Cellulase

Endpoint summary

Description of key information

Acute toxicity via the oral route has been tested. No signs of toxicity were observed in five male and five female rats treated with a single oral dose of 2960 mg enzyme concentrate dry matter/kg bw. The test was conducted according to OECD guidelines and GLP standards.

Acute toxicity via inhalation route has been tested. Cellulase causes only minimal evidence of toxicity in rats after 4 hours of inhalation of a concentration of 4.86 mg/L (corresponding to 4.72 mg enzyme concentrate dry matter/L). The LC50 for Cellulase is in excess of 4.72 mg enzyme concentrate dry matter/L. The test was conducted according to OECD guidelines and GLP standards.

Acute toxicity via dermal route is waived based on exposure considerations and the known properties of the substance.

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 September 20, 1994 to October 12, 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed according to GLP and the procedures were according to the OECD test guideline 401 (1987).

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.

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: approximately 21 hrs 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 112-121 g (males), 108- 127 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: 5 days
- Temperature (°C): 19-21°C
- Humidity : 33-80 %

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Remarks:

Test batch used as is. Control animals were dosed with tap water.

Details on oral exposure:

Undiluted test material, dose volume 20 mL/kg bw, corresponds to 2.96 g enzyme concentrate dry matter/kg body weight (limit test), based on the conservative assumption that the specific gravity of the undiluted test batch is 1 g/mL.

No. of animals per sex per dose:

5

Control animals:

yes

Details on study design:

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

Statistics:

No

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 960 mg/kg bw

Based on:

other: enzyme concentrate dry matter

Mortality:

Male: 2960 mg enzyme concentrate dry matter/kg bw; Number of animals: 5; Number of deaths: 0
Female: 2960 mg enzyme concentrate dry matter/kg bw; Number of animals: 5; Number of deaths: 0
No control animals died during the study.

Clinical signs:

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

Gross pathology:

Effects on organs:
No treatment related findings were observed in the dose group or the control group. Histopathology was not performed.

Interpretation of results:

other: Data insufficient for classification as 2960 mg enzyme concentrate dry matter/kg bw corresponds to 1512 mg active enzyme protein matter/kg bw

Conclusions:

In conclusion, the acute oral lethal dosage (LD50) of cellulase was greater than 2960 mg enzyme concentrate dry matter/kg bw.

Executive summary:

The study was conducted in accordance with the OECD Guideline No 401, “Acute Oral Toxicity”. The limit test procedure was used. The test item was supplied as a brown liquid ready to use. The dose volume administered was 20 mL/kg of the undiluted test material.

Five male and five female Wistar rats were dosed a volume of 20 mL/kg bw corrosponding to 2960 mg enzyme concentrate dry matter/kg bw. A control group was dosed with tap water at the same dose volume. 

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 2960 mg enzyme concentrate dry matter/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 960 mg/kg bw

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 have 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 May 28, 1991 to June 12, 1991

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

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
- Weight at time of dosing: 187-224 g (females), 283- 315 g (males)
- Housing: In animal room with control of temperature and humidity
- Diet: Rat Modified No 1 Diet expanded from Special Diets Service Ltd., Essex, ad libitum, except during the 4 hours exposure
- Water: Tap water ad libitum, except during the 4 hours exposure
- Acclimation period: 18 days
- Temperature (°C): 18-22°C
- Humidity : 34-65 %

IN-LIFE DATES: From: May 28, 1991 To: June 12, 1991

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

clean air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: ADG Instruments Ltd., England
- Exposure chamber volume: 41.5 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 21 L/min. The volume of air was measured continuously using flowmeters and was recorded at 30 min intervals.
- Method of conditioning air: Filtered, oil-free compressed air for the production of the test atmosphere was supplied by Hydrovane compressors.
- System of generating particulates/aerosols: A rotating brush aerosol generator ( RBG - 1000, Palas, Germany) scraped the powdered test material from a canister to micronize it and a stream of air ducted the test material to the exposure chamber.
- Method of particle size determination: Particle size was estimated twice during exposure using a Marple Cascade Impactor (Anderson Samplers Inc., Model 296) capable of fractionating the aerosol into the size range 0.25 -> 10 µm. Samples were taken from the breathing zone. The material collected on the impaction stages of the sampler was weighed before and after sampling to determine the particle size distribution in the atmosphere.
- Temperature, humidity, pressure in air chamber: 23-26°C, 24-67% humidity, normal pressure of the atmosphere.

