Laccase

Endpoint summary

Description of key information

The acute oral, inhalation and dermal toxicity of Laccase have been tested. The studies were short-term toxicity test procedures according to OECD or EU standard procedures, and in compliance with GLP. The conclusion was that Laccase is non-toxic by oral, dermal and inhalation exposure.

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 23, 1996 to March 07, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Annex to Commission Directive 92/69/EEC of 31 July 1992. B1. L 383 A/110. OECD Guidelines for Testing of Chemicals, 401, 1987.

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Møllegaard Breeding Center, Ejby, Denmark.
- Fasting period before dosing: Overnight
- Housing: Five animals per cage, transparent polycarbonate cages Type IV, 590 MAK dimensions 59 x 38 x 20 cm
- Weight at time of dosing: between 139-153 g (males), 114- 118 g (females)
- Housing: In animal room with control of temperature and humidity
- Diet: Standard diet ad libitum
- Water: Acidified tap water ad libitum
- Acclimation period: 6 days
- Temperature (°C): 19-25°C
- Humidity : 30-70 %

Route of administration:

oral: gavage

Vehicle:

other: Test batch used as is.

Details on oral exposure:

Undiluted test material, dose volume 12 mL/kg bw, corresponding to 2.07 g TOS/kg body weight (limit test)

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations for clinical signs of effect: 1.5 and 4 hours after dosing and then once a day. Weighing on Days 1, 8 and 15
- Animals were fasted from the evening before dosing and until 3 hrs after dosing
- Necropsy of survivors performed: yes

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 070 mg/kg bw

Based on:

other: TOS (Total Organic Solids)

Mortality:

Male: 2070 mg TOS/kg bw; Number of animals: 5; Number of deaths: 0
Female: 2070 mg TOS/kg bw; Number of animals: 5; Number of deaths: 0

Clinical signs:

other: Signs of toxicity related to dose levels: No clinical signs were observed in the dose group.

Gross pathology:

Effects on organs:
No significant findings regarding necropsy were observed in the dose group. Histopathology was not performed.

Interpretation of results:

GHS criteria not met

Conclusions:

In conclusion, the acute oral lethal dosage (LD50) of laccase was greater than 2070 mg Total Organic Solids (TOS)/kg.

Executive summary:

The study was conducted in accordance with the OECD Guideline No 401, “Acute Oral Toxicity”. The limit test was used. The test item was supplied as a brown liquid ready to use. The dose volume administered was 12 mL/kg of the undiluted test material.

No clinical signs were observed and the overall body weight gain during the study was considered to be normal. The post-mortem inspection revealed no abnormalities.

In conclusion, no signs of toxicity were observed among the rats treated with a single oral dose of 2070 mg Total Organic Solids (TOS)/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 070 mg/kg bw

Quality of whole database:

Study is in accordance with GLP and Klimisch 1.
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, 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 thus be considered of high quality.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From November 6, 1996 to June 19, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River UK Ltd, Kent UK.
- Housing: Five animals per cage, dimensions 53 x 35 x 25 cm
- Weight at time of dosing: Between 207-221 g (females), 257- 273 g (males)
- Housing: In animal room with control of temperature and humidity
- Diet: Standard diet ad libitum
- Water: Tap water ad libitum
- Acclimation period: 5 days
- Temperature (°C): 18-24°C
- Humidity: 40-70 %

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: ADG Developments
- Exposure chamber volume: 30 L
- Method of holding animals in test chamber: Snout only
- Source and rate of air: Clean dried air was provided by the aerosol generator at a flow rate of 10 L /min
- Method of conditioning air: A syringe with undiluted test material was via a syringe pump connected to the aerosol generator at a flow rate of 0.4 mL/min.
- System of generating particulates/aerosols: Stainless steel atomiser
- Method of particle size determination: Gravimetric analysis using a marble cascade impactor at 90, 150 and 225 min after start of exposure. The material collected on the stages of the sampler was weighed to determine the particle size distribution in the atmosphere.
- Treatment of exhaust air: Through an absolute filter
- Temperature, humidity, pressure in air chamber: 20 C, 91% humidity, normal pressure of the atmosphere

TEST ATMOSPHERE
- Brief description of analytical method used: Five air samples were taken during exposure, at 2 L/min, through weighed glass fibre filters (Whatman GF/A). The collected material was weighed to determine the concentration of test material in the exposure chamber. The volume of air was measured with a wet-type gas meter.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: 91% respirable (< 7 um)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): 2.7 um (GSD 2.04)

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: The flow rate of 0.4 mL/min of the undiluted material was from experience expected to provide a concentration of enzyme in excess of 5 mg/L.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

>= 4 h

Concentrations:

5.16 mg/L

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observations for clinical signs of effect: During exposure, immediately after and with hourly intervals during the first 2 hours after the exposure and subsequently every day in the 14-day observation period. Weighing: Daily
- Necropsy of survivors performed: yes

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 5.16 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality.

