both sexes, though not in the controls. This sign is commonly observed in rodent studies where the test item is administered by gavage and is associated with taste (palatability) of the test item. It was considered of no toxicological significance.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One male (No. 18) receiving 435 KMTU/kg bwt/day (Group 4) died after the first dose on Day 25. The animal appeared stressed when removed from the cage prior to dosing, showing vocalisation, having dark eyes and appearing cyanotic (blue colouration) around its nose. This animal was returned to its cage, without being dosed, and after a short interval the animal was handled and dosed with no apparent difficulty, though the coloration around the nose and dark eyes were still evident. Approximately ten minutes after dosing breathing became very irregular and a decision was made to kill the animal for welfare reasons, but the animal subsequently convulsed and died. Necropsy revealed the presence of dark fluid in the trachea and stomach and the main histopathological findings were moderate diffuse alveolar haemorrhage in the lungs, minimal periaortic haemorrhage and minimal myocardial inflammatory cell infiltrate, minimally increased extramedullary haemopoiesis and increased cellularity of the white pulp in the spleen and slight multifocal haemorrhage in the thymus. The cause of death was undetermined, but as there were no similar findings in animals receiving higher doses, the death of this animal was not attributed to treatment.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Overall body weight gains (Days 0 to 28) were approximately 29% higher than the controls, in females receiving 6841 KMTU/kg bwt/day (Group 7). Males were unaffected. There was no effect of treatment on the body weight gain for males and females receiving 3420 KMTU/kg bwt/day or below.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Overall food consumption (Weeks 1 to 4) was approximately 13% higher than that of the controls by females receiving 6841 KMTU/kg bwt/day (Group 7) but this represented a trend that was evident during the final week of the acclimatization period and, consequently, was not attributed to treatment.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Water consumption in Week 3 was slightly high, when compared to the controls and pretreatment values, for males receiving 6841 KMTU/kg bwt/day (Group 7). This was not a consistent finding in the three-day measurement periods and there was no dose-response, since the next highest water intake was observed at 67 KMTU/kg bwt/day (Group 2) and the high dose females were unaffected.
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
All inter-group differences from controls, including those that attained statistical significance, were minor, occurred in one sex only or were without dose-relationship and were therefore considered to represent normal biological variation.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
All inter-group differences from controls, including those that attained statistical significance, were minor, occurred in one sex only, were due to high/low values in a few animals within the group or were without dose-relationship and were therefore considered to represent normal biological variation.
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The analysis of the urine after 4 weeks of treatment revealed slightly low urinary pH at all doses and in both sexes but there was no dose-response and the majority of individual pH values were within or just below the historical control ranges at these laboratories (90-percentile range 6.2 to 8.4 (n=413) for males and 5.9 to 7.6 (n=408) for females).
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Some variations were attributed to normal biological variation.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
The organ weight analysis after 4 weeks of treatment did not identify any differences from controls that were attributable to treatment.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The macroscopic examination performed after 4 weeks of treatment revealed the presence of depressions in the glandular mucosa of the stomach of two males and four females which received 6841 KMTU/kg bwt/day (Group 7), with two of the affected females also having thickening of the non-glandular mucosa. The incidence and distribution of all other macroscopic findings were considered to be unrelated to treatment and were of the type encountered normally in Sprague-Dawley rats at these laboratories.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with SP387/TL-1 were seen in the liver and stomach.
Periportal vacuolation was seen in males which received 67, 870 or 6841 KMTU/kg bwt/day (Groups 2, 5 and 7) and in females which received 67 KMTU/kg bwt/day and above (Groups 2 to 7). In females the severity was higher in the groups which received 67, 870 or 6841 KMTU/kg bwt/day (Groups 2, 5 and 7).
Epithelial hyperplasia and erosion of the non-glandular region was seen in males and females which received 6841 KMTU/kg bwt/day (Group 7).
The incidence and distribution of all other microscopic findings were considered to be unrelated to treatment. This included the incidence and severity level of inflammatory cell infiltrates in the liver which were consistent with the common background of microscopic changes seen in Sprague-Dawley rats at these laboratories (historical control data provided in Annex 3) and were not considered to be related to treatment.
Histopathological findings: neoplastic:
no effects observed

