Pullulanase

Endpoint summary

Description of key information

The acute oral and inhalation toxicity of pullulanase 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. The conclusion was that pullulanase is non-toxic by acute oral and inhalation exposure (GHS Toxicity category V). Based on weight of evidence, pullulanase 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:

March 28 - August 3, 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: ICH S4A - Single dose toxicity

Version / remarks:

1991

Deviations:

no

Principles of method if other than guideline:

A limit acute oral toxicity test was performed. A preliminary group of one male and one female rat received a single dose of the undiluted test material in a volume of 20 mL/kg body weight. These animals were observed for 7 days and since the dosing was well tolerated, additionally five males and females were dosed at 20 mL/kg body weight followed by a 14 days observation period, where clinical conditions and bodyweight were followed. At the end, the animals were killed and macroscopic examination was performed.

GLP compliance:

yes

Test type:

other: ICH S4A - Single dose toxicity, 1991

Limit test:

yes

Species:

rat

Strain:

other: Crl:CD® (SD)IGS BR rats

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River (UK) Ltd
- Age: 35 to 41 days at the day of treatment
- Fasting period before dosing: overnight fasting prior to dosing
- Housing: Barriered rodent facility with control of temperature and humidity. Five of the same sex in cages made of a polycarbonate body and floor with a stainless steel mesh lid. Lignocel type 3/4 wood shavings were provided as bedding. Aspen chew block for environmental enrichment.
- Weight at the end of the acclimatisation period: Males 149 - 168 g, females 116 -130 g.
- Diet: Pelleted standard rodent diet (Rat and Mouse No. 1 Maintenance Diet) ad libitum
- Water: Water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23 °C
- Humidity (%): 40-70%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Undiluted test material

Doses:

Dose volume was 20 mL/kg bodyweight (equivalent to 2.012 g TOS/kg bodyweight, 31230 PUN-RPA/kg)

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: soon after dosing, at least twice during the first hour after dosing (approximately 30 minutes apart) and subsequently at approximately one-hour intervals for the remainder of day 1. On subsequent days animals were inspected at least twice daily. Weighing was done immediately before dosing at day 1, and on day 8 and day 15.
- Necropsy of survivors performed: yes

Statistics:

No

Key result

Sex:

male/female

Dose descriptor:

other: Fixed dose method - no effects were seen

Effect level:

> 20 mL/kg bw

Based on:

test mat.

Key result

Sex:

male/female

Dose descriptor:

other: Fixed dose method - no effects were seen

Effect level:

> 2 012 mg/kg bw

Based on:

other: Total Organic Solids (TOS)

Mortality:

No animals died during the study.

Clinical signs:

No animals died and no sign of toxicity or ill health was seen at the routine physical examination.

Body weight:

No effect was observed on the body weights.

Gross pathology:

Effects on organs:
Macroscopic examination of animals killed on Day 15 of the observation period did not reveal any treatment-related findings.

Other findings:

Punctate clear cysts were found on the left kidney of female No. 11. This is a common finding in laboratory rodents and was considered to be unrelated to a single administration of Pullulanase, PPY 25645.

Interpretation of results:

GHS criteria not met

Conclusions:

No signs of toxicity were observed among the rats treated with a single oral dose of 2012 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 study was conducted as a limit test in accordance with the International Conference on Harmonisation (ICH) S4A – Single dose toxicity guidance, which is principally similar to the OECD Guideline No 420, "Acute Oral Toxicity - Fixed dose Method".

The test item was supplied as a brown liquid. The dose volume administered was 20 mL of the undiluted test material per kg body weight, corresponding to a dosage of 2.012 g Total Organic Solids (TOS)/kg bw. A group of five male and five female rats received the test material by a single oral administration (gavage). The animals were subjected to clinical observations daily for a fourteen day observation period and at termination of the study, gross necropsy of all animals was carried out.

 

No clinical effects 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 20 mL of the undiluted test material per kg body weight (= 2.012 g TOS/kg bw).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 012 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:

June 25 - July 9, 1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

(1981)

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, the sexes being kept separately
- Weight at time of dosing: Between 155-167 g (females), 195-202 g (males)
- Housing: In animal room with control of temperature and humidity
- Diet: Standard diet ad libitum
- Water: Tap water ad libitum
- Temperature (°C): 18-24°C
- Humidity: 45-70 %

