Mannanase, endo-1,4-β-

Endpoint summary

Description of key information

The acute oral and acute inhalation toxicity of mannanase has been tested. Both studies 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 mannanase is non-toxic by acute oral or inhalation exposure. Based on weight of evidence, mannanase does not exert any acute dermal toxicity under foreseeable realistic exposures for either workers or 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:

From January 21, 1991 to August 29, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.1 (Acute Toxicity (Oral))

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 U.K. Limited, Margate, Kent, England.
- Fasting period before dosing: Overnight prior to and approximately 4 hours after dosing.
- Housing: Five animals per cage, metal cages with wire mesh floors.
- Weight at time of dosing: Between 114-134 grams
- Housing: In animal room with control of temperature and humidity
- Diet: Standard diet ad libitum
- Water: Tap water ad libitum
- Acclimation period: 8 days
- Temperature (°C): 20-22°C
- Humidity: 68% (mean daily relative humidity value)

Route of administration:

oral: gavage

Vehicle:

other: Distilled water

Details on oral exposure:

Diluted at a concentration of 50% w/v in distilled water.

Doses:

3.32 g total protein/kg bogyweight, 10 mL/kg bodyweight

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days after dosing
- Frequency of observations and weighing: soon after dosing, every 5 hours for the remainder of the day of dosing, twice a day for the rest of the study period. Weighing on Day of dosing (Day 1), Day 8, and Day 15.
- Necropsy of survivors performed: yes (macroscopic post mortem examination)

Statistics:

No

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 3.32 other: g/kg bw

Based on:

other: Total protein

Mortality:

Male: 5 g/kg bw; Number of animals: 5; Number of deaths: 0
Female: 5 g/kg bw; Number of animals: 5; Number of deaths: 0

Clinical signs:

other: Pilo-erection was observed in all rats within two minutes after dosing and throughout the remainder of Day 1 (day of dosing). No clinical signs were observed by Day 2.

Gross pathology:

Effects on organs:
No treatment related findings were observed. Histopathology was not performed.

Interpretation of results:

study cannot be used for classification

Conclusions:

In conclusion, the acute oral lethal dosage (LD50) to rats of the present Mannanase enzyme, Mannase EM, was greater than 3.32 g total protein/kg bw.

Executive summary:

The study was performed according to GLP and the procedures were according to EEC Methods for the determination of toxicity, Directive 84/449/EEC (OJ No. L251, 19.09.84), Part B, Method B.1. Acute Toxicity (oral).

The test item was supplied as a beige powder and was prepared at a concentration of 50% w/v in distilled water. The dose volume administered was 10 mL/kg.

The only clinical sign observed was piloerect coat in all rats within two minutes after dosing and throughout the remainder of Day 1 (day of dosing). No clinical signs were observed by Day 2. The overall body weight gain during the study was considered to be normal. The post-mortem inspection revealed no abnormalities.

In conclusion, the acute lethal oral dose to rats of the present mannanase enzyme, Mannase EM, was found to be greater than 3.32 g total protein/kg bw.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Value:

3 320 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 August 16, 1999 to November 30, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: 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

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, Manston Road, Margate, Kent UK.
- Housing: Five animals per cage
- Housing: In holding cages (size 35 cm x 53 cm x 25 cm height) in animal room with control of temperature and humidity
- Diet: SDS rat and mouse diet (RM1), ad libitum, except during the 4 hr exposure
- Water: Tap water ad libitum, except during the 4 hr exposure
- Acclimation period: 8 days
- Temperature (°C): 20-26°C
- Humidity: 44-66 %

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

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: Filtered, oil-free compressed air for the production of the test atmosphere was supplied by compressors.
- System of generating particulates/aerosols: The test substance was supplied to the generator via the feed line from a syringe driven at a constant rate by a syringe pump (Precidor® type 5003).
- 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. The volume of air sampled was measured using a wet-type gas meter (Model DM3B, G. H. Seal Ltd., London, England).
- Temperature, humidity, pressure in air chamber: Mean temp: 19.9°C (control group) and 19.6°C (test group), relative humidity: 49% (control group) and 95% (test group).

TEST ATMOSPHERE
- Brief description of analytical method used: Seven 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 15 minutes in an oven at 30-35°C, and then re-weighed for gravimetric analysis of the test aerosol.

