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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 (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November 26, 2001 to January 18, 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Test conducted following internationally accepted testing guidelines and performed according to the GLP.
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes
Type of study:
Buehler test
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Justification of Species: the guinea pig is conventionally used in skin sensitization studies to provide information on which human hazard can be judged, and is preferred by the regulatory agencies.
- Source: Charles River Laboratories; Wilmington, MA.
- Weight at study initiation: males 380-542 g; females 413-520 g
- Housing: suspended, wire bottom, stainless steel. 1-4 per cage (males separate from females).
- Diet: PMI Feeds, Inc.TM Guinea Pig Diet #5025; available ad libitum.
- Water: municipal water supply analyzed by TNRCC Water Utilities Division; available ad libitum from water bowls.
- A uarantine: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature:20 ± 3 °C
- Humidity: 30-70 %
- Air changes: 10-12 air changes/hour
- Photoperiod: 12-hour light/dark cycle

IN-LIFE DATES
Animlas were received by the laboratory at November 26 and December 10, 2001. They were treated with the test substance for first induction on December 19, for second induction on January 02 and for challenge on January 16, 2002. The in-life portion of the study was termined on January 02, 2002.

No contaminants were expected to have been present in the feed or water which would have interfered with or affected the results of the study.
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
Induction: 400 mg moistened w/0.5 ml corn oil
Challenge: 400 mg moistened w/0.5 ml corn oil
Concentration / amount:
Induction: 400 mg moistened w/0.5 ml corn oil
Challenge: 400 mg moistened w/0.5 ml corn oil
No. of animals per dose:
2 males and 2 females (Range-finding)
15 males and 15 females (definitive, of which 10 treated and 5 used as naive control)
Details on study design:
IRRITATION SCREENING
Two male and two female albino guinea pigs were selected for irritation screening to determine both the maximum dose producing no more than moderate irritation, and the maximum non¬irritating dose. Tested in the screening were 400 mg of test substance moistened wich com oil, and 75 %, 50 % and 25 % w/v concentrations of the test substance in corn oil.

PREPARATION of ANIMALS
On the day prior to each treatment, the animals were prepared by clipping the back of the trunk free of hair to expose a longitudinal area at least 8 x 10 cm of each animal. Individual body weights were recorded on Days 0 and 28.

MAIN STUDY
A. INDUCTION EXPOSURE
- Application: for each induction treatment, animals were treated by introducing the test substance beneath a 4 ply, 2.5 x 2.5 cm surgical gauze patch. Each gauze patch was placed laterally from the midline of the back on the left front quadrant of the exposure area and secured with a strip of non-irritating adhesive tape. A strip of clear polyethylene film was placed over the patch and securely taped.
- Exposure period: each animal was placed in a restrainer for approximately six hours. At the end of the exposure period, the animals were removed from the restrainers, the wrappings and patches were removed, and the animals were returned to their cages.
- Frequency of applications: animals were treated once weekly for three weeks. Induction treatments were on Days 1, 8 and 15.
- Concentrations: 400 mg of test substance moistened with 0.5 ml of com oil.
- Control group: control group (5 animals) remained untreated during the induction phase of the study.

B. CHALLENGE EXPOSURE
After a two-week rest period, all animals (treated and untreated) were each challenged at a virgin test site.
- Concentrations: 400 mg of test substance moistened with 0.5 ml of com oil.
- Day of challenge: on day 29
- Application: as for the induction treatment, animals were treated by introducing the test substance beneath a 4 ply, 2.5 x 2.5 cm surgical gauze patch. Each gauze patch was placed laterally from the midline of the back on the right front quadrant of the exposure area and secured with a strip of non-irritating adhesive tape. A strip of clear polyethylene film was placed over the patch and securely taped.

OBSERVATIONS and SCORING METHOD
Observations for skin reactions at each test site were made approximately 24 hours after each treatment. In addition, observations for skin reactions were made approximately 48 hours after the first induction treatment and 48 hours after the challenge treatment.

An average score for each time period was obtained by adding all of the scores for each time period and dividing by the number of test sites scored for that tie period. The test substance is considered a sensitizer if the mean irritation scores, the total number of animals with scores, and/or the total number of scores for the virgin test site ion the test group after the challenge treatment are appreciably greater than those for the naive challenge group.
The Buehler sensitisation scoring scale was used, based on the erythema observed:
no reaction: score 0
Very faint, usually nonconfluent: score 0.5
Faint, usually confluent: score 1
Moderate: score 2
Strong, with or without oedema: 3
Positive control substance(s):
yes
Remarks:
1-chloro-2,4-dinitrobenzene (DNCB). Conc. administered: induction 0.4 ml of 1 % w/v solution in 80 % ethanol; challenge 0.4 ml of 0.1 % w/v solution in acetone

The test substance produced no irritation in the naive control animals after the single treatment at challenge.

