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Description of key information

The acute oral and inhalation toxicity of alpha-amylase has been tested. The acute oral toxicity test and the acute inhalation toxicity test were short-term toxicity tests conducted according to OECD guidelines, and in compliance with GLP. No acute dermal toxicity test was conducted but a 28-day repeated dose dermal toxicity study in rabbits has been performed.  The conclusion was that alpha-amylase is non-toxic by acute oral and inhalation exposure (GHS Toxicity category V). Based on weight of evidence, alpha-amylase does not exert any acute dermal toxicity under foreseeable realistic exposures for both workers and consumers.

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:
June 29 to August 31, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
Dec. 2001
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
female
Details on test animals and environmental conditions:
- Source: Charles River, Germany
- Fasting period before dosing: Overnight
- Housing: A maximum of 6 animals per sex per cage, transparent macrolon cages
- Weight at time of dosing: between 168-174 g
- Housing: In animal room with control of temperature and humidity
- Diet: Standard diet ad libitum
- Water: Tap water ad libitum
- Acclimatization period: 5 days
- Temperature (°C): 20-24°C
- Humidity : 45-70 %
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
MAXIMUM DOSE VOLUME APPLIED: 20 mL/kg

Doses:
Undiluted test material 20 mL/kg body weight, corresponding to 1911 mg total Total organic solids (TOS)/kg body weight (limit testing)
No. of animals per sex per dose:
6 (only females)
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: within 1 hour and within 4 hours after dosing and at least once daily throughout the observation period. Weighing: just prior to dosing on day 0 and on days 3, 7 and 14
- Necropsy of survivors performed: yes
Key result
Sex:
female
Dose descriptor:
LD0
Effect level:
1 911 mg/kg bw
Based on:
other: Total organic solids
Sex:
female
Dose descriptor:
other: Limit test - no effects were seen
Effect level:
> 20 mL/kg bw
Based on:
test mat.
Mortality:
No mortality.
Clinical signs:
No clinical signs.
Body weight:
Body weights and body weight gains normal.
Gross pathology:
No abnormalities.
Interpretation of results:
GHS criteria not met
Conclusions:
No signs of toxicity were observed among the rats treated with a single oral dose of 1911 mg total organic solids/kg, which was the highest possible dose at dose volume 20 mL/kg, using the undiluted test item.
Executive summary:

The objective of this study was to assess the acute toxicity of Alpha-amylase when administered as a single oral dose to rats followed by an observation period of 14 days. The purpose of the study was to satisfy regulatory demands because the enzyme is used for production of food in EU.

The study was conducted in accordance with the OECD Guideline No 423, “Acute Oral Toxicity – Acute Toxic Class method”. The design of the limit test was used. The test item was supplied as a brown liquid ready to use. The dose volume administered was 20 mL/kg body weight corresponding to 1911 mg/kg body weight, based on the Total Organic Solids (TOS) content of the test substance.

This was the highest possible dose level at dose volume 20 mL/kg body weight, using the undiluted test item. No mortality or clinical signs were observed after treatment and the overall body weight gain during the study was considered to be normal. The necropsy revealed no abnormalities.

In conclusion, no signs of toxicity were observed among the rats treated with a single oral dose of 1911 mg TOS/kg body weight, which was the highest possible dose at dose volume 20 mL/kg, using the undiluted test item.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
1 911 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 June 9 to July 14, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Harlan UK Ltd., Oxon, UK.
- Housing: Five animals of same sex per cage, (polypropylene cage)
- Weight at time of dosing: between 202-231 g (females), 244- 264 g (males)
- Housing: In animal room with control of temperature and humidity
- Diet: Standard diet ad libitum
- Water: Tap water ad libitum
- Acclimatization period: At least 5 days
- Temperature (°C): 19-25°C
- Humidity : 30-70 %
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Radleys, Saffron Walden, Essex, UK
- Exposure chamber volume: 30 L, flow rate 45 L/min providing 90 air changes per hour at an oxygen concentration of 20.8%.
- Method of holding animals in test chamber: Snout only
- Source and rate of air: Compressed air was supplied by an oil free compressor and passed through a water trap and respiratory quality filters before introduced to the nebuliser, the aerosol generator at a flow rate of 10 L /min.
- Method of conditioning air: The nebuliser was connected to a glass syringe attached to an infusion pump, which provided a continuous supply of test material under pressure, and to a metered compressed air supply.
- System of generating particulates/aerosols: The test material was aerosolised using a glass concentric jet nebuliser located on the top of the exposure chamber.
- 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 a high efficiency filter to a metered exhaust system
- Temperature, humidity, pressure in air chamber: Temperature and relative humidity were measured in the animals breathing zone and recorded every 30 minutes during the 4 hour exposure period; 17-19 degrees of C during exposure, 63-100% humidity during exposure, normal pressure of the atmosphere