Clinical signs:

other: No clinical signs. Immediately following exposure, the rats exhibited a wet and unkempt appearance due to the method of restraint but this cleared within 2 h after the end of exposure.

Body weight:

Reduced food consumption and body weight gain were recorded for 1 day following exposure. Apart from this, all body weights and body weight gains were normal.

Gross pathology:

No abnormalities.

Interpretation of results:

GHS criteria not met

Conclusions:

Laccase causes only minimal evidence of toxicity in rats after 4 hours of inhalation of a concentration of 5.16 mg/L (corresponding to 0.85 mg TOS/L). The LC50 for laccase is in excess of 5.16 mg/L.

Executive summary:

In accordance with OECD guideline No. 403, a Limit Test was performed with one group of rats consisting of 5 females and 5 males.

The animals were exposed by snout only exposure for 4 hours to air containing aerosolised Laccase, batch PPX 5720, at a concentration of 5.16 mg/L (equivalent to 0.85 mg TOS*/L).

Particle size measurements revealed that the respirable fraction (% of aerosol mass < 7um) was 90%. The mass median aerodynamic diameter was 2.7 µm, in total a very high respirable fraction. The animals were observed during exposure, immediately after and with half hourly intervals during the first 2 hours after the exposure and subsequently every day in the 14-day observation period. After the observation period, the animals were sacrificed and examined pathologically.

Immediately following exposure, the rats exhibited a wet and unkempt appearance due to the method of restraint but this cleared within 2 h after the end of exposure. Reduced food consumption and body weight gain were recorded for 1 day following exposure. During the rest of the observation period, all rats were normal in appearance and behaviour. No animals died during the observation period, and the pathological examination revealed no abnormalities related to the exposure.

 

In conclusion, laccase causes only minimal evidence of toxicity in rats after 4 hours of inhalation of a concentration of 5.16 mg/L (corresponding to 0.85 mg TOS/L). The LC₅₀ for laccase is in excess of 5.16 mg/L.

 

* Total Organic Solids

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

5 160 mg/m³ air

Quality of whole database:

Study is in accordance with GLP and Klimisch 1.
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, 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 thus be considered of high quality.

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From October 14, 1996 to March 07, 1997

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Møllegaard Breeding Center, Ejby, Denmark.
- Housing: One animal per cage, transparent polycarbonate cages Type III, 420 MAK dimensions 42 x 26 x 15 cm
- Weight at time of dosing: Between 189-205 g (females), 234- 250 g (males)
- Housing: In animal room with control of temperature and humidity
- Diet: Standard diet ad libitum
- Water: Acidified tap water ad libitum
- Acclimation period: 6 days
- Temperature (°C): 19-25°C
- Humidity: 30-70 %

Type of coverage:

semiocclusive

Vehicle:

unchanged (no vehicle)

Details on dermal exposure:

TEST SITE
- Area of exposure: 4x5 cm2
- % coverage: 10% of the body surface
- Type of wrap if used: The test substance was applied to four layers of gauze measuring 5x5 cm and this was covered with 3M Micropore Surgical tape 1530-3 3M.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): Yes, with water
- Time after start of exposure: 24 hrs

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1.2 mL per 100 g bw, corresponding to 2.07 g TOS*/kg bw
- Concentration (if solution): Undiluted test material, containing 16.6% w/w TOS; specific density 1.039 g/mL
- Constant volume or concentration used: yes

* Total Organic Solids

Duration of exposure:

24 hrs

Doses:

12 mL/ kg bw, corresponding to 2.07 g TOS*/kg bw
* Total Organic Solids

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: The animals were observed clinically after dosing, particularly at day 1-2, and subsequently every day during the entire 14-day observation period. The skin on the site of application was scored after 2 and 24 hours after removal of the patch. The animals were weighed at days 1, 8 and 15.
- Necropsy of survivors performed: yes
- Other examinations performed: The skin reactions were scored according to the guideline.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 070 mg/kg bw

Based on:

other: Total Organic Solids (TOS)

Mortality:

No mortality.

