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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From March 23, 2009 to 05 August, 2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to GLP and according to OECD test guideline 429 (2002).
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
The non-radiolabelled 5-bromo-2-deoxyuridine (BrdU) was used to determine the cell proliferation.
Principles of method if other than guideline:
The present LLNA test used the non-radioactive modification, which utilised non-radiolabelled 5-bromo-2-deoxyuridine (BrdU) instead of 3H-methyl thymidine, and the LNC suspensions were analyzed by flow cytometry for BrdU incorporation and the total number of LNC. This is a fully valid and accepted procedure.
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Jackson Lab., Bar Harbor, ME.
- Age at study initiation: approx. 8-12 weeks old
- Weight at study initiation: 18 - 23 g (main study)
- Housing: Individually in suspended wire cages
- Diet (e.g. ad libitum): ad libitum PMI Rodent Chow (Diet #5001)
- Water (e.g. ad libitum): Ad libitum mains tap water
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Animal room was temp controlled
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 2009-03-26 To: 2009-03-31
Vehicle:
propylene glycol
Concentration:
50 %, 25% and 10% of the test material in PG.
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: Propylene Glycol (PG) was found to be suitable for dosing.
- Irritation: The test material, 25 µL of 10%, 25% and 50% in PG, was applied to the dorsal surface of both ears of two mice per group for three consecutive days. Any signs of toxicity or local irritation was recorded. No irritation was noted at any of the concentrations tested.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA) according to OECD 429
- Criteria used to consider a positive response: The stimulation index (SI) for each group was calculated by dividing the proliferative response (i.e. the BrdU+ lymph node cells, LNC) of each test article treated animal with the mean proliferative response of the corresponding vehicle control group. The mean SI and the standard deviation was calculated for each group. According to the OECD guideline, substances at a given concentration with a SI ≥ 3 should be classified as a skin contact sensitiser.

TREATMENT PREPARATION AND ADMINISTRATION:
The treatment groups consisted of five animals each, dosed with the test material at concentrations of 10%, 25% and 50% in PG. The induction phase of each group comprised a daily topical application of 25 µL test material spread over the entire dorsal surface of both ear lobes for three consecutive days. A negative control group receiving the vehicle, PG, and a positive control group receiving 25% alpha-hexylcinnamaldehyde in PG were included. 5 days after initial application, each mouse received an intraperitoneal injection of 200 µL Dulbeccos phosphate buffered saline (DPBS) containing a dose of approx 150 mg 5-bromo-2’-deoxyuridine (BrdU) per kg bw. Five hours later, the mice were euthanized and the draining auricular lymph node of each ear was excised. A single cell suspension of lymph node cells was prepared from each mouse in fetal bovine serum and fixed with 85% ethanol.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Students' t-test was used to compare each group to the control group.
Positive control results:
The positive control material, alpha-hexyl cinnamic aldehyde (HCA), 25% v/v in PG, gave a positive response, SI = 14.5, and the study was therefore accepted as valid.
Parameter:
SI
Remarks on result:
other: (see Table 1 below)

Table 1. The results of the LLNA, expressed as mean number of proliferating cells in the lymph nodes of each treatment group and the stimulation index (SI) for each group.

Test material

Lymphocyte proliferation

(# BrdU+)

SI

Vehicle, PG

10267

1.0

25% HCA in PG

148989

14.5

Lipase 3, 10% in PG

7242

0.7

Lipase 3, 25% in PG

14078

1.4

Lipase 3, 50% in PG

8921

0.9

 

 

Interpretation of results:
GHS criteria not met
Remarks:
Migrated information
Conclusions:
The enzyme Lipase is classified as a dermal non-sensitiser.
Executive summary:

The study was performed according to OECD test guideline 429, using the non-radioactive modification, which utilised non-radiolabelled 5-bromo-2-deoxyuridine (BrdU) instead of the normally used tritiated methyl thymidine. The lymph node cell (LNC) suspensions were analyzed by flow cytometry for BrdU incorporation and the total number of LNC. The dermal irritation potential of the test article at 10%, 25% and 50% in Propylene glycol (PG) was determined first in three groups of two female CBA/J mice per group. No irritation was noted and concentrations of 10%, 25% and 50% in PG were selected for the main sensitization study.

