lysophospholipaseIUBMB 3.1.1.5

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 April 2003 to 6 February 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

revised 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Active enzyme protein of lysophospholipase ( (EC no.618-333-0, CAS no. 9001-85-8, EC name Lysophospholipase, Enzyme class no 3.1.1.5 )

Molecular formula:

Not available

IUPAC Name:

Active enzyme protein of lysophospholipase ( (EC no.618-333-0, CAS no. 9001-85-8, EC name Lysophospholipase, Enzyme class no 3.1.1.5 )

Constituent 2

Reference substance name:

Inorganic salts as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available.

IUPAC Name:

Inorganic salts 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.

IUPAC Name:

Carbohydrates constituent of enzyme deriving from the fermentation or extraction process

Constituent 4

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 5

Reference substance name:

Lipids as a constituent of enzyme deriving from the fermentation or extraction process

Molecular formula:

Not available.

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.: PPW22320
- Expiration date of the lot/batch: 2013-02-19
- Stability under test conditions: The test material is stable for at least 24 hours at room temperature
- Storage condition of test material: Frozen (-20°C)

Test animals

Species:

rat

Strain:

other: Crl:CD® (SD)IGS BR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, UK, Ltd
- Age at study initiation: 45 to 49 days
- Weight at study initiation: 226 to 266 g for males; 158 to 203 g for females
- Fasting period before study: None
- Housing: 5 animals of one sex per cage.
- Diet: Standard rodent diet Rat and Mouse No. 1 Maintenance Diet from Special Diets Services Ltd., Witham, Essex, England), ad libitum, except overnight before routine blood sampling
- Water: Tap water ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature : 19-23°C
- Humidity : 40-70 % RH
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2003-06-02 To: 2003-09-02

OTHER: Before the start of treatment, one male with non-resolving ophthalmic lesions, one female with ill health and three males and two females with bodyweights at the extreme of the weight range were replaced with spare animals of suitable weight from the same batch.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE: Purified water obtained by reverse osmosis.
- Concentration in vehicle: 10, 33 and 100% (equivalent to 0.24, 0.80 or 2.42g TOS/kg/day).
- Amount of vehicle (if gavage): At the same volume-dosage.
- Purity: Water for formulation

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Significant differences were found in the low dose group of week 6 when compared to the same group in week 1. The measured activity in the low dose group in week 6 was indeed 13% lower than expected but 12% variation is accepted on this analytical method. This is only a small deviation from the expected value and this is considered as having no significance. No activity was found in any of the analysed control samples. It was further investigated if the enzyme activity for the high dose group was equal to the activity in the test material. No significant differences were found between the test material and the high dose group.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 other: mL/kg/day

Remarks:

Equivalent to 0.24 g TOS/kg/day

Doses / concentrations 2

Dose / conc.:

3.3 other: mL/kg/day

Remarks:

Equivalent to 0.80 g TOS/kg/day

Doses / concentrations 3

Dose / conc.:

10 other: mL/kg/day

Remarks:

Equivalent to 2.42 g TOS/kg/day

No. of animals per sex per dose:

10

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 previous work with this compound where it was concluded that oral administration of lysophospholipase, PPW 22320, to CD rats at dosages up to 10.0 ml/kg/day for two weeks was well-tolerated and did not produce any toxicologically significant changes. The lower doses were selected using an approximate ratio of 3.3 between doses.

Positive control:

Not included

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: The animals were inspected daily for evidence of ill-health.
Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily during the first week of treatment, twice weekly during Weeks 2 to 4 and weekly thereafter. Detailed observations were recorded in relation to dose administration.

BODY WEIGHT: Yes
- Time schedule for examinations: At the day that treatment commenced (Week 0), weekly throughout the treatment period, and before necropsy.

FOOD CONSUMPTION:
- Food consumption for each cage, i.e. sum of five animals, was determined and mean daily diet consumption per group calculated as g feed/rat/week: Yes

FOOD EFFICIENCY:
- Body weight gain in g/food consumption in g per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE: Yes, visual.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment started and during week 13
- Dose groups that were examined: Before treatment all animals. During week 13 Group 1 (control) and Group 4 (highest dose group)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13 (before dosing)
- Anaesthetic used for blood collection: Yes (isoflurane anaesthesia)
- Animals fasted: yes, overnight
- How many animals: From all animals
- Parameters checked:
Haematocrit (Hct)
Haemoglobin concentration (Hb)
Red blood cell count (RBC)
Mean cell haemoglobin (MCH)
Mean cell haemoglobin concentration (MCHC)
Mean cell volume (MCV)
Total white cell count (WBC)
Differential WBC count
Neutrophils (N)
Lymphocytes (L)
Eosinophils (E)
Basophils (B)
Monocytes (M)
Large unstained cells (LUC)
Platelet count (PLT)
Prothrombin time (PT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13 (before dosing)
- Animals fasted: yes, overnight
- How many animals: From all animals (80)
- Parameters checked:
Alkaline phosphatase (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Gamma-glutamyl transpeptidase (gGT)
Total bilirubin (Bili)
Urea
Creatinine (Creat)
Glucose (Gluc)
Total cholesterol (Chol)
Triglycerides (Trig)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Calcium (Ca)
Inorganic phosphorus (Phos)
Total protein (Total Prot)
Albumin (Alb)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations: During week 12
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / : yes

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, full macroscopic examination
HISTOPATHOLOGY: Yes. Adrenals, brain, femur with joint, heart, ileum, kidneys, liver, lungs, salivary glands, spinal cord, sternum, stomach, thyroid and uterus were preserved and for the control group and the high dose group these tissues were examined.