TEST ATMOSPHERE
- Brief description of analytical method used: Seventeen air samples of 2 L during the 4 hour exposure period were taken, at 2 L/min, through weighed glass fibre filters. The collected material was weighed to determine the concentration of test material in the exposure chamber.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: 73.1% inhalable (< 4 um)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): MMAD=2.12 µm, GSD=2.82 µm

Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
>= 4 h
Concentrations:
4.96 mg/L (mean achieved atmosphere concentration of test item as received)
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: Hourly intervals during exposure, immediately and one hour after termination of exposure and subsequently once daily in the 14-day observation period. Weighing: Prior to treatment on the day of exposure and on Days 7 and 14.
- Necropsy of survivors performed: yes
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 4.96 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No mortality.
Clinical signs:
Clinical signs of hunched posture and piloerection were observed following exposure. This is commonly noted in animals due to the method of restraint. During and following exposure, the rats exhibited a wet appearance also commonly seen due to the method of restraint. In addition increased respiratory rate was noted in all animals during exposure, on removal from the chamber and one hour post-exposure. Animals recovered quickly to appear normal from the first day post-exposure.
Body weight:
Normal body weight development was noted during the study.
Gross pathology:
No abnormalities.
Interpretation of results:
GHS criteria not met
Conclusions:
In a group of ten rats no deaths occured following 4 hours of inhalation of alpha-amylase at a mean achieved atmosphere concentration of 4.96 mg/L, based on test material as received. It is therefore considered that the LC50 for alpha-amylase is greater than 4.96 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 alpha-amylase at a concentration of 4.96 mg/L (test material as received).

Particle size measurements revealed that the respirable fraction (% of aerosol mass < 4 µm) was 73.1%. The mass median aerodynamic diameter was 2.7 µm.

The animals were observed for clinical signs at hourly intervals during exposure immediately and one hour after termination of exposure and subsequently once daily in the 14-day observation period. After the observation period, the animals were sacrificed and examined pathologically.

No deaths occured during the study period. Clinical signs of hunched posture and piloerection were observed following exposure. This is commonly noted in animals due to the method of restraint. During and following exposure, the rats exhibited a wet appearance also commonly seen due to the method of restraint. In addition increased respiratory rate was noted in all animals during exposure, on removal from the chamber and one hour post-exposure. Animals recovered quickly to appear normal from the first day post-exposure.

No effect was observed on the body weight, and the pathological examination revealed no abnormalities.

 

Clinical signs of hunched posture and piloerection were observed following exposure. This is commonly noted in animals due to the method of restraint. During and following exposure, the rats exhibited a wet appearance also commonly seen due to the method of restraint. In addition increased respiratory rate was noted in all animals during exposure, on removal from the chamber and one hour post-exposure. Animals recovered quickly to appear normal from the first day post-exposure. No effect was observed on the body weight and necropsy revealed no abnormalities.

In conclusion, no deaths occured in rats after 4 hours of inhalation of a mean achieved atmosphere concentration of alpha-amylase of 4.96 mg/L. LC50 for alpha-amylase is therefore considered to be greater than 4.96 mg/L.

 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LC50
4 960 mg/m³
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

The acute oral and inhalation toxicity of alpha-amylase has been tested. The acute oral toxicity test and the acute inhalation toxicity test were short-term toxicity tests conducted according to OECD guidelines, and in compliance with GLP.

No acute dermal toxicity test was conducted, but dermal toxicity of alpha-amylase has been tested in a 28-day dermal toxicity study in rabbits (section on repeat dose dermal). The overall conclusion is that alpha-amylase does not exert any acute oral, dermal or inhalation toxicity.

 

Acute Oral Toxicity: No signs of toxicity were observed among the rats treated with a single oral dose of 1911 mg/kg body weight (expressed in mg total organic solids), which was the highest possible dose at dose volume 20 mL/kg, using the undiluted test item.

Acute Inhalation Toxicity: Increased respiratory rate was noted in all animals during exposure, on removal from the chamber and one hour post-exposure. Animals recovered quickly to appear normal from the first day post-exposure. No other effects related to the test compound were observed. In conclusion, no deaths occured in rats after 4 hours of inhalation and LC50 was therefore considered to be greater than 4.96 mg/L (of the test item as received).

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).

 

Acute Dermal Toxicity: No acute dermal toxicity study has been conducted but a 28 day repeated dose dermal toxicity study in rabbits has been conducted with no dermal toxicity of significance (one dose level : 12.4 mg/kg of the test item as received).

In general, enzymes are of very low toxicity due to ready biodegradability and very low bioavailability. 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 alpha-amylases are proteins with molecular weight above 10,000 D, with a low logPow value, 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 dermel 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 alpha-amylase, 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, alpha-amylase should not be classified.