Mucorpepsin

Endpoint summary

Description of key information

Rennilase SP 252 did not cause any effects on Wistar rats administered a single dose of 14.2 g enzyme concentrate dry matter/kg bw followed by a 14 -day observation period. Therefore, it was considered as relatively harmless (according to Guidebook: Toxic Substance Control Act, edited by George Dominquez and published by CRC Press, Inc. in 1977).

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:

supporting study

Study period:

January 6, 1982 - March 15, 1982

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Principles of method if other than guideline:

- Principle of test: Acute toxicity of Rennilase SP 252 given once orally to rats followed by an observation period of 14 days.
- Short description of test conditions: The rats were deprived of food approximately 18 hours before dosing once orally by gavage. Rennilase SP 252 was dosed in 2.5 g/kg BW, 5 g/kg BW, 10 g/kg BW and 15 g/kg BW (equivalent to 2.4, 4.7, 9.5 and 14.2 g enzyme concentrate dry matter/kg BW). A control group was also included. All rats were carefully observed in the first two hours after dosing and daily in the following 14 days whereupon a gross post mortem examination was performed. All the rats were weighed on the day of dosing (day 1), on day 7 and on the day of termination (day 14).
- Parameters analysed / observed: Clinical observations, post mortem observations and body weight.

GLP compliance:

no

Remarks:

Before GLP was established. Internal quality check was used under this study.

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:

TEST ANIMALS
- Source: Møllegaard Breeding Centre Ltd., Ejby, Denmark
- Females nulliparous and non-pregnant: Yes
- Weight at study initiation: 47 - 62 g
- Fasting period before study: approximately 18 hours before.
- Housing: Macrolon cages (Type IV)
- Diet: Ad libitum (Brood Stock Feed for Rats and Mice - R3 - Astra Ewos)
- Water: Ad libitum (Adjusted to pH 3 with citric acid)

ENVIRONMENTAL CONDITIONS
Not specified

IN-LIFE DATES: From: 1982-01-28 To: 1982-02-11

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

VEHICLE
- Amount of vehicle (if gavage): 20 mL/kg bw

MAXIMUM DOSE VOLUME APPLIED: 15 g/kg bw

Doses:

2.4, 4.7, 9.5 and 14.2 g enzyme concentrate dry matter/kg BW

No. of animals per sex per dose:

10

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: 1 day, 7 day and 14 day
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight, organ weights, histopathology, other: microscopic and microscopic examination.

Statistics:

Body weights were compared between groups and no significant intergroup difference (p<0.05) was seen.

Key result

Sex:

male/female

Dose descriptor:

LD0

Effect level:

>= 14 200 mg/kg bw

Based on:

other: Enzyme concentrate dry matter

Mortality:

No animals died during the observation period.

Clinical signs:

other: No clinical signs were observed during the study period.

Gross pathology:

Two animals (1 in control and 1 in the group with highest dosage) showed pneumonia like areas, but microscopic findings omitted pneumonia. Perirenal edema was seen in animal from the control group.

Other findings:

Microscopic findings:
Control animal: In the lung, no abnormalities, only a slightly agonal blood aspiration. In the kidney, few degenerative tubuli in the outer zone of cortex predominantly due to pressure from the perirenal edema.
Animal from the highest dosage group: Agonal blood aspiration (no signs of inflammation).

Interpretation of results:

other: Data adequate, but insufficient for classification

Conclusions:

Rennilase SP 252 did not cause any effects on Wistar rats administered a single oral dose of 14.2 g enzyme concentrate dry matter/kg bw (equivalent to 1356 mg active enzyme protein/kg bw) followed by a 14-day observation period. Therefore, it can be considered as relatively harmless (according to Guidebook: Toxic Substance Control Act, edited by George Dominquez and published by CRC Press, Inc. in 1977).

Executive summary:

The object of this study was to evaluate the acute toxicity in rats of Rennilase SP 252, an enzyme of microbial origin. The test substance, a yellow-brown powder, was dissolved in tap water and given once orally in doses of 2.4, 4.7, 9.5 and 14.2 g enzyme concentrate dry matter/kg BW to groups of 10 male and 10 female Wistar rats. Tap water was given to another group of 10 male and 10 female Wistar rats as a control. The rats were carefully observed in the first two hours after dosing and subsequently daily in the following 14 days. On day 14 they were sacrificed and post mortem examined. The rats were weighed on the day of dosing, on day 7 and on the day of termination. No animals died during the study.

Rennilase SP 252 did not cause any effects on Wistar rats administered a single oral dose of 14.2 g enzyme concentrate dry matter/kg bw (equivalent to 1356 mg active enzyme protein/kg bw) followed by a 14-day observation period. Therefore, it was considered as relatively harmless (according to Guidebook: Toxic Substance Control Act, edited by George Dominquez and published by CRC Press, Inc. in 1977).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

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

Data adequate, but insufficient for classification.