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Description of key information

Acute toxicity via the oral route has been tested. No signs of toxicity were observed at the highest dose toxicity of 2000 total protein mg/kg bw. The test was conducted according to OECD guidelines and GLP standards. Acute toxicity via the inhalation and 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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 420 (Acute Oral Toxicity - Fixed Dose Method)
Deviations:
no
GLP compliance:
yes
Test type:
fixed dose procedure
Limit test:
yes
Species:
rat
Strain:
other: HanTac:WH
Remarks:
SPF (Specific Pathogen Free)
Sex:
female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic M&B A/S, Dk-8680 Ry, Denmark
- Females (if applicable) nulliparous and non-pregnant: not reported
- Age at study initiation: not reported
- Weight at study initiation: 140-151 g
- Fasting period before study: Overnight; after treatment, feed was withheld for a further 3 hours
- Housing: Transparent polycarbonate cages (macrolone type III, floor area 810 cm2). The cages were cleaned and the bedding changed at least twice a week.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±3°C
- Humidity (%): 55±15%
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light
Route of administration:
oral: gavage
Vehicle:
other: distilled water
Details on oral exposure:
VEHICLE: Distilled water

MAXIMUM DOSE VOLUME APPLIED: 16 mL/kg body weight

DOSAGE PREPARATION (if unusual): Prior to use, the test substance was thawed in the refrigerator overnight. The test substance dose formulations were prepared by diluting the test substance with vehicle. The dose formulations were prepared on the respective days of treatment and stored at room temperature until used.
Doses:
2000 mg/kg
No. of animals per sex per dose:
5
Control animals:
yes
Remarks:
For the animal welfare of the sighting study animal, a female companion animal was housed with the sighting study animal. The companion animal was observed for clinical behaviour and subjected to gross necropsy (results not included in report).
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Animals were weighed prior to treatment on Day 1, and on Days 2, 3, 8, and 15; each rat was observed 15 minutes and 1, 3, and 6 hours after administration and thereafter daily for a period of 14 consecutive days.
- Necropsy of survivors performed: yes
Statistics:
None
Key result
Sex:
female
Dose descriptor:
LD50
Effect level:
> 2 000 other: mg total protein/kg bw
Based on:
test mat.
Mortality:
No mortality was observed during the study.
Clinical signs:
Sighting Study: At the observations 1 and 3 hours after treatment, the sighting animal appeared less active. Furthermore, the animal had piloerection 3 hours after treatment. However, the animal appeared normal at all other observations.

Main Study: At the observations 1, 3, and 6 hours after treatment, all animals had piloerection. At all other observations all 4 animals appeared normal.
Body weight:
Sighting Study: The sighting animal had a normal body weight gain from treatment until termination of the study.

Main Study: All 4 animals had a normal body weight gain from treatment until termination of the study.
Gross pathology:
No macroscopic abnormality of tissues or organs was noted at necropsy for the sighting study or the main study.
Interpretation of results:
GHS criteria not met
Conclusions:
LD50: >2000 mg total protein/kg bw
Executive summary:

The acute oral toxicity of the test substance was studied in rats after administration of a single oral dose followed by an observation period of 14 days. A preliminary sighting study, using one female animal, was included in order to estimate the dose effect for toxicity and to provide information on dose selection for the main study. The study was conducted in accordance with OECD Guideline 420. The study was initiated with a sighting study in one female rat, in which 2000 mg total protein/kg body weight was examined in order to provide information on dose selection for the main study. On the basis of the results of the sighting study, the main study was performed in 4 additional female rats given a dose of 2000 mg total protein/kg body weight.

All animals survived the treatment. Slight signs of toxicity (piloerection and less activity) were observed among the animals on the day of treatment. However, no signs of toxicity were observed on any of the following days. All four animals had a normal body weight gain from treatment until termination of the study. The post-mortem inspection revealed no abnormalities. Under the experimental conditions, it was found that the minimal lethal dose of the test substance was above 2000 mg total protein/kg body weight.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 000 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. The database can thus be considered of high quality.

Acute toxicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Acute toxicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The acute oral toxicity of aspergillopepsin I has been tested, while the acute inhalation and dermal toxicity were waived.

 

Acute Oral Toxicity:

The acute oral toxicity was an acute toxicity study in accordance with OECD guideline, and in compliance with GLP. The conclusion was that aspergillopepsin I should not be classified.

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 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 for both workers and consumers are low and without any toxicological relevance. Commonly, the occupational exposure limit (OEL) value for workers is 60 ng active enzyme protein/m3(8 hour time-weighted average value) in EU countries. The dose level of enzyme needed to perform an acute inhalation assay is much higher and irrelevant to all known exposure scenarios. In the few cases where LC50 values could be established, the LC50 values were more than a factor of 100,000 times higher than the actual OEL value of 60 ng/m3based on pure active enzyme protein (ref. 4), indicating that concentrations 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:

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 of the enzyme. This group of enzymes are proteins with high molecular weight (ref. http://www.brenda-enzymes.info), they have a low logPow value (<0 i.e. low lipophilicity), indicating that they have no bioaccumulation potential and can be anticipated to be readily biodegradable. 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. 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 aspergillopepsin I, 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, it should not be classified.