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Description of key information

The acute oral and inhalation toxicity of xylanase 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 of the performed studies was that xylanase is non-toxic by acute oral and inhalation exposure (GHS Toxicity category V and IV, respectively). Based on weight of evidence, xylanase 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 04 - August 13, 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
The purpose of the study was to satisfy regulatory demands because the enzyme is used for food in China.
Qualifier:
according to
Guideline:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
Adopted 2001
Deviations:
no
GLP compliance:
yes
Test type:
acute toxic class method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: In-house bred animals
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 10 weeks
- Weight at study initiation: 189.64 - 203.03 g
- Fasting period before study: The animals were not fasted prior to dose administration
- Housing: Three animals were housed in a standard Polysulfone cage (size: L 430 x B 285 x H; 200 mm) with stainless steel mesh top grill. Clean sterilized paddy husk was provided as bedding material. Sterilized paper shreds were provided as nesting material for enrichment.
- Diet (e.g. ad libitum): Nutrilab rodent feed (Manufactured by Provimi Animal Nutrition India Pvt Ltd.) ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 7-9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 to 22.4°C
- Humidity (%): 50 to 64%
- Air changes (per hr): 12 to 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 22 June 2015 To: 15 July 2015
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Undiluted test material
Doses:
The animals were given two dosages of 10.3 mL/kg body weight with an interval of 4 hours between each administration (equivalent to 2102 mg TOS/kg body weight)
No. of animals per sex per dose:
6
Control animals:
no
Details on study design:
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Observation for clinical signs of toxicity and mortality at 30 to 40 min, 1 hr (±10 min), 2 hrs (±10 min), 3 hrs (±10 min) and 4 hrs (±10 min) after each administration on Day 1 and thereafter, once daily for clinical signs of toxicity and twice daily for mortality during the 14 days observation period. Individual animal body weight was recorded on Day 1 (before test item administration for first time), Day 7 and Day 14 during the observation period.
- Necropsy of survivors performed: yes
Statistics:
No
Sex:
female
Dose descriptor:
other: Fixed dose method - no effects were seen
Effect level:
> 20.6 mL/kg bw
Based on:
test mat.
Key result
Sex:
female
Dose descriptor:
other: Fixed dose method - no effects were seen
Effect level:
> 2 102 mg/kg bw
Based on:
other: Total Organic Solids (TOS)
Mortality:
No animals died during the study.
Clinical signs:
No clinical signs of toxicity and mortality were observed during the 14 days observation period.
Body weight:
No effect was observed on the body weights.
Gross pathology:
Effects on organs:
No gross pathological changes observed
Interpretation of results:
Category 5 based on GHS criteria
Conclusions:
No signs of toxicity were observed among the rats treated with a single oral dose of 2102 mg total organic solids/kg, which was the highest possible dose at dose volume 20.6 mL/kg, using the undiluted test item.
Executive summary:

The objective of this study was to assess the acute toxicity of Xylanase when administered as a single oral dose to six 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 China.

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 20.6 mL/kg body weight corresponding to 2102 mg/kg body weight, based on the Total Organic Solids (TOS) content of the test substance.

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 2102 mg TOS/kg body weight, which was the highest possible dose at dose volume 20.6 mL/kg, using the undiluted test item.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LD50
Value:
2 102 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 October 14, 1999 to February 15, 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 403 (Acute Inhalation Toxicity)
Version / remarks:
1981
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test type:
standard acute method
Limit test:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd, Manston Road, Margate, Kent UK.
- Age at arrival: 7-8 weeks old
- Housing: Five animals per cage. In holding cages (size 35 cm x 53 cm x 25 cm height).
- Diet (e.g. ad libitum): SDS rat and mouse diet (RM1), ad libitum, except during the 4 hr exposure
- Water (e.g. ad libitum): Tap water ad libitum, except during the 4 hr exposure
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20%
- Air changes (per hr): 12-15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: October 27, 1999 To: November 16, 1999
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
nose only
Vehicle:
air
Mass median aerodynamic diameter (MMAD):
>= 3.1 µm
Geometric standard deviation (GSD):
>= 2.19
Remark on MMAD/GSD:
Approximately 85% of the particles were considered of a respirable size (< 7 µm aerodynamic diameter).
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: ADG Developments Ltd., Hitchin, Hertfordshire, England
- Exposure chamber volume: 30 L
- Method of holding animals in test chamber: Snout only
- Source and rate of air: A supply of clean dried air was connected to the aerosol generator and the supply pressure was adjusted to give a flow rate of 15 litres/minute measured at the outlet of the generator. An in-line flow meter was used to monitor airflow throughout the exposure.
- Method of conditioning air and generate aerosols: A syringe filled with the test substance was fitted to a syringe pump and connected to a generator via Teflon tubing. To generate a chamber concentration at a target of 5 mL/L of air an initial feed rate of 1.4 mL/minute was selected.
- Method of particle size determination: Two air samples were taken during the exposure at a sampling rate of 2 L/minute using a Marple cascade impactor (Model 296, Graseby Andersen Inc., Atlanta, USA) to determine particle size distribution.
- Temperature, humidity, pressure in air chamber: Mean temp: 20.9°C (control group) and 21.1°C (test group), relative humidity: 41% (control group) and 98% (test group).