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 3 420 other: The result is given in enzymatic activity
Based on:
act. ingr.
Remarks:
KMTU/kg bwt/day
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
6 841 other: KMTU/kg bw/day
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes

Applicant's summary and conclusion

Conclusions:
It was concluded that the twice-daily oral administration of SP387/TL-1 to Sprague-Dawley rats for 4 weeks at a range of enzyme activities up to a total daily dose of 6841 KMTU/kg bwt/day was generally well-tolerated, though at the highest dose level there were stomach erosions that were considered responses to an irritant test material and these findings were considered adverse. In view of the presence of these erosions in the high dose animals, the no-observed-adverse-effect level (NOAEL) was considered to be 3420 KMTU/kg bwt/day (1.96 g TOS/kg bw/day).
Executive summary:

The objective of this study was to assess and compare the systemic toxic potential of a range of percentage enzyme activities of SP387/TL-1 (an enzyme used in the food industry), after twice-daily oral (gavage) administration to Sprague-Dawley rats for 4 weeks. The control group received the vehicle (reverse osmosis water) at a total volume-dose of 20 mL/kg body weight (bwt). The test concentrations were 67, 217, 435, 870, 3420, and 6841 KMTU/kg bw/day.

During the study, clinical condition, detailed physical examination and arena observations, sensory reactivity observations, grip strength, motor activity, body weight, food and water consumption, hematology (peripheral blood), blood chemistry, urinalysis, organ weight, macropathology and histopathology investigations were undertaken.

There were no deaths associated with SP387/TL-1. The general appearance and behavior of the animals and sensory activity, grip strength and motor activity were unaffected by treatment. There was no effect of treatment upon body weight and on food and water consumption. Females receiving 6841 KMTU/kg bwt/day showed an approximately 29% higher weight gain, than the controls but this was due to their higher food intake, compared to controls, which was a trend that was present before treatment commenced. There were no treatment-related haematology or blood chemistry findings. Urine analysis revealed slightly low pH at all doses and in both sexes but there was no dose-response and the findings were considered to be incidental and of no toxicological importance.

The statistical analysis of organ weights indicated high liver weights in males and females which received 6841 KMTU/kg bwt/day and in females which received 67 or 870 KMTU/kg bwt/day. In addition, there was a small increase of spleen weights in males which received 67 or 6841 KMTU/kg bwt/day and of heart weights in females at 6841 KMTU/kg bwt/day. There were no associated histopathological findings, the values were within concurrent or historical control ranges and these organ weight variations were considered to be due to normal biological variation not related to treatment.

The macroscopic examination revealed the presence of depressions in the glandular mucosa of the stomach of two males and four females given 6841 KMTU/kg bwt/day, with two of these females also having thickening of the non-glandular mucosa. This correlated with epithelial hyperplasia and erosion of the non-glandular region of the stomach and these findings were considered related to treatment and adverse.

In the liver there was a minor but treatment-related histopathology finding to just above background levels, in the form of periportal vacuolation in males given 67, 870 or 6841 KMTU/kg bwt/day and at all doses in females; in females the extent of this finding was greatest at 67, 870 and 6841 KMTU/kg bwt/day. As these groups received the highest dose in terms of g TOS, they represented expected (and reversible) findings in response to the administration of high doses of protein and, as such, were considered toxicologically insignificant and non-adverse.

It was concluded that the twice-daily oral administration of SP387/TL-1 to Sprague-Dawley rats for 4 weeks at a range of enzyme activities up to a total daily dose of 6841 KMTU/kg bwt/day was generally well-tolerated, though at the highest dose level there were stomach erosions that were considered responses to an irritant test material and these findings were considered adverse. In view of the presence of erosions in the high dose animals the no-observed-adverse-effect level (NOAEL) was considered to be 3420 KMTU/kg bwt/day (corresponding to 1.96 g TOS/kg bw/day).