Lyase, pectin

Endpoint summary

Description of key information

The acute median lethal oral dose (LD50) to rats of pectin lyase PPJ34366 was demonstrated to be greater than 2000 mg/kg bodyweight. There were no deaths during the study. There were no clinical signs of reaction to treatment throughout the study. All animals were considered to have achieved satisfactory bodyweight gains throughout the study. No abnormalities were noted in any animal at the macroscopic examination at study termination on Day 15.

Pectin lyase PPJ34366 is included in Category 5 or unclassified according to the Globally Harmonised System (GHS).

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:

03-01-2013 to 05-03-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

Adopted 17 December 2001.

Deviations:

no

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

other: Crl:CD(SD) albino rats

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 218-240 g
- Fasting period before study: overnight fasting prior to dosing and approximately four hours after dosing.
- Housing: They were housed in groups of three rats of the same sex, in solid bottomed polycarbonate cages with a stainless steel mesh lid. Each cage contained a quantity of autoclaved wood flake bedding. Cages, food hoppers, water bottles and bedding were changed at appropriate intervals.
- Diet: standard rodent diet (Rat and Mouse No. 1 Maintenance Diet) ad libitum
- Water: ad libitum
- Acclimation period: 6 days

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

IN-LIFE DATES: From: 2013-01-16 To: 2013-02-01

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The appropriate dose volume of the test substance was administered to each rat by oral gavage using a plastic syringe and plastic catheter. A record of the weight of each formulation dispensed and the amount remaining after dosing was made. The balance of these two weights was compared with the predicted usage as a check that the doses had been administered correctly. Formulations were stirred before and throughout the dosing procedure.

Doses:

Dose volume was 16 mL/kg bodyweight (equivalent to 2000 mg TOS/kg bodyweight).

No. of animals per sex per dose:

6 (female only)

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Cages of rats were checked at least twice daily for any mortalities. Observations for clinical signs of effect: soon after dosing, and at frequent intervals for the remainder of Day 1. On subsequent days, animals were observed once in the morning and again at the end of the experimental day (with the exception of Day 15 - morning only). The weight of each rat was recorded on Days 1 (prior to dosing), 8 and 15.
- Necropsy of survivors performed: All animals were humanely killed on Day 15 by carbon dioxide asphyxiation. All animals were subject to a macroscopic examination which consisted of opening the cranial, thoracic and abdominal cavities. The macroscopic appearance of all examined organs was recorded.

Statistics:

No

Key result

Sex:

female

Dose descriptor:

LD0

Effect level:

> 2 000 mg/kg bw

Based on:

other: Total Organic Solids (TOS)

Mortality:

There were no deaths during the study.

Clinical signs:

There were no clinical signs of reaction to treatment throughout the study.

Body weight:

All animals were considered to have achieved satisfactory bodyweight gains throughout the study.

Gross pathology:

No abnormalities were noted in any animal at the macroscopic examination at study
termination on Day 15.

Interpretation of results:

GHS criteria not met

Conclusions:

On the basis of the results for the present study, it was concluded that the acute median lethal oral dose (LD50) to rats of pectin lyase, batch PPJ34366 was demonstrated to be greater than 2000 mg/kg bodyweight.

Executive summary:

The study was performed to assess the acute oral toxicity of pectin lyase, batch PPJ34366 to the rat. A group of three fasted female rats received a single oral gavage dose of the test substance, ‘as supplied’ at a dose level of 2000 mg/kg bodyweight. As results at this dose level indicated the acute lethal oral dose of the test substance to be greater than 2000 mg/kg bodyweight, in compliance with the study guidelines, a further group of three fasted females was similarly dosed at 2000 mg/kg bodyweight to complete the study.

There were no deaths during the study. There were no clinical signs of reaction to treatment throughout the study. All animals were considered to have achieved satisfactory bodyweight gains throughout the

study. No abnormalities were noted in any animal at the macroscopic examination at study termination on Day 15.

The acute median lethal oral dose (LD50) to rats of pectin lyase, batch PPJ34366 was therefore demonstrated to be greater than 2000 mg/kg bodyweight.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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

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

GHS criteria not met.