Other examinations:

FAECAL ANALYSIS: No

Weight of individual organs: Yes
- Time schedule for collection of organs: At necropsy

Statistics:

Statistical evaluation of grip strength, motor activity, bodyweight, haematology, blood chemistry, organ weights and any data derived from these was performed. Significant differences between Control and treated groups were expressed at the 5% (p<0.05) or 1% (p<0.01).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Description (incidence):

Following routine blood sampling during Week 13 of treatment, one male receiving 10.0 ml/kg/day sustained physical trauma to the eye and was killed for humane reasons. The death of this animal was clearly incidental, and not attributed to treatment.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

no effects observed

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

no effects observed

Description (incidence and severity):

Water consumption was visual.

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Description (incidence and severity):

There were no haematological changes in Week 13 that could, with any confidence, be attributed to treatment.
Prothrombin times were increased, when compared with Controls, for females receiving 3.3 or 10.0 mL/kg/day with the differences attaining statistical significances (p<0.05). In veiw of the small difference from Controls, and the absence of a similar finding amongst the males, this finding is of doubtful toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Biochemical examination of the plasma during Week 13 indicated, when compared with the Controls, high glucose concentration for males and females receiving 10.0 mL/kg/day. Plasma potassium concentrations were increased for animals receiving 10.0 mL/kg/day. In the absence of any change in food intake or histopathology of the liver, the change in glucose concentration is not considered toxicologically significant. The change in potassium concentration was not associated with any histopathological change in the kidney and is therefore also not considered toxicologically significant.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Two males in each of the groups receiving 1.0 or 3.3 mL/kg/day and three males receiving 10.0 mL/kg/day failed to show a touch response (Grade 1) and three males receiving 10.0 ml/kg/day displayed a weak (Grade 2) auditory startle reflex. The findings for the auditory startle reflex were within historical background control data, though the incidence of animals failing to show a touch response was slightly higher than the historical control data. Since all males showed a normal response to the tail pinch test, and in the absence of any similar findings in the females, these findings are attributed to inter-animal variation.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

When compared with Controls, absolute and adjusted brain weights were increased for females given 10.0 ml/kg/day, attaining statistical significance (p<0.05). The difference from Control was small and was attributed to normal biological variation.

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

An increased incidence of prominent ultimobranchial cysts in the thyroids in males that received 10.0 mL/kg/day attained statistical significance (p<0.05). This is a congenital finding often seen in the thyroids of rats. The increased incidence in males treated at 10.0 mL/kg/day was considered fortuitous and not related to treatment.

Histopathological findings: neoplastic:

no effects observed

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 420 mg/kg bw/day (actual dose received)

Based on:

other: total organic solids (TOS)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

It is concluded that oral administration of lysophospholipase, PPW 22320 to CD rats at dosages up to 10.0 mL/kg/day for 13 weeks was well-tolerated and did not produce any toxicological significant change. Consequently the no-observed-adverse-effect-level (NOAEL) in this study was considered to be the highest dose administered 10.0 mL/kg/day, which is the equivalent to 2.42g TOS/kg/day.

Executive summary:

The repeated dose oral toxicity study was a subchronic toxicity test conducted according to OECD guideline 408 (revised 1997), and in compliance with GLP.

The systemic toxic potential of lysophospholipase, PPW 22320 a food enzyme, to Crl:CD® (SD)IGS BR rats by oral administration was assessed over a period of 13 weeks. Three groups of ten male and ten female rats received lysophospholipase, PPW 22320 by gavage at dosages of 1.0, 3.3 or 10.0 mL/kg/day (equivalent to 0.24, 0.80 or 2.42g TOS/kg/day). A similarly constituted Control group received the vehicle (purified water obtained by reverse osmosis) at the same volume-dosage. During the study, clinical condition, detailed physical and arena observations, sensory reactivity, grip strength, motor activity, bodyweight, food consumption, haematology, blood chemistry, organ weight, macroscopic and microscopic pathology investigations were undertaken.

There were no signs related to treatment and the functional observation battery investigations also did not indicate any changes that were attributable to treatment. One male died during the treatment period, though this was not attributed to treatment, but rather was done for humane reasons. Bodyweight gain, food consumption and food conversion efficiency were unaffected by treatment. There were no treatment related ophthalmic findings. Haematological investigations in Week 13 did not indicate any treatment-related changes. Biochemical examination of the plasma during Week 13 indicated increased glucose and potassium concentration for animals receiving 10.0 mL/kg/day, but it was considered not to be toxicologically significant. There were no treatment-related organ weight, macroscopic or histopathological changes after 13 weeks of treatment.

It is concluded that oral administration of lysophospholipase, PPW 22320 to CD rats at dosages up to 10.0 mL/kg/day for 13 weeks was well-tolerated and did not produce any toxicological significant change. Consequently the no-observed-adverse-effect-level (NOAEL) in this study was considered to be the highest dose administered 10.0 mL/kg/day, which is the equivalent to 2.42 g TOS/kg/day.