In the sensitization study, the test article was applied at 10%, 25% or 50% in PG to the dorsum of each ear, once daily for three consecutive days to three groups of 5 female mice. Vehicle (PG) and positive control (25% HCA) were applied in the same manner to two groups of 5 female mice. Five days after the initial application, all animals received an intraperitoneal injection of BrdU. Five hours after the intraperitoneal injection, all mice were sacrificed, the auricular lymph nodes were isolated, single-cell suspensions of LNC were generated and the LNC suspensions were analyzed by flow cytometry for BrdU incorporation. The amount of BrdU-positive LNC was determined as a measure of the proliferative response of the local lymph node. The stimulation index (SI) was calculated by dividing the proliferative response of each treated animal by the mean proliferative response of the vehicle control group. The mean SI was calculated for each group. A stimulation index (SI) equal or above 3 indicates a positive response.

The SI for the positive control (25% HCA) and vehicle control (PG) groups were 14.5 and 1.0, respectively, validating the sensitivity of this local lymph node assay. The group SI for the test article at 10%, 25% and 50% in PG were 0.7, 1.4 and 0.9 indicating that the test article is not a skin sensitizer.

 

GHS Classification: Not classified

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Lipase was shown to have no sensitisation potential (a dermal non-sensitizer) when tested in the Local Lymph Node Assay using a suitable vehicle.

 

In support of this, the skin sensitisation potential of enzymes has been reviewed recently by Basketter et al. (ref. 1) and in publications 2, 3, 10 and 11, revealing that enzymes should not be considered skin sensitizers. In addition, there is an unequivocal statement from the trade organization of enzyme manufacturers AMFEP (www.amfep.org) on this topic showing that enzymes do not have skin sensitising potential. The lack of skin sensitising potential is substantiated by evidence from robust human experimental data and extensive in-use human studies performed with detergents containing enzymes (ref. 4-8; 11-16). All of these studies including those conducted with atopic individuals confirmed that the presence of enzymes in the detergents did not result in contact skin sensitisation.

 

References

1) Basketter,D.A., English,J.S., Wakelin,S.H., and White,I.R. (2008) Enzymes, detergents and skin: facts and fantasies. British journal of dermatology 158, 1177-118

2) HERA Human and environmental risk assessment on ingredients of household cleaning products - alpha-amylases, cellulases and lipases. 2005.

3) HERA Human and environmental risk assessment on ingredients of household cleaning products - Subtilisins (Proteases). Edition 2.0. 2007.

4) Bannan,E.A., Griffith,J.F., Nusair,T.L., and L.J.Sauers (1983) Skin testing of laundered fabrics in the dermal safety assessment of enzyme containing detergents. Journal of Toxicology - Cutaneous and Ocular Toxicology 11, 327-339

5) Griffith,J.F., Weaver,J.E., Whitehouse,H.S., Poole,R.L., and Newmann EANixon,G.A. (1969) SAFETY EVALUATION OF ENZYME DETERGENTS ORAL AND CUTANEOUS TOXICITY IRRITANCY AND SKIN SENSITIZATION STUDIES. Food and Cosmetics Toxicology 7, 581-593

6) Rodriguez,C., Calvin,G., Lally,C., and LaChapelle,J.M. (1994) Skin effects associated with wearing fabrics washed with commercial laundry detergents. Journal of Toxicology - Cutaneous and Ocular Toxicology 13, 39-45

7) Cormier,E.M., Sarlo,K., Scott,L.A., MacKenzie,D.P., Payne,N.S., Carr,G.J., Smith,L.A., Cua-Lim,F., Bunag,F.C., and Vasunia,K. (2004) Lack of type 1 sensitization to laundry detergent enzymes among consumers in the Philippines: results of a 2-year study in atopic subjects. Annals of Allergy Asthma and Immunology 92, 549-557

