Laccase

Endpoint summary

Description of key information

The repeated dose oral toxicity of the enzyme laccase has been tested. The repeated dose inhalation and dermal toxicity were waived based on exposure considerations and the properties of the substance.

In the repeated dose oral toxicity study in rats, the No Observed Adverse Effect Level (NOAEL) was the highest dose level tested, equivalent to 10 mL of the undiluted laccase batch/kg bw/day or 1720 mg Total Organic Solids (TOS)/kg bw/day.

Based on repeated dose oral and weight of evidence, laccase does not exert any repeated dose inhalation or dermal toxicity to workers or consumers.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 27 November 1996 to 18 December 1997

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 1981

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Limited, Kent, UK
- Age at study initiation: 30-37 days
- Weight at study initiation: 82-105g
- Fasting period before study: None
- Housing: 5 animals per cage, separate sex
- Diet : ad libitum
- Water : ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature : 19-25°C
- Humidity : 40-70 % RH
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1996-12-06 To: 1997-03-08

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Purified water obtained by reverse osmosis

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
VEHICLE
- Concentration in vehicle: 1, 10 and 100% (undiluted), corresponding to 1.7, 17 and 172 mg total organic solids (TOS)/mL
- Amount of vehicle (if gavage): constant volume 10 mL/kg b.w.
- Purity: Purified water obtained by reverse osmosis

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of achieved concentration with regard to the enzyme activity was performed on samples taken once during weeks 1 and 13. The mean achieved enzyme activities were found to be within the acceptance criteria, demonstrating satisfactory formulation.

The analysis was performed according to the current standard operating procedure, KAL-SM-0002.01/01.
The analysis principle is described as follows:
An automated Cobas Fara method was used for the determination of laccase activity(LAMU), based on the following principle: Laccase catalyses under aerobic conditions the oxidation of the substrate chromophore syringsaldazine under formation of tetrametoxy azo bis methylene quinone. Using a kinetic measurement, the formed violet colour is measured spectrophotometrically at 530 nm. One LAMU is the amount of enzyme that converts 1 umol Syringaldazine per min under stated analytical conditions.
The laccase activity is measured in laccase units (LAMU) relative to an enzyme standard of declared activity.
- Detection limits (LOD, LOQ) (indicate method of determination/calculation): The limit of determination is 0.005 LAMU/mL ; limit of quantification is 0.01 LAMU/mL
- Linearity range: 0.01 - 0.044 LAMU/mL
The samples were analysed as double weigh out determinations on one standard curve.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0.1, 1.0 or 10.0 mL/kg/day, equivalent to 17, 172 and 1720 mg total organic solids (TOS)/kg/day
Basis:
nominal in water

Dose / conc.:

17 mg/kg bw/day (nominal)

Remarks:

Concentration given in total organic solids (TOS)

Dose / conc.:

172 mg/kg bw/day (nominal)

Remarks:

Concentration given in total organic solids (TOS)

Dose / conc.:

1 720 mg/kg bw/day (nominal)

Remarks:

Concentration given in total organic solids (TOS)

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 a 2-wk oral dose range finder study. Groups of 5 male and 5 female CD rats were dosed for 2 weeks with Laccase, batch PPX 5720, orally via gavage at dose levels of 0, 0.17, 0.57 and 1.72 g Total organic solids (TOS)/kg body weight/day, at a constant dose volume of 10 ml per kg body weight. There were no obvious effects of treatment with laccase at dose levels up to 1.72 g TOS/kg bw/day. The highest dose (10 mL/kg/day) represents administration of the enzyme, as received, at a volume-dose of 10 mL/kg bodyweight, which is the maximum practical volume-dose for repeat dose oral administration. The lower doses were selected as 10-fold dilutions to make sure that the biological differences could be seen in case of any effects at the highest dose.