TEST ATMOSPHERE
- Brief description of analytical method used: Six air samples were taken during exposure. Chamber air was drawn at a measured rate of 2 L/minute, through a pre- weighed glass fibre filter (Whatman GF/A) mounted in an open face filter holder. Filters were dried for 20 minutes in an oven at 35-40°C, and then re-weighed for gravimetric analysis of the test aerosol.

TEST ATMOSPHERE (if not tabulated)
- Particle size distribution: 85% respirable (< 7 µm in aerodynamic diameter)
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): MMAD: 3.1µm. GSD: 2.19
Analytical verification of test atmosphere concentrations:
yes
Duration of exposure:
>= 4 h
Concentrations:
4.95 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: At the end of the chamber equilibration period, at 0.25, 0.5 and 1.0 hours then at hourly intervals during the exposure, immediately following completion of the exposure and then at 1.0 and 2.0 hours post-exposure. Subsequent, daily observations: Body weight: Twice during the week prior to exposure, immediately before exposure (day 0) and weekly during the observation period.
- Necropsy of survivors performed: yes
- Other examinations performed: Organ weights: The lungs, liver and kidneys of each animal were weighed.
Statistics:
Not performed.
Key result
Sex:
male/female
Dose descriptor:
LC50
Effect level:
> 4.95 mg/L air (analytical)
Based on:
test mat.
Exp. duration:
4 h
Mortality:
No mortality.
Clinical signs:
other: During exposure exaggerated breathing was first noted in test rats after 15 minutes of exposure, and after 2 hours of exposure in all test rats. The exaggerated breathing persisted in 1 test female up to 2 hours post exposure. The fur of all test rats wa
Body weight:
The mean bodyweight gain for male and female test rats were 81 g and 20 g compared with 95 g and 23 g for control male and female rats during the 14-days observation period respectively.
Gross pathology:
No abnormalities.
Interpretation of results:
Category 4 based on GHS criteria
Conclusions:
There were no unscheduled deaths or evidence of a toxic response following exposure of rats for 4 hours to a droplet aerosol generated from Xylanase, PPQ 6460 at a chamber concentration of 4.95 mg/L in air.
Executive summary:

The present study was performed in rats, in accordance with GLP and in compliance with EEC, OECD and US EPA (Health Effects Test Guidelines, OPPTS 870, 1300, Acute Inhalation Toxicity, 5 August 1998) and JMAFF test guidelines for acute inhalation studies. One control group and one test group each consisting of 5 females and 5 males were included.

The animals in the test group were exposed by snout-only exposure for 4 hours to air containing a liquid droplet aerosol generated from the test substance, Xylanase, PPQ 6460, at a concentration of 4.95 mg/L. In total 85% of the particles of Xylanase, PPQ 6460, were < 7 µm and had a mass median aerodynamic diameter of 3.1 µm.

The animals were observed during exposure, for two hours after the exposure and daily during the 14-day observation period. After the observation period, the animals were sacrificed and examined pathologically.

During exposure exaggerated breathing was noted in all animals. The exaggerated breathing persisted in 1 test female up to 2 hours post exposure. Fur/skin soiled with excreta was observed in all test and control rats immediately following exposure, persisting in 1 control male up to 2 hours post exposure. Wet fur (whole body) was noted in a proportion of control and test rats immediately following exposure, persisting up to 1-hour post exposure. Brown staining on the head was seen on a proportion of control and test rats up to 2 hours after exposure. These were temporary signs and were considered to be associated with the tube restraint for inhalation exposure.

The mean bodyweight gain for male and female test rats were 81 g and 20 g compared with 95 g and 23 g for control male and female rats during the 14-days observation period respectively.

In conclusion, as there were no unscheduled deaths or evidence of a toxic response following exposure of rats for 4 hours to a droplet aerosol generated from Xylanase, PPQ 6460 at a chamber concentration of 4.95 mg/L in air, the LC50 for Xylanase is in excess of 4.95 mg/L based on test material.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
LC50
Value:
4 950 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 xylanase have 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 overall conclusion of the performed studies was that xylanase does not exert any acute oral or inhalation toxicity. Based on physico-chemical properties and data from other enzymes, xylanase is not expected to present any hazard with regard to acute dermal toxicity under foreseeable realistic exposures for both workers and consumers.

 

Acute Oral Toxicity: No signs of toxicity were observed among the rats treated with a single oral dose of 2102 mg/kg body weight (expressed in mg total organic solids).

Acute Inhalation Toxicity: No clinical effects related to the test compound were observed and no animals died. In conclusion, the LC50 was therefore considered to be greater than 4.95 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. 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 xylanases are proteins with molecular weight above 7,700 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).

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 selection of acute toxicity – oral endpoint
Key study selected as most relevant study of highest concern.

Justification for selection of acute toxicity – inhalation endpoint
Key study selected as most relevant study of highest concern.

Justification for classification or non-classification

Based on the low acute oral toxicity of xylanase, 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, xylanase should not be classified.