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Description of key information

No signs of toxicity were observed among the rats dosed with a single oral dose of up to 3000 mg/kg bw. Therefore, a LD50 could not be estimated.

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:
From December 14, 1983 to March 27, 1984
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline available
Principles of method if other than guideline:
- Principle of test: Rats were deprived of food approximately 18 hours before dosing. Rats were dosed once orally by gavage and observed 30 minutes, 2 hours and daily after dosing for a period of seven days. Control and the group receiving the highest dose were observed daily for a period of 14 days after dosing. All rats were given the same volume 20 mL/kg b.w.
- Short description of test conditions: Animals weighed 69-81g at the time of dosing. Rats were caged in Macrolon cages (type IV) at 22-26 degrees centigrade and 24-38% relative humidity. They were offered a standard rodent diet (Brood Stock Feed for Rats and Mice - R3- Ewos) and tap water (adjusted to approximately pH 3.0 with citric acid) ad libitum.
- Parameters analysed / observed: 30 minutes and 2 hours after dosing rats were observed for any signs of reaction to dosing, especially the following symptoms: Death, increased and decreased respiration, ptosis, salivation, lachrymation, piloerection, head drop, tail constantly erected, increased or decreased motoric activity, abduced hind legs, unsteady gait, convulsion, writhing movements, tremor, high gait and diarrhea. Followed by daily observations for seven days. Control and high dose group were observed for another seven days and thus for a period of 14 days, whereas animals of all other groups were sacrificed after seven days.
GLP compliance:
no
Test type:
fixed dose procedure
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Møllegaard Breeder Center Ltd, Skensved, Denmark
- Fasting period before dosing: 18 hours fasting prior to dosing.
- Housing: Groups of 5, 2, 2, 2, 2 and 5 males and females respectively were caged in standard Macrolon cages (type IV).
- Weight at the time of dosing: 69 - 81 g
- Diet: Standard rodent diet (Brood Stock Feed for rats and mice - R3 - Ewos) ad libitum.
- Water: Tap water (adjusted to approximately pH 3.0 with citric acid) ad libitum.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-26 °C
- Humidity (%): 24-38%

IN-LIFE DATES: From: 1983-12-14 To: 1983-12-28
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
VEHICLE
- Concentration of test substance in vehicle: 0, 8.3, 16.7, 25.0, 50.0 or 100.0% (v/v)
Doses:
Dose volume was 20 mL/kg bw for all animals.
Concentration of test substance were: 0, 8.3, 16.7, 25.0, 50.0 or 100.0% (v/v) corresponding to mg test substance given in dry matter/kg bw: 0, 250, 500, 750, 1500 and 3000 mg/kg bw.
No. of animals per sex per dose:
5, 2, 2, 2, 2 and 5 males and females, respectively for the dosage groups 0, 250, 500, 750, 1500 and 3000 mg dry matter/kg bw.
Control animals:
yes
Details on study design:
- Duration of observation period following administration: 7 days.
- Frequency of observations and weighing: 30 minutes and 2 hours after dosing.
- Necropsy of survivors performed: Yes.
Statistics:
No
Key result
Sex:
male/female
Dose descriptor:
LD0
Effect level:
>= 3 000 mg/kg bw
Remarks on result:
other: No effects were observed
Mortality:
No animals died during the study.
Clinical signs:
No clinical signs were observed in any of the rats.
Body weight:
No changes in group mean body weights and group mean body weight gains were seen during the study.
Gross pathology:
The post mortem examination showed no macroscopic findings.
Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
No signs of toxicity were observed among the rats dosed with a single oral dose of up to 3000 mg/kg bw. Therefore, a LD50 could not be estimated.
Executive summary:

The objective of this study was to evaluate the acute oral toxicity of the enzyme TS-25-Amylase batch ABF7 in rats. The liquid test substance was diluted with tap water and given once orally in doses of 0, 250, 500, 750, 1500 and 3000 mg/kg bw to groups of 5, 2, 2, 2, 2 and 5 male and female Wistar rats, respectively.

Thirty minutes and 2 hours after dosing and daily in the following period of the study the rats were observed for any signs of response to treatment. The rats were weighed at weekly intervals. After 7 days the four intermediate groups were sacrificed and a macroscopic examination was performed. All rats from the highest dose level and the control group were observed for another 7 days and sacrificed and macroscopically examined.

No rats died during the study and no signs of toxicity or reactions to treatment were seen. Group mean values of body weight and body weight gain showed no differences in the period of the study. No dose-related organ changes were seen in any of the rats.

No signs of toxicity were observed among the rats treated with a single oral dose of up to 3000 mg/kg bw. Therefore, a LD50 could not be calculated.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

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.