Registration Dossier

Administrative data

Description of key information

No deaths, clinical symptoms or macroscopic findings were seen in any of the mice, but a decrease in body weight gain was seen in the highest dosed males.

As no deaths occured the LD50 value was estimated to be over 15000 mg/kg for both males and females and Novozym 230 could be classified "relatively harmless" according to Guidebook: Toxic Substances Control Act, George Dorninquez, 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:
key study
Study period:
20-03-1982 to 29-03-1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
according to
Guideline:
other: Toxic Substances Control Act, George Dominquez, 1977
Deviations:
no
GLP compliance:
no
Test type:
acute toxic class method
Limit test:
no
Species:
mouse
Strain:
NMRI
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Bred by Novozymes A/S (previously known as Novo Industri A/S)
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: Not stated
- Weight at study initiation: 18-22 g
- Fasting period before study: 18 hour fasting prior to dosing
- Housing: Barriered rodent facility with control of temperature, humidity and lighting. The cages were solid bottomed polycarbonate cages with a stainless steel mesh lid. Softwood bark-free fibre were provided as bedding. Aspen chew block and plastic shelter for environmental enrichment.
- Diet: standard diet (Brood Stock Feed for Rats and Mice - R3 - Astra Ewos) ad libitum
- Water: tap water (adjusted to approximately pH 3.0 with citric acid) ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23°C
- Humidity (%): 45-70%

IN-LIFE DATES: From: 1982-01-20 To: 1982-02-04
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Undiluted test material for the top dose.
Doses:
Doses were 1000, 2000, 5000, 15000 mL/kg bodyweight.
No. of animals per sex per dose:
Three groups of 5 male and 5 female and 2 groups of 2 male and 2 female NMRI mice.
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 15 days.
- Frequency of observations and weighing: Thirty minutes after dosing and daily in the following 9 or 15 days the mice were observed for any signs of reaction. The body weight was recorded just before dosing day 1 (all groups) and day 8 (group 1, 4 and 5), but due to a mistake no body weights were recorded day 15 before sacrifice.
- Necropsy of survivors performed: A macroscopic post mortem examina.tion was perforrned on all mice.
Statistics:
Body weight and body weight gain were subjected to statistical anal ysis using Student's t-test comparing group 1 (control) with group 4 and 5.
Key result
Sex:
female
Dose descriptor:
LD0
Effect level:
15 000 mg/kg bw
Based on:
other: Total Organic Solids (TOS)
Mortality:
No deaths or clinical symptoms were seen at anytime during the observation period.
Clinical signs:
No clinical symptoms were seen at anytime during the observation period.
Body weight:
A decrease in body weight and body weight gain was seen in males given the highest dose, 15000 mg/kg, as the only intergroup difference.
Gross pathology:
No dose related macroscopic findings were seen in any of the mice.
Interpretation of results:
GHS criteria not met
Conclusions:
No deaths clinical symptoms or macroscopic findings were seen in any of the mice, but a decrease in body weight gain was seen in the highest dosed males.
As no deaths occured the LD50 value was estimated to be over 15000 mg/kg for both males and females and Novozym 230 could be classified "relatively harmless" according to Guidebook: Toxic Substances Control Act, George Dorninquez, 1977.
Executive summary:

The objective of this study was to evaluate the acute oral toxicity in mice of the enzyme Novozym 230 (batch PPZ1281). The test substance was dissolved in tap water and given once orally in doses of 0, 1000 , 2000, 5000 and 15000 mg/kg body weight to groups of 2 or 5 male and 2 or 5 female NMRI mice. Thirty minutes after dosing and daily in the following 9 or 15 days, the mice were observed for any signs of reaction. The mice were weighed at weekly intervals. They were sacrificed at the end of the observation period and a macroscopic post mortem examination was performed on all the mice.

No deaths clinical symptoms or macroscopic findings were seen in any of the mice, but a decrease in body weight gain was seen in the highest dosed males.

As no deaths occured the LD50 value was estimated to be over 15000 mg/kg for both males and females and Novozym 230 could be classified "relatively harmless" according to Guidebook: Toxic Substances Control Act, George Dorninquez, 1977.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
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

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
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

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.