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Administrative data

Description of key information

The animals at all the tested doses (1060, 1591 and 2111 mg TOS/kg body weight) did not reveal any clinical signs of toxicity throughout the observation period and no mortality occurred. The body weight and percent change in body weight of animals at all the steps increased with respect to Day 1. A complete gross pathological examination was carried out for the animals and there were no gross pathological changes observed in any of the animals.

Based on the results of the experiment and under experimental conditions employed, it can be concluded that the test item Serine Endopeptidase, batch PPA 26797 cannot be classified based on the GHS criteria.

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:
25-06-2015 to 27-08-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
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:
Sprague-Dawley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BIONEEDS INDIA PRIVATE LIMITED (in-house bred)
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 10 weeks
- Weight at study initiation: 160.07 g to 170.45 g
- Fasting period before study: No
- Housing: Three animals were housed in a standard Polysulfone cage (size: L 430 x B 285 x H 200 mm) with stainless steel mesh top grill having facilities for holding pelleted food and drinking water in water bottle fitted with stainless steel sipper tube. Clean sterilized paddy husk was provided as bedding material. Sterilized paper shreds were provided as nesting material for enrichment.
- Diet: Ad libitum. Nutrilab rodent feed (Manufactured by Provimi Animal Nutrition India Pvt Ltd)
- Water: Ad libitum. Deep bore-well water passed through activated charcoal filter and exposed to ultraviolet rays in Aquaguard water filter purifier was provided in plastic water bottles with stainless steel sipper tubes.
- Acclimation period: Healthy and young adult animals used for step I, step II and step III were acclimatized for seven, nine and eleven days, respectively.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 to 22.4°C
- Humidity (%): 50-61%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 02 July 2015 To: 20 July 2015
Route of administration:
oral: gavage
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
Undiluted test material for the top dose.
Doses:
The acute toxicity test was conducted with starting dose 1060 mg TOS/kg body weight, followed by 1591 mg TOS/kg body weight and the highest dose level of 2111 mg TOS/kg body weight. Three animals were used per step (total nine animals).
Fixed dose volume of 5.3 mL/kg body weight was used with increased frequency of administration to gain the final dose volume.
No. of animals per sex per dose:
3 (female only)
Control animals:
no
Details on study design:
The total quantity of test item to be administered per day was administered in divided doses of two (Step I), three (Step II) and four (Step III) times with approximately 4 hour interval between each administration. Test item administration was sequential and allowing at least 24 hours before dosing the next set of animals. The time interval between dosing at each level was determined by the onset, duration and severity of toxic signs.
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: All the animals were observed for clinical signs of toxicity and mortality at 30 to 40 min, 1 hr (±10 min), 2 hrs (±10 min), 3 hrs (±10 min) and 4 hrs (±10 min) after each administration on Day 1 and thereafter, once daily for clinical signs of toxicity and twice daily for mortality during the 14 days observation period.
- Necropsy of survivors performed: At the end of observation period, all the animals were sacrificed by exsanguination in deep Isoflurane anaesthesia. A complete gross pathological examination was carried out for terminally sacrificed animals.
Statistics:
No
Key result
Sex:
female
Dose descriptor:
LD0
Effect level:
>= 2 111 mg/kg bw
Based on:
other: Total Organic Solids (TOS)
Mortality:
No mortality during 14 days of observation period.
Clinical signs:
The animals did not reveal any clinical signs of toxicity and mortality during 14 days of observation period.
Body weight:
There were no treatment related changes observed in body weight and percent change in body weight of animals dosed at 1060, 1591 and 2111 mg TOS/kg body weight for step I, step II and step III, respectively with respect to day 1. All the animals exhibited
normal increase in body weight on Day 7 and Day 14 with respect to Day 1.
Gross pathology:
There were no gross pathological changes observed in the animals dosed at 1060, 1591 and 2111 mg TOS/kg body weight for step I, step II and step III, respectively during necropsy.
Interpretation of results:
GHS criteria not met
Conclusions:
The animals at all the tested doses (1060, 1591 and 2111 mg TOS/kg body weight) did not reveal any clinical signs of toxicity throughout the observation period and no mortality occurred. The body weight and percent change in body weight of animals at all the steps were increased with respect to Day 1. A complete gross pathological examination was carried out for the animals and there were no gross pathological changes observed in any of the animals.
Based on the results of the experiment and under experimental conditions employed, it was concluded that the test item serine endopeptidase, batch PPA26797 could not be classified based on the GHS criteria.
Executive summary:

The test item serine endopeptidase, batch PPA26797 was evaluated for Acute Oral Toxicity in Sprague Dawley Rats. The acute toxicity test was conducted with starting dose 1060 mg TOS/kg body weight, followed by 1591 mg TOS/kg body weight and the highest dose level of 2111 mg TOS/kg body weight. Three animals were used per step (total nine animals). Fixed dose volume of 5.3 mL/kg body weight was used with increased frequency of administration to gain the final dose volume.

All the animals were observed for clinical signs of toxicity at 30 to 40 minutes, 1 hr (±10 min), 2 hr (±10 min), 3 hr (±10 min) and 4 hr (±10 min) after each administration on Day 1 and once daily thereafter for clinical signs and twice daily for mortality.

The animals at all the tested doses (1060, 1591 and 2111 mg TOS/kg body weight) did not reveal any clinical signs of toxicity throughout the observation period and no mortality occurred.

Individual animal body weight was recorded at receipt, Day 1 before test item administration, Day 7 and Day 14 during the observation period for step I, step II and step Ill animals. The body weight and percent change in body weight of animals at all the steps were increased with respect to Day 1.

All the animals (step I, step II and step Ill) were observed for 14 days, sacrificed by exsanguination in deep Isoflurane anaesthesia on Day 15 and subjected to necropsy. A complete gross pathological examination was carried out for the animals and there were no gross pathological changes observed in any of the animals.

Based on the results of the experiment and under experimental conditions employed, it was concluded that the test item serine endopeptidase, batch PP A26797 could not be classified based on the GHS criteria.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

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.