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: An exposure chamber connected to 2 Wright dust feeders (Wright, B. M., 1950, J. Sci. Instr., 27, 12.) and two Broomwade compressors (type CAR 31) to supply compressed air.
- Exposure chamber volume: 41.5 L
- Method of holding animals in test chamber: Nose only
- Source and rate of air: Air was supplied in a volume of 18.0 L/min to prepare and disperse the dust.
- Method of conditioning air: The dust feeders were positioned at the base of the chamber. An extract duct at the top of the chamber was connected by way of a high efficiency filter to a metered vacuum system. The test compound particles were dispersed evenly throughout the chamber and exited through a series of small holes connected to a filtered vacuum line operating at a flow rate of approximately 2.0 L/min greater that the input flow rate (to reduce risk of contamination of the environment). The exposure chamber provided a single pass of the freshly generated dust.
- System of generating particulates/aerosols: The test atmosphere was produced by 2 Wright dust feeders. A scraper blade remoced powder from a prepacked canister at a steady rate. The loose powder was removed from the canister by a stream of clean, dry, compressed air. The dust passed through a jet and was impinged on a baffled plate to break up any aggregates. The air containing the finely dispersed dust was passed directly into the exposure chamber.
- Method of particle size determination: The particle size was estimated twice during the exposure period by using an Andersen Mini Sampler (2000 Inc., Salt Lake City, Utah, USA). The device consists of 4 metal impaction plates and a back-up glass fiber filter housed within an anodized aluminium can. The sampler was positioned and temporarily sealed in a port in the exposure chamber in the animal’s breathing zone. Chamber air was drawn through the sampler at a rate of 1.4 L/min using an Andersen 2000 vacuum pump for a recorded time period. Each impaction plate together with the back-up filter was weighed before and after sampling and the weight of material collected on each stage calculated by difference. From the results obtained the weight distribution of 5 size ranges of particles was calculated.
- Treatment of exhaust air: An extract duct at the top of the chamber was connected by way of a high efficiency filter to a metered vacuum system. The particles dispersed evenly throughout the chamber, and were exited through a series of small holes connected to a filtered vacuum line operation at a flow rate of approximately 2. 0 L/min greater than the input flow rate and therefore reducing risk of contamination of the environment.
- Temperature, pressure in air chamber: 21oC ± 2oC, normal pressure of the atmosphere

TEST ATMOSPHERE
- Brief description of analytical method used: The chamber concentration was estimated gravimetrically during the exposure periods at regular intervals. The gravimetric method used employed pressed glass fiber filters placed in a filter holder. The conical input side of the holder was positioned and temporarily sealed in a port in the exposure chamber in the animal’s breathing zone. Chamber air was drawn through the filter at a measured rate of 2.5 L/min using a vacuum pump. Seven air samples (volume between 15-37.5 L) during the 4 hour exposure period were taken. 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: 25.71% respirable (< 4.7 um)
- MMAD (Mass mean aerodynamic diameter) / GSD (Geometric st. dev.): MMAD=15.0 µm, GSD=4.1 µm

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

>= 4 h

Concentrations:

2.14 mg/L

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: During exposure, immediately after and frequently for the first 4 hours post exposure, and at least twice daily during the subsequent 14 day post exposure period. Weighing: Immediately before dosing and on days 2, 3, 4, 7, 10 and 14 post exposure.
- Necropsy of survivors performed: yes

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 2.14 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality.

Clinical signs:

other: All animals showed initial struggling with increased urination and defecation during the early part of the exposure period. Test animals showed a slight reduction in respiratory rate during exposure. In addition a clear nasal secretion and red nasal encru

Body weight:

No effects on body weight gain were observed.

Gross pathology:

No abnormalities.

Interpretation of results:

GHS criteria not met

Conclusions:

Pullulanase causes only minimal evidence of toxicity in rats after 4 hours of inhalation of a concentration of 2.14 mg/L. Thus it was concluded that the LC50 for pullulanase is in excess of 2.14 mg/L.

Executive summary:

In accordance with OECD guideline No. 403, a Limit Test was performed with two groups, one control group and one test group. Both groups consisted of 5 females and 5 males rats.

The animals were exposed by snout only exposure for 4 hours to air containing dust of Pullulanase, batch PPY1323, at a concentration of 2.14 mg/L.

Particle size measurements revealed that the respirable fraction (% of aerosol mass < 4.7 um) was 25.71%. The animals were observed during exposure, immediately after and frequently for the first 4 hours post exposure, and at least twice daily during the subsequent 14 day post exposure period. They were weighed immediately before dosing and on days 2, 3, 4, 7, 10 and 14 post-exposure. After the observation period, the animals were sacrificed and examined pathologically.

The clinical signs observed during exposure of the test material were confined to a slightly depressed respiratory rate, a clear nasal secretion after 30 minutes exposure and red nasal encrustation after 3 hours. No animals died during exposure or during the observation period, and the pathological examination revealed no abnormalities.

In conclusion, the LC50 (4 h) for pullulanase in Sprague-Dawley rats was not demonstrated other than an indicuation of the value being in excess of 2.14 mg/L.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

2 140 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

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

The acute oral and inhalation toxicity of pullulanase 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. Due to the low likelihood of absorption of the enzyme through the skin due to the physico-chemical properties the overall conclusion is that pullulanase 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 2012 mg/kg body weight (expressed in mg total organic solids (TOS)), which was the highest possible dose at dose volume 20 mL/kg, using the undiluted test item.

 

Acute Inhalation Toxicity: Slight reduction in respiratory rate was noted in the test animals during exposure. A clear nasal secretion and red nasal encrustations were observed during exposure and subdued appearance was noted for all treated animals following exposure. Normal grooming activity was observed on return to the cages. In conclusion, no deaths occurred in rats after 4 hours of inhalation and LC50 was therefore considered to be greater than 2.14 mg/L.

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.

However, 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 pullulanases are proteins with molecular weight above 53,000 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 dermal 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).

 

The overall conclusion is that pullulanase does not exert any acute oral, dermal or inhalation toxicity.

 

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 pullulanase, 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, pullulanase should not be classified.