TEST ATMOSPHERE
- Brief description of analytical method used: 16 air samples were taken during exposure. Chamber air was drawn at a measured rate of 1 L/min using a vacuum pump, with 2 liters of air sampled. The gravimetric method used employed pressed glass fiber filters (Whatman GF/B) placed in a filter holder. The collected material was weighed before and after sampling to determine the concentration of test material in the exposure chamber in relation to the air flow in liters.

- Samples taken from breathing zone: yes

TEST ATMOSPHERE
- Particle size distribution: 89% respirable (< 10 um)

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

>= 4 h

Concentrations:

4.86 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 the exposure (ca 1-2 hrs) and subsequently at least every day in the 14-day observation period. Body weights: Just before dosing and on days 2, 3, 4, 7, 10 and 14.
- Necropsy of survivors performed: Yes
- Other examinations performed: Organ weights: The lungs of each animal were weighed and the lung:body weight ratio determined. Histopathology was performed on the lungs of all animals.

Statistics:

Not performed.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 4.86 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 4.72 mg/L air

Based on:

other: enzyme concentrate dry matter

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 hrs after the end of exposure and all animals were normal the following day.

Body weight:

All body weights and body weight gains were normal throughout the study.

Gross pathology:

No abnormalities.

Other findings:

Lung:body weight ratios were within normal limits for all animals.
Histological examination: Slightly increased number of mucous cells in the bronchial epithelium of 2 animals, one male and one female. Otherwise, the changes seen were all compatible with the ususal background level seen in Sprague-Dawley rats of this age at the present contract research laboratory.

Table 1. Cellulase: Acute inhalation toxicity study in rats, particle size distribution

 

Group/ Conc. (mg/L)	Sample	Stage	Particle size range (um)	Amount collected (g)	% of total	% respirable (<0.9 um)	% respirable (< 6um)	% respirable (< 10um)
1 (4.86)	1	1 2 3 4 5 6 Filter back up	>10 6-10 3.5-6 2.0-3.5 0.9-2.0 0.5-0.9 0.25-0.5	0.27 0.91 0.51 0.57 0.07 0.03 -	11.4 38.6 21.6 24.2 3.0 1.3 -	1.3	50.1	88.7
	2	1 2 3 4 5 6 Filter back up	>10 6-10 3.5-6 2.0-3.5 0.9-2.0 0.5-0.9 0.25-0.5	1.31 1.45 0.74 0.72 0.20 0.14 -	28.7 31.8 16.2 15.8 4.4 3.1 -	3.1	39.5	71.3
Mean						2.2	44.8	80.0

 

Particle size measurements revealed that the respirable fraction (% of aerosol mass < 10um) was 80% (44.8% of the particles were < 6 um).

 

Interpretation of results:

other: Data insufficient for classification since the highest dose < 5 mg active enzyme protein/L.

Conclusions:

Cellulase causes only minimal evidence of toxicity in rats after 4 hours of inhalation of a concentration of 4.86 mg/L (corresponding to 4.72 mg enzyme concentrate dry matter/L). The LC50 for Cellulase is in excess of 4.72 mg enzyme concentrate dry matter/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 freeze-dried powder of Cellulase, batch PPC 3425, at a concentration of 4.86 mg/L (equivalent to 4.72 mg enzyme concentrate dry matter/L).

Particle size measurements revealed that the respirable fraction (% of aerosol mass < 10um) was 80% (44.8% of the particles were < 6 um). The animals were observed during exposure, for two 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. Body weight and body weight gain were unaffected by 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, Cellulase causes only minimal evidence of toxicity in rats after 4 hours of inhalation of a concentration of 4.86 mg/L (corresponding to 4.72 mg enzyme concentrate dry matter/L). The LC50 for Cellulase is in excess of 4.72 mg enzyme concentrate dry matter/L.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LC50

Value:

4 720 mg/m³ air

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 have 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

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Acute toxicity via the oral route has been tested. No signs of toxicity were observed in five male and five female rats treated with a single oral dose of 2960 mg enzyme concentrate dry matter/kg bw. The test was conducted according to OECD guidelines and GLP standards.