TEST ATMOSPHERE
- Particle size distribution: 87% respirable (< 7 µm in aerodynamic diameter)

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

>= 4 h

Concentrations:

0.45 mg enzyme concentrate dry matter/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: Just before exposure 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:

> 0.45 mg/L air (analytical)

Based on:

other: Enzyme concentrate dry matter

Exp. duration:

4 h

Mortality:

No mortality.

Clinical signs:

other: No clinical signs. Fur/skin soiled with excreta was observed in all test and control rats immediately after exposure, but was not present 1 hour later. Brown staining on the head was seen in 2 male and female control rats and 2 male test rats up to 1 hour

Body weight:

The mean bodyweight gain for male test rats was 69 g compared with 47 g for control rats during the 14-day observation period; however, all body weights and body weight gains were within the expected range throughout the study.

Gross pathology:

No abnormalities.

Other findings:

The mean liver weight of test males was higher (+14%) than the control values. This was considered to be associated with the slightly heavier bodyweights of the test rats.

Interpretation of results:

study cannot be used for classification

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 Mannanase, PPE 6432 at a chamber concentration of 0.45 mg enzyme concentrate dry matter/L.

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, Mannanase, PPE 6432, at a concentration of 0.45 mg enzyme concentrate dry matter/L. Mass median aerodynamic diameter and percentage of particles < 7 µm of Mannanase, PPE 6432, were 2.9 µm and 87%, respectively.

 

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.

 

Fur/skin soiled with excreta was observed in all test and control rats immediately after exposure, but was not present 1 hour later. Brown staining on the head was seen in 2 male and female control rats and 2 male test rats up to 1 hour 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 test rats was 69 g compared with 47 g for control rats during the 14-day observation period; however, all body weights and body weight gains were within the expected range throughout the study period. Moreover, the mean liver weight of test males was higher (+14%) than the control values. This was considered to be associated with the slightly heavier bodyweights of the test rats.

 

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 Mannanase, PPE 6432 at a chamber concentration of 0.45 mg enzyme concentrate dry matter/L in air, and the LC₅₀ for mannanase is thus in excess of 0.45 mg enzyme concentrate dry matter/L.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Value:

450 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:

the study does not need to be conducted because the physicochemical and toxicological properties suggest no potential for a significant rate of absorption through the skin

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In general, enzymes are of very low toxicity due to ready biodegradability and very low bioavailability. In traditional acute 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 on the existing toxicokinetic knowledge of enzymes, which due to their relatively large molecular weight, are not expected to be absorbed through the skin (Basketter et al. 2008, Smith Pease et al. 2002). Therefore, it can be safely assumed that technical enzymes do not exert any acute dermal toxicity (Basketter et al 2012). This conclusion is confirmed by the toxicological data available. Sub-acute dermal toxicity studies with protease in rabbits (Novozymes, unpublished data) did not provide evidence for systemic effect to enzymes. This finding is confirmed by data from acute dermal toxicity studies (Novozymes, unpublished data) of other enzyme products in both rats and rabbits. None of these studies revealed any acute toxic effect through the dermal administration route. No clinical signs or adverse effects due to systemic exposure could be observed. 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 (Basketter et al 2010). In vivo acute dermal toxicity studies will not add any value and cannot be expected to provide valuable knowledge and are considered scientifically and ethically unjustified. Therefore, in accordance with column 2 of REACH Annex VIII acute toxicity testing by the dermal route is inappropriate.  

References:

- Basketter DA, English JS, Wakelin SH, White IR (2008). Enzymes, detergents and skin: facts and fantasies.Br. J. Dermatol., 158 (6):1177-1181.

- Smith Pease CK, White IR, Basketter DA (2002). Skin as a route of exposure to protein allergens.Clin. Exp. Dermatol., 27(4):296-300.  

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

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

Justification for classification or non-classification

Both acute oral and acute inhalation toxicity studies have been conducted testing mannanase. Both studies were conducted according to OECD guidelines and in compliance with GLP. The conclusions were that mannanase did not give rise to any toxicological concerns following either acute oral exposure (3.32 g total protein/kg bw) and inhalation exposure (0.45 mg enzyme concentrate dry matter/L). However, data is not suffecient for classification.