8) White,I.R., Lewis,J., and el,A.A. (1985) Possible adverse reactions to an enzyme-containing washing powder. Contact Dermatitis 13, 175-179

9) Basketter D., Berg N., Broekhuizen C., Fieldsend M., Kirkwood S., Kluin C., Mathieu S. and Rodriguez C.Enzymes in Cleaning Products: An Overview of Toxicological Properties and Risk Assessment/Management. 2012a. Reg. Toxicol. Pharmacol, 64/1: 117-123

10) Basketter D.; N. Berg; F. Kruszewski; K. Sarlo; B. Concoby. The Toxicology and Immunology of Detergent Enzymes. 2012b. J. Immunotox., 9, 320-326.

11) Andersen,P.H., Bindslev-Jensen,C., Mosbech,H., Zachariae,H., and Andersen,K.E. (1998) Skin symptoms in patients with atopic dermatitis using enzyme-containing detergents. A placebo-controlled study. Acta dermato-venereologica 78, 60-62

12) Belsito,D.V., Fransway,A.F., Fowler,J.F., Jr., Sherertz,E.F., Maibach,H.I., Mark,J.G., Jr., Mathias,C.G., Rietschel,R.L., Storrs,F.J., and Nethercott,J.R. (2002) Allergic contact dermatitis to detergents: a multicenter study to assess prevalence. Journal of the American Academy of Dermatology 46, 200-206

13) Lee,M.Y., Park,K.S., Hayashi,C., Lim,H.H., Lee,K.H., Kwak,I., and Laurie,R.D. (2002) Effects of repeated short-term skin contact with proteolytic enzymes. Contact Dermatitis 46, 75-80

14) Pepys,J., Wells,I.D., D'souza,M.F., and Greenberg,M. (1973) CLINICAL AND IMMUNOLOGICAL RESPONSES TO ENZYMES OF BACILLUS-SUBTILIS IN FACTORY WORKERS AND CONSUMERS. Clinical Allergy 3, 143-160

15) Peters,G., Johnson,G.Q., and Golembiewski,A. (2001) Safe use of detergent enzymes in the workplace. Appl.Occup Environ.Hyg. 16, 389-39

16) Zachariae,H., Thomsen,K., and Rasmussen,O.G. (1973) Occupational enzyme dermatitis. Results of patch testing with Alcalase. Acta dermato-venereologica 53, 145-148

 


Migrated from Short description of key information:
The Local Lymph Node Assay (LLNA) study was performed according to OECD test guideline 429. The group SI for the test article at 10%, 25% and 50% in propylene glycol were 0.7, 1.4 and 0.9, respectively, which indicate that the test article is not a skin sensitizer.
Enzyme products including lipase are further well documented not to be skin sensitizers in humans.

Justification for selection of skin sensitisation endpoint:
Key study selected as most relevant study of highest concern.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

For enzyme protein respiratory allergens, a DMEL for workers and consumers has been summarized and discussed in a recent publication (Ref. 1). The conclusion is drawn from a thorough review of existing occupational and consumer data on exposure by inhalation from the involved industrial partners in combination with medical data. As no valid animal models exist to test and rank respiratory sensitizers, the human surveillance data are the core of such evaluation. Any sub-categorization based on relative potency is not feasible (Ref. 2).

REFERENCES

1.Basketter DA, Broekhuizen C, Fieldsend M, Kirkwood S, Mascarenhas R, Maurer K, Pedersen C, Rodriguez C, Schiff HE: Defining occupational and consumer exposure limits for enzyme protein respiratory allergens under REACH. Toxicology. 268:165-170, 2010.

2. Basketter D.A., Kimber I. (2011) Assessing the potency of respiratory allergens: Uncertainties and challenges. Regul. Toxicol. Pharmacol., 61, 365-372.


Migrated from Short description of key information:
From occupational data it is well known that active enzymes including lipase are potential respiratory sensitizers. However, decades of experience has shown that enzymes can be used safely by ensuring that exposure is limited, supported by the DMEL for workers and consumers.

Justification for classification or non-classification

Lipase should not be classified as a skin sensitiser.

Lipase is classified as a respiratory sensitiser.