Lyase, pectate

Endpoint summary

Description of key information

The acute toxicity of pectate lyase was tested by administration by gavage as a single oral dose to one group of five male and five female rats followed by an observation period of 14 days.

The dose volume administered was 2x12 mL/kg bodyweight of the undiluted test material. No clinical signs were observed and the overall body weight gain during the study was 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 2064 mg Total Organic Solids (TOS)/kg. abnormalities. In conclusion, no signs of toxicity were observed among the rats treated with a single oral dose of 2064 mg Total Organic Solids (TOS)/kg.

 

The acute inhalation toxicity of pectate lyase (batch number PPE 6345) was studied by nose-only exposure of one group of five male and five female rats each for a 4-hour period to a test atmosphere containing an aerosol of pectate lyase at a concentration of 5.07 ± 0.11 g/m3. The mass median aerodynamic diameter of the particles in the aerosol was 2.8 μm and the geometric standard deviation was 2.0. It was concluded that the 4-hour LC50 value of an aerosol of pectate lyase (Batch number PPE 6345) was larger than 5.07 g/m3 for both sexes.

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:

February 22, 1999 to May 5, 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC directive 92/69/EEC

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Møllegaard Avlslaboratorium, Ejby, DK
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Not specified
- Fasting period before dosing: 20 hours prior to dosing and until 1 hour after the last dosing procedure.
- Housing: Barriered rodent facility with control of temperature and humidity. Five of the same sex per cage in transparent plastic boxes with wire grid tops with aspen wood chips as bedding.
- Weight at the end of the acclimatisation period: Males 172-184 g, females 137-147 g.
- Diet: Altromin rat/mouse Breeding 1320 diet pellets ad libitum
- Water: Tap water added citric acid to pH 2-3 ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3 °C
- Humidity (%): 30-70%
- Air changes/hr: 8-12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 1999-02-26 To: 1992-03-12

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Undiluted test material.

Doses:

Dose volume was 24 mL/kg bodyweight. The animals were dosed twice with 12 mL/kg bodyweight within 2 hours.
24 mL/kg bodyweight is equivalent to 2064 mg TOS/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 symptoms after the first and second dosing procedure and 2 hours after the last dosing. Thereafter clinical observations were carried out once a day for the following 14 days. Animals were weighed on day 1 (before dosing) and on day 8 and before necropsy on day 15.
- Necropsy of survivors performed: yes

Statistics:

No

Sex:

male/female

Dose descriptor:

LD0

Effect level:

>= 24 mL/kg bw

Based on:

test mat.

Key result

Sex:

male/female

Dose descriptor:

LD0

Effect level:

>= 2 064 mg/kg bw

Based on:

other: Total Organic Solids (TOS)

Mortality:

No animals died during the observation period.

Clinical signs:

other: No clinical symptoms were seen during the observation period.

Gross pathology:

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

Interpretation of results:

GHS criteria not met

Conclusions:

No signs of toxicity were observed for pectate lyase tested on rats treated with a single oral dose of 2064 mg total organic solids/kg, which was the highest possible dose at dose volume 24 mL/kg, using the undiluted test item.

Executive summary:

The objective of the study was to assess the acute toxicity of pectate lyase when administered by gavage as a single oral dose to one group of five male and five female rats followed by an observation period of 14 days.

The study was conducted in accordance with the OECD Guideline No 401, 1987 “Acute Oral Toxicity”. The limit test was used. The test item was supplied as a brown liquid ready to use. The dose volume administered was 2x12 mL/kg bodyweight of the undiluted test material.

No clinical signs 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 2064 mg Total Organic Solids (TOS)/kg.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 064 mg/kg bw

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:

March 5, 1999 to April 20, 1999

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:

Adopted 12 May 1981.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 6-7 weeks
- Mean body weight just before exposure: 203 g (males), 149 g (females)
- Fasting period before study: Not specified
- Housing: In animal room in suspended stainless steel cages with wire-mesh floor and front.
- Diet (e.g. ad libitum): Standard diet ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20.5 - 23.0 °C
- Humidity: 38 - 60 %
- Air changes: 10 per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

IN-LIFE DATES: From: 1999-03-17 To: 1999-03-31

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks:

The test atmosphere was generated by nebulizing the test material into small droplets by using a compressed air driven nebulizer.

Mass median aerodynamic diameter (MMAD):

2.8 µm

Geometric standard deviation (GSD):

2

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Nose-only inhalation chamber from ADG Developments Ltd., Codicote, Hitchin, Herts, United Kingdom
- Exposure chamber volume: 50 L
- Method of holding animals in test chamber: Nose only
- Source and rate of air: Air was supplied in a volume of 53.1 L/min
- Method of conditioning air and generate aerosols: The test atmosphere was generated by nebulizing the test material into droplets by using as compressed air driven nebulizer (Schlick, Coburg, Germany, type 970/S).
- Method of particle size determination: Once during preliminary tests and once during exposure using a 10-stage cascade impactor (Anderssen, Atlanta, USA) with the largest cut-off size of 32 µm.
- Temperature and humidity in air chamber: Mean temperature during exposure 21.1 ± 0.2 °C (range 20.9 - 21.5 °C), mean relative humidity 53 ± 1 (range 51-54 %).

TEST ATMOSPHERE
- Brief description of analytical method used: Concentration of the test material in the test atmosphere was determined approx. once per hour by gravimetric analysis. 10 L test atmosphere samples were passed through fibre glass filters (Sartorius) at 5 L/min. The collected material was weighed to determine the concentration of test material in the exposure chamber.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE
- Particle size distribution: 80% contained in particles with an aerodynamic diameter equal to or smaller than 5 µm
- MMAD (Mass median aerodynamic diameter) / GSD (Geometric st. dev.): MMAD= 2.8 µm, GSD=2.0 µm

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

>= 4 h

Concentrations:

5.07 mg/L

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: Just before exposure, during exposure, shortly after and at least daily during the observation period. Weighing: Prior to exposure (day 0), and on days 7 and 14.
- Necropsy of survivors performed: yes

Key result

Sex:

male/female

Dose descriptor:

LC0

Effect level:

>= 5.07 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Mortality:

No mortality.

Clinical signs:

other: A decreased breathing rate could be seen in all animals. Its severity increased from slight to moderate over the 4 hour exposure period. Abnormalities could not be seen shortly after exposure or during the 14-days observation period.

Body weight:

No effects on body weight gain were observed.

Gross pathology:

No abnormalities.

Interpretation of results:

not classified

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 pectate lyase, PPE 6345 at a chamber concentration of 5.07 mg/L in air. Thus, it was concluded that the LC50 for pectate lyase is greater than 5.07 mg/m3.

Executive summary:

The acute inhalation toxicity of pectate lyase (Batch number PPE 6345) was studied by nose-only exposure of one group of five male and five female rats each for a 4-hour period to a test atmosphere containing an aerosol of pectate lyase at a concentration of 5.07 ± 0.11 g/m3. The mass median aerodynamic diameter of the particles in the aerosol was 2.8 μm and the geometric standard deviation was 2.0. After exposure, the animals were kept for a 14-day observation period. During exposure, breathing abnormalities were restricted to a decreased breathing rate. No abnormalities were seen shortly after exposure or during the 14-day observation period. No mortality occurred. Weight gain in the animals was considered normal for animals of this strain and age. No abnormalities were seen at necropsy.

It was concluded that the 4-hour LC50 value of an aerosol of pectate lyase (Batch number PPE 6345) was greater than 5.07 g/m3 for both sexes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

5 070 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:

other:

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

Based on the acute toxicity tests outcome, pectate lyase cannot be classified.