Clinical signs:

other: No significant clinical signs. No erythema or oedema of the skin was observed in any animals.

Gross pathology:

No abnormalities.

Interpretation of results:

GHS criteria not met

Conclusions:

The acute percutaneous median lethal dosage (LD50) of laccase was found to be greater than 2070 mg Total Organic Solids (TOS)/kg bw.

Executive summary:

In accordance with EU guideline B3, a limit test was performed with one group of rats consisting of 5 females and 5 males.

The dose was applied to the closely clipped dorsum of each animal in a total volume of 12 ml undiluted test material per kg bw (equivalent to 2.07 g Total Organic Solids (TOS)/kg), and was covered by an semiocclusive dressing for 24 hours.

The animals were observed after dosing, particularly at day 1-2, and subsequently every day during the entire 14-day observation period. The skin on the site of application was scored after 2 and 24 hours after removal of the patch. At the end of the observation period, the animals were sacrificed and examined pathologically.

 

No deaths occurred and no clinical effects (including local skin effects) were seen relating to the test substance. There were no abnormal findings at necropsy.

 

Under the conditions of this study, the acute percutaneous median lethal dosage (LD50) of laccase was greater than 2070 mg TOS/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 070 mg/kg bw

Quality of whole database:

Study is in accordance with GLP and Klimisch 1.
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, 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 thus be considered of high quality.

Additional information

The acute oral, inhalation and dermal toxicity of Laccase has been tested. The studies were short-term toxicity test procedures performed according to OECD or EU standard procedures, and in compliance with GLP. The conclusion was that Laccase is non-toxic if swallowed or inhaled or by contact with the skin. The conclusion regarding the classification is based on limit tests, i.e. only one dose level to save the number of animals used, however, LD50 is expected to be > 5g/kg (oral, dermal) or 5mg/L (inhalation). The test material Laccase is not expected to exert any acute oral, dermal or inhalation toxicity under foreseeable realistic exposures for workers or consumers.

Further information regarding the Acute Inhalation Toxicity:

Due to the fact that enzymes are respiratory allergens, DMEL (Derived Minimum Effect Level) values have to be established to ensure that enzymes can be used safely (ref. 3 below). Appropriate exposure limits have been being established to protect consumers, professionals and workers (ref. 3 below). Respiratory allergy is considered the most sensitive endpoint for enzymes. However, when the exposure limit recommendations are followed, this will ensure that exposure levels are low and without any toxicological relevance. Commonly, occupational exposure limit (OEL) values for workers are between 40-60 ng enzyme protein/m3 (8 hour time-weighted average values) in EU countries. More than 30 studies on acute inhalation toxicity in rodents revealed that for the majority of enzymes, no harmful effect could be detected at concentrations up to several mg/l air or g/m3 representing the highest possible concentrations administered and equivalent to nuisance dust levels. In the few cases where LC50 values could be established, the values were more than a factor of 10^6 above the actual OEL value, indicating that the concentrations normally used in acute inhalation toxicity studies are irrelevant to all known exposure scenarios.

The industry has further taken measures to minimize occupational exposure. Workers safety is assured through proper work practices, effective cleaning, engineering controls, and use of personal protective equipment (ref. 5).

 

 

 

Justification for selection of acute toxicity – oral endpoint
Key study selected based on dose, quality and highest concern.

Justification for selection of acute toxicity – inhalation endpoint
Key study selected based on dose, quality and highest concern.

Justification for selection of acute toxicity – dermal endpoint
Key study selected based on dose, quality and highest concern.

Justification for classification or non-classification

Acute Oral Toxicity: Laccase is not classified, LD50 is > 2070 mg Total Organic Solids (TOS)/kg.

 

Acute Dermal Toxicity: Laccase is not classified, LD50 is > 2070 mg Total Organic Solids (TOS)/kg.

 

Acute Inhalation Toxicity: Laccase is not classified, LD50 is > 5160 mg/m3 (equivalent to 850 mg Total Organic Solids (TOS)/m3)