glutamyl endopeptidase

Endpoint summary

Description of key information

Glutamyl endopeptidase was not tested, but a similar enzyme, subtilisin, was tested in rats.

The LD50 value was 1.8 g/kg. The main clinical symptoms and the causes of death were ascribed to gastrointestinal disturbances. From other studies, it is known that when the proteolytic activity of subtilisin is inactivated by treatment with hydrochloric acid, the toxicological potential is decreased significantly. Thus, the proteolytic activity contributes essentially to the toxic effect.

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:

31-08-1984 to 08-02-1985

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Due to the similarity between the two enzymes, similar results are expected for glutamyl endopeptidase.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

1987

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Møllegaards Breeding Center Ltd., Ll. Skensved, Denmark
- Fasting period before dosing: 18 hours
- Housing: 5 animals per cage, Macrolon type IV, separate sex
- Weight at time of dosing: between 80 - 104 g
- Housing: In animal room with control of temperature and humidity
- Diet: Standard diet ad libitum
- Water: Tap water ad libitum
- Acclimation period: 4 days
- Temperature (°C): 17-26C
- Humidity: 31-55%

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

Tap water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 0, 50, 100 and 200 mg/mL, corresponding to 0, 29, 58 and 116 mg TOS/mL
- Amount of vehicle (if gavage): constant volume 20 mL/kg b.w.
- Justification for choice of vehicle: The test material is water soluble and any human exposure will be in aqueous solutions.
- Purity: tap water

MAXIMUM DOSE VOLUME APPLIED: 20 mL/kg

Doses:

0, 1000, 2000 and 4000 mg/kg bw, corresponding to 0, 580 mg, 1160 mg and 2320 mg TOS/kg body weight

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: 30 min., 2 hrs and daily after dosing. Weighing: once weekly
- Necropsy of survivors performed: Yes
- Other examinations performed: Clinical signs, body weight

Statistics:

LD50 was determined by iterative probit method from log-dose response (Finney DJ. Probit Analysis. Cambridge University Press, 1971).

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

1 800 mg/kg bw

Based on:

test mat.

95% CL:

> 1 200 - < 2 300

Mortality:

All animals in the top dose group died within 2 hours after dosing. In the mid dose group, four males and three females died within five hours after dosing and one female rat died 23 hours after dosing. See also table below.

Clinical signs:

Affected animals showed decreased activity, head drop and diarrhoea. No clinical signs in low dose group and negative controls.

Body weight:

No difference in body weights and body weight gains between the negative control group and the treated groups (surviving animals).

Gross pathology:

The animals that died shortly after dosing all showed extensive gastrointestinal bleedings, some also bleedings from the nostrils and anus. Surviving animals sacrificed at day 15 showed no abnormalities.

Other findings:

- Potential target organs: No dose related organ changes were found in the surviving animals.

Table showing the mortality

Dosage mg/kg	Group size		Mortality %
	Males	Females	Males	Females
4000 2000 1000 0	5 5 5 5	5 5 4* 5	100 80 0 0	100 80 0 0

* One female excluded due to misdosing

Interpretation of results:

Category 4 based on GHS criteria

Conclusions:

The LD50 value was 1.8 g/kg. The main clinical symptoms and the causes of death were ascribed to gastrointestinal disturbances. From other studies, it is known that when the proteolytic activity of Subtilisin is inactivated by treatment with hydrochloric acid, the toxicological potential is decreased significantly. Thus, the proteolytic activity contributes essentially to the toxic effect (Please see the HERA document attached to the summary Toxicological Information) .

Executive summary:

The acute toxicity of Subtilisin, batch PPA 1619, was investigated according to the principles of the later OECD test guideline 401. Four groups of five male and five female Wistar rats received the test material at a dosage of 0, 1000, 2000 and 4000 mg test material per kg body weight by oral administration (gavage). The animals were subjected to clinical observations daily for a fourteen-day observation period. Gross necropsy was carried out on all rats that died during the study or were sacrificed at termination of the study. All animals in the top dose group died within 2 hours after dosing. In the mid dose group, four males and four females died within 5 - 23 hours after dosing. Main clinical signs were decreased activity, head drop and diarrhoea. All decedents showed extensive gastrointestinal bleedings, some also bleedings from the nostrils and anus. The low dose group and the negative controls showed no clinical signs. All surviving animals had normal body weights and body weight gains. Surviving animals sacrificed at day 15 showed no abnormalities at necropsy. The oral LD50 value was determined to be 1800 mg/kg body weight for both male and female rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LD50

Value:

1 800 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

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Endpoint conclusion

Endpoint conclusion:

no study available

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

Classified as category 4 for acute toxicity.