Positive control:

Not included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily

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

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each cage, i.e. sum of five animals, was determined and mean weekly 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 : No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Before treatment started and during week 12
- Dose groups that were examined: control and highest dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: Yes (halothane/nitrous oxide anaesthesia)
- Animals fasted: yes, overnight
- How many animals: From all animals
- Parameters checked:
Packed cell volume (PCV)
Haemoglobin (Hb)
Erythrocyte 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 (Plat)
Normoblasts
Prothrombin time (PT)


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13
- Animals fasted: yes, overnight
- How many animals: From all animals
- Parameters checked:
Alkaline phosphatase activity (ALP)
Alanine aminotransferase (ALT)
Aspartate aminotransferase (AST)
Ornithine carbamyl transferase activity (OCT)
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)
Electrophoretic protein fractions (albumin, alpha-globulin, beta-globulin, gamma-globulin)
Albumin (Alb)/globulin ratio


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 7)
HISTOPATHOLOGY: Yes (see table 8)

Other examinations:

FAECAL ANALYSIS: No

Weight of individual organs: yes
- Time schedule for collection of organs: At necropsy
- Results presented in table 6

Statistics:

Statistical evaluation of bodyweights, haematology, blood chemistry, organ weights and any data derived from these was performed. The evaluation included the following elements: Exclusion of outliers, test for homogeneity of variance (and transformation if significant), followed by analysis of covariance (ANOCOVA) for the final in-life bw, with the bw on day 0 as covariate, and organ weights with the autopsy bw as covariate; ANOVA and Students t-test was used for all other individual numerical data. Kruskall-Wallis and if significant a Mann-Whitney U-test was used for data found significant after the various transformations. Attribute data was analused, where considered appropriate, using Fisher's Exact Probability test. The level of probability chosen as significant was p<0.05.

Clinical signs:

no effects observed

Mortality:

no mortality observed

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):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

There were no effects of treatment on clinical signs, body weight, food consumption, food conversion efficiency, ophthalmic findings, haematology and clinical chemistry profiles, or on organ weights and macroscopic or microscopic findings. Neurobehaviour was not specifically investgated as the study was performed before this parameter was introduced to the guideline, but no clinical effects was seen.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 720 mg/kg bw/day (nominal)

Based on:

other: Total organic solids (TOS)

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Key result

Critical effects observed:

not specified

 

 

 

Conclusions:

The No Observed Adverse Effect Level (NOAEL) in rats is 10 mL of the undiluted laccase batch/kg bw/day equivalent to 1720 mg Total Organic Solids (TOS)/kg bw/day.

Executive summary:

The repeated dose oral toxicity was a subchronic toxicity test conducted according to OECD guideline 408 (adopted 1981), and in compliance with GLP. 

Oral administration (by gavage) of Laccase, PPX 5720, to rats at dosages of up to the highest dose level tested, equivalent to 10 mL of the undiluted laccase batch/kg bw/day or 1720 mg Total Organic Solids (TOS)/kg bw/day for thirteen weeks, was well-tolerated and did not produce any toxicologically significant changes. Necropsy and the following microscopic examination revealed no treatment related.

Consequently, the No Observed Adverse Effect Level (NOAEL) is considered to be the highest dose level administered, equivalent to 10 mL undiluted laccase batch/kg bw/day or 1.72 g Total Organic Solids (TOS)/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 720 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Toxicological data has been generated within the enzyme producing industry during the last 40 years. Substantial documentation on the safety of the production strains has been generated, and the enzyme test materials are thoroughly characterized. High quality studies for all relevant endpoints for in vivo studies as well as in vitro studies show that industrial enzymes from well-known and well characterized production strains have very similar safety profiles across the catalytic activities. Read-across can therefore be applied for the majority of toxicological endpoints. The database can therefore be considered of high quality.

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

The repeated dose oral toxicity of laccase has been tested, while the repeated dose inhalation and dermal toxicity were waived.

- The dermal study was waived because of the low likelihood of absorption of enzymes through the skin due to the physico-chemical properties of the enzyme protein.

- The inhalation study was waived because exposure is too low to exert any toxicity. Potential exposure by inhalation to an amount of enzyme, which is toxicologically relevant, is unrealistic due to the stringent work practices and the formulation of enzymes, enforced because of the risk of sensitization by inhalation.

- The repeated dose oral toxicity was a subchronic toxicity test conducted according to OECD guideline 408 (adopted 1998), and in compliance with GLP. The conclusion was that the No Observed Adverse Effect Level (NOAEL) in rats was the highest dose level tested, equivalent to 10 mL of undiluted test material/kg bw/day or 1720 mg Total Organic Solids (TOS)/kg bw/day.

 

Based on repeated dose oral and weight of evidence, laccase does not exert any repeated dose oral, dermal or inhalation toxicity to workers or consumers.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The present key study was selected based on quality, duration and GLP status.

Justification for classification or non-classification

Based on repeated dose oral study and weight of evidence laccase does not exert any repeated dose oral, dermal or inhalation toxicity to workers or consumers.