The test substance likewise produced no irritation in the test animals after the challenge treatment and therefore did not elicit a sensitizing reaction in guinea pigs.

Group Mean challenge scores
Neive control 0.0
Test group 0.0
Interpretation of results:
not sensitising
Remarks:
Migrated information according to the CLP Regulation Criteria used for interpretation of results: EU
Conclusions:
Not sensitising
Executive summary:

A skin sensitization study was conducted on 15 male and 15 female short-haired albino guinea pigs to determine if test substance produced a sensitizing reaction. Males and females were assigned to each of two goups, designated Groups I (5/sex) and II (10/sex). Group I animals remained untreated during the induction phase of the study and served as a negative control group. Group II animals, the test group, were treated with 400 mg of test substance moistened with 0.5 ml of corn oil (selected from previous screening). The animals were treated once weekly for three weeks, for a total of three treatments. After a two week rest period, all animals (Groups I and II) were challenged at a virgin test site with an application of 400 mg of test substance moistened with 0.5 ml of corn oil. The test substance produced no irritation in the naive control animals (Group I) after the single treatment at challenge. The test substance likewise produced no irritation in the test animals (Group II) after the challenge and therefore did not elicit a sensitizing reaction in guinea pigs.

Conlusion

Not sensitising

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

A skin sensitization study was conducted on 15 male and 15 female guinea pigs. Males and females were assigned to each of two groups, designated Groups I (5/sex) and II (10/sex). Group I animals remained untreated during the induction phase of the study and served as a negative control group. Group II animals, the test group, were treated with 400 mg of test substance moistened with 0.5 ml of corn oil (selected from previous screening). The animals were treated once weekly for three weeks, for a total of three treatments. After a two week rest period, all animals (Groups I and II) were challenged at a virgin test site with an application of 400 mg of test substance moistened with 0.5 ml of corn oil. The test substance produced no irritation in the naive control animals (Group I) after the single treatment at challenge. The test substance likewise produced no irritation in the test animals (Group II) after the challenge and therefore did not elicit a sensitizing reaction in guinea pigs.

Furthermore, the Environment Directorate General of the European Commission commissioned a collection of information about enzymes (European Communities, 2002); the publication provides an in-depth view of enzymes, their production, use and properties, as well as enzyme regulation in some non-EU countries. Based on literature review, it is stated that there are no indications that enzymes are skin sensitizers. Therefore, testing the skin sensitizing properties of enzymes is not considered to be relevant.


Migrated from Short description of key information:
Not skin sensitising

Justification for selection of skin sensitisation endpoint:
Test conducted following internationally accepted testing guidelines and performed according to the GLP.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Specific tests about the respiratory sensitisation potential were not conducted on the lysozyme hydrochloride.

The scientific literature indicates that enzymes have the potential for sensitisation of the respiratory tract. At present, no specific test exists to determine the lysozyme hydrochloride sensitisation potential via the inhalation route.

The Environment Directorate General of the European Commission commissioned a collection of information about enzymes (European Communities, 2002); the publication provides an in-depth view of enzymes, their production, use and properties, as well as enzyme regulation in some non-EU countries. It also outlines potential hazards to the environment and human health and gives recommendations as to how enzymes could be regulated in future. Concerning the respiratory sensitisation potential of enzymes, it is proposed to apply the precautionary principle and generally label enzymes with R42 „May cause sensitisation via inhalation”, unless convincing evidence to the contrary is provided. Potential for respiratory sensitisation has to be considered as a serious concern, in particular when applying enzymes in personal care and cosmetics products and for occupational exposure.

In the EFSA review (EFSA, 2010) on the potential of microorganisms, microbial products and enzymes to induce respiratory sensitization was reported that enzymes have been a cause of concern for several decades in food and feed industry, albeit at lower levels than in the bakery or detergent industry (Vanhanen et al., 2001).

The most reported cause of respiratory sensitization by enzymes in the food and feed industry is the case of alpha-amylase and baker’s asthma, but other enzymes such as proteases, cellulase and glucose oxydase are also a cause of concern. To compound the problem, industrial enzyme grades are often poor and contain other enzymes from the same production organism, leading to unexpected sensitization and cross-reactions (Vanhanen et al., 1996).

In the remarks of the EFSA document (EFSA, 2010), it was reported that the inherent properties of an enzyme may not be decisive, as to whether it is going to create inhalation allergies and that it is not known whether sensitization always lead to clinical allergy, but based on industrial hygiene experiences and knowledge from other areas of allergy, it must be assumed that sensitization would be a very strong risk factor for also developing symptoms upon continued exposure.