Acute toxicity via inhalation route has been tested. Cellulase causes only minimal evidence of toxicity in rats after 4 hours of inhalation of a concentration of 4.86 mg/L (corresponding to 4.72 mg enzyme concentrate dry matter/L). The LC50 for Cellulase is in excess of 4.72 mg enzyme concentrate dry matter/L. The test was conducted according to OECD guidelines and GLP standards.

Moreover, 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 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).

Acute Dermal Toxicity: No acute dermal toxicity study has been conducted. Acute toxicity via dermal route is waived based on exposure considerations and the known properties of the substance.

Investigations of percutaneous absorption of peptides, proteins and other molecules of large size revealed that percutaneous absorption of proteins is extremely low and of no toxicological relevance (ref. 1, 2, 4). This is further supported by the physico-chemical data, as cellulases are proteins with molecular weight above 13,000 D, with a low logPow value (<0), indicating that it has no bioaccumulation potential and can be anticipated to be readily biodegraded. Thus, systemic exposure following enzyme exposure at occupational exposure levels is without toxicological significance. In traditional acute dermal toxicity testing, mortality has been the endpoint. However, because enzymes show very low toxicity, extremely high doses that are far above human exposure levels typically have been applied. Therefore, acute toxicity studies are not considered to provide appropriate knowledge and are as such not a relevant test system for enzymes. Systemic exposure by the dermal route is unlikely based upon the existing toxicokinetic knowledge of enzymes, which due to their relatively large molecular weight, are not expected to be absorbed through the skin. Therefore, it can be assumed with high certainty that non-protease enzymes do not exert any acute dermal toxicity. Data waivers will further be established through exposure scenarios, i.e. no significant dermal exposure to consumers and professionals due to the toxicologically insignificant enzyme concentrations in end products and in the case of workers due to occupational hygiene measures associated with the prevention of respiratory allergy which includes protective clothing. In conclusion, toxicokinetic data together with evidence from animal studies and historical human experience derived from the use of detergent enzymes for decades confirm that exposure to technical enzymes will not result in any toxicologically relevant uptake by dermal route. Acute systemic exposure to a toxicologically significant amount of enzymes by this route can therefore be excluded and will further be prohibited by the obligatory setting of a DMEL value for enzymes, resulting in negligible exposure to enzymes (ref. 3).

References

1) Basketter,D.A., English,J.S., Wakelin,S.H., and White,I.R. (2008) Enzymes, detergents and skin: facts and fantasies. British journal of dermatology 158, 1177-1181

2) Pease,C.K.S., White,I.R., and Basketter,D.A. (2002) Skin as a route of exposure to protein allergens. Clinical and experimental dermatology 27, 296-300

3) D.A. Basketter, C. Broekhuizen, M. Fieldsend, S. Kirkwood, R. Mascarenhas, K. Maurer, C. Pedersen, C. Rodriguez & H.E. Schiff: Defining occupational and consumer exposure limits for enzyme protein respiratory allergens under REACH, Toxicology 268: 165-170, 2010.

4) Basketter D., Berg N., Broekhuizen C., Fieldsend M., Kirkwood S., Kluin C., Mathieu S. and Rodriguez C.Enzymes in Cleaning Products: An Overview of Toxicological Properties and Risk Assessment/Management. 2012. Reg. Toxicol. Pharmacol, 64/1: 117-123

5) US SDA. Risk assessment guidance for enzyme-containing products. 2005. Washington, Soap and Detergent Association

Justification for classification or non-classification

Based on the low acute oral toxicity of cellulase, the low likelihood of absorption of enzymes through the skin due to the physico-chemical properties of the enzyme and the low exposure to enzymes by inhalation enforced by the respiratory allergy exposure limits, cellulase should not be classified.