Some literature data about the lysozyme respiratory sensitisation cases reported are available:

- Egg lysozyme. A case report: a worker in a company manufacturing egg lysozyme powder for use in the pharmaceutical industry showed respiratory sensitization (asthma symptoms) (Vanhanen, 2001 and Bernstein et al., 1993).

- Serratial peptidase and lysozyme.A case report from pharmaceutical industry: One worker sensitized(asthma symptoms) (Vanhanen, 2001 and Park and Nahm, 1997)

- The patient sifted 50 mg of powdered enzyme back and forth between porous trays; lactose powder as placebo test; FEV1.0 and FVC recorded; for possible late reaction, a mini-Wright flowmeter for home measurements.Symptoms: Immediate asthmatic reaction (Vanhanen, 2001 and Bernstein et al., 1993)

- Interesting case reports on amylase include a double sensitization to lysozyme and amylase in a baker with rhinoconjunctivitis and asthma (EFSA, 210 and Santaolalla et al., 2002)

Considering the high concern related to the sensitization reaction and the information available about reported cases, it is appropriate to consider lysozyme as potentially able to cause allergy or asthma symptoms or breathing difficulties if inhaled.

REFERENCE

Bernstein JA, Kraut A, Bernstein DI, Warrington R, Bolin T, Warren CPW, Bernstein IL (1993). Occupational asthma induced by inhaled egg lysozyme. Chest 1993;103:532–535.

EFSA (2010). Scientific/technical report submitted to EFSA. Cyril Martela, Gunnar D. Nielsenb, Adriano Maric, Tine Rask Lichta, Lars K. Poulsena. Bibliographic review on the potential of microorganisms, microbial products and enzymes to induce respiratory sensitization. CFP/EFSA/FEEDAP/2009/02. Accepted for Publication on 22nd October 2010.

European Communities, (2002). Final ReportCollection of Information on Enzymes Contract No B4-3040/2000/278245/MAR/E2.

Park HS, Nahm DH (1997). New occupational allergen in a pharmaceutical industry: serratial peptidase and lysozyme chloride. Ann Allergy Asthma Immunol 1997;78:225–229.

Santaolalla M, De BM, De FC, Gandolfo M, Zubeldia J, et al. (2002) Double sensitization to enzymes in a baker. Allergy 57: 957.

Vanhanen M, Tuomi T, Tiikkainen U, Tupasela O, Tuomainen A, et al. (2001) Sensitisation to enzymes in the animal feed industry. Occupational and Environmental Medicine 58: 119-123.

Vanhanen M, Tuomi T, Hokkanen H, Tupasela O, Tuomainen A, et al. (1996) Enzyme exposure and enzyme sensitisation in the baking industry. OccupEnvironMed 53: 670-676.

Vanhanen Markku (2001). Exposure, sensitization and allergy to industrial enzymes. People and Work Research Reports 46. Finnish Institute of Occupational Health Department of Pulmology, Helsinki University Central Hospital Helsinki.


Migrated from Short description of key information:
Respiratory sensitizer

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.4 Respiratory or skin sensitisation section, skin sensitizer means a substance that will lead to an allergic response following skin contact.

The criteria to classify a substance as skin/respiratory sensitizer are reported into the second adaptation to technical progress*.

Based on the Buehler guinea pig test results, a substance in considered a skin sensitizer when:

- at least the 15 % of the animals respond at ≤ 0.2 % topical induction dose; or

- at least 60 % of the animals respond to the topical induction dose greater than 0.2 % - lower/equal to 20 %; or

- a number of animals equal/higher than 15 % to 60 % respond to a topical induction dose in the range of 0.2 - 20 %; or

- at least the 15 % of the animals respond at higher 20 % topical induction

The test substance produced no irritation in the test animals after the challenge treatment and did not elicit a sensitizing reaction in guinea pigs.

Substances shall be classified as respiratory sensitisers (Category 1, where data are not sufficient for sub-categorisation) if there is evidence in humans that the substance can lead to specific respiratory hypersensitivity.

The scientific literature indicates that enzymes in general, and lysozyme in particular, have the potential for sensitisation of the respiratory tract.

In conclusion, lysozyme hydrochloride is not classified as skin sensitizer; on the other hand, a classification as respiratory sensitiser (Resp. Sens. 1, H334) is proposed, according to the CLP Regulation (EC 1272/2008).

*Commission Regulation (EU) No 286/2011 of 10 March 2011, amending, for the purposes of its adaptation to technical and scientific progress, Regulation (EC) No 1272/2008 of the European Parliament and of the Council on classification, labelling and packaging of substances and mixtures