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Skin sensitisation

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Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
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: J.H. Draize in the F. & D.A. handbook, Appraisal of the Safety of Chemicals in Food, Drugs and Cosmetics (1959)
Deviations:
not applicable
Remarks:
Test was performed before actual guideline was established
Principles of method if other than guideline:
Test was performed before actual guideline was established.
GLP compliance:
no
Type of study:
Draize test
Justification for non-LLNA method:
Historical in vivo data available
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 300-500 g
- Diet: ad libitum
- Water: ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): thermostatically controlled, not further specified
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
0.1 % active ingredient
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
0.1 % active ingredient
No. of animals per dose:
6
Details on study design:
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 10 (1x0.05 ml; 9x0.1 ml)
- Test groups: Test substance in saline
- Site: back, not further specified
- Frequency of applications: every 2 days
- Duration: 0-18 days
- Concentrations: 0.1 % active ingredient


B. CHALLENGE EXPOSURE
- No. of exposures: 1 (0.05 ml)
- Day(s) of challenge: 32
- Test groups: Test substance
- Site: back, not further specified
- Concentrations: 0.1 % active ingredient
- Evaluation (hr after challenge): 4 and 24
Reading:
1st reading
Hours after challenge:
4
Group:
test group
Dose level:
0.1 % active ingredient
No. with + reactions:
0
Total no. in group:
6
Clinical observations:
Injection site pink with white centre, 7.0 mm mean diameter, 1.0 mm mean height.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 4.0. Group: test group. Dose level: 0.1 % active ingredient. No with. + reactions: 0.0. Total no. in groups: 6.0. Clinical observations: Injection site pink with white centre, 7.0 mm mean diameter, 1.0 mm mean height..
Reading:
2nd reading
Hours after challenge:
24
Group:
test group
Dose level:
0.1 % active ingredient
No. with + reactions:
0
Total no. in group:
6
Clinical observations:
Injection site pink with white centre, 9.0 mm mean diameter, 1.0 mm mean height; the reaction produced by the final challenge did not appear to be any greater in diameter, height or colour than the reaction produced by any of the previous injections.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0.1 % active ingredient. No with. + reactions: 0.0. Total no. in groups: 6.0. Clinical observations: Injection site pink with white centre, 9.0 mm mean diameter, 1.0 mm mean height; the reaction produced by the final challenge did not appear to be any greater in diameter, height or colour than the reaction produced by any of the previous injections..
Group:
negative control
Remarks on result:
not measured/tested
Group:
positive control
Remarks on result:
not measured/tested
Interpretation of results:
not sensitising
Remarks:
Migrated information
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. Additional rechallenge 2 weeks after primary challenge which increases validity of the study.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
Rechallenge 2 weeks after primary challenge
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
Rechallenge 2 weeks after primary challenge
GLP compliance:
yes (incl. certificate)
Type of study:
Buehler test
Justification for non-LLNA method:
Historical in vivo data available
Species:
guinea pig
Strain:
other: Himalayan spotted (Ibm: GOHI)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: RCC Ltd, Laboratory Animal Services, Fuellinsdorf, Switzerland
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 346-432 g
- Housing: Individually
- Diet (e.g. ad libitum): Pelleted standard Provimi Kliba 3418, batch no. 28/03, guinea pig breeding/maintenance diet, containing Vitamin C (Provimi Kliba Ag, Kaiseraugst, Switzerland), ad libitum
- Water (e.g. ad libitum): Community tap water from Fuellinsdorf, ad libitum
- Acclimation period: 2 weeks


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Induction 5%; Challenge 1 %
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Induction 5%; Challenge 1 %
No. of animals per dose:
Test group: 20 females, Control group: 10 females
Details on study design:
RANGE FINDING TESTS:
Two irritation screening tests were performed to determine the appropriate test substance concentrations for induction and challenge. Patching was performed in the same way as in the actual test. The following concentrations were tested: 100, 75, 50, 25 % (Irritations screening I). As the skin of these animals was seriously burnt at all concentrations, and they had to be killed in extremis, a second screening with 5, 3, 1 and 0.3 % (Irritation screen II) was performed. The most representative concentration to stimulate a state of immune hypersensitivity was 5 % used in the induction phase. The highest non-irritating concentration for the challenge was determined as the concentration of 1 % in purified water.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 2 weeks
- Test group: test substance
- Control group: untreated
- Site: left shoulder
- Frequency of applications: once per week (first application on d 0)
- Duration: for 6 hours each
- Concentrations: 5% in purified water


B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 29 and 43
- Test group: test substance
- Control group: test substance
- Site: 1st challenge (test and control group): left posterior quadrant of the side and back (2 patches); rechallenge (test group): right flank
- Concentrations: 1% in purified water
- Evaluation (hr after challenge): 24 and 48 hours after removal of the patches
Challenge controls:
Actually the control group was a challenge control, as the animals had not been treated in any way during the induction phase.
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde
Positive control results:
Twenty (at the 24-hour reading) and 19 (at the 48-hour reading) out of 20 test animals were observed with discrete/patchy to moderate/confluent eythema after the challenge treatment with the highest non-irritating concentration of alpha-hexylcinamaldehyde at 5 % in PEG 300. No skin effect was observed in the control group.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
1%
No. with + reactions:
11
Total no. in group:
20
Clinical observations:
Discrete/patchy erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1%. No with. + reactions: 11.0. Total no. in groups: 20.0. Clinical observations: Discrete/patchy erythema.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
1%
No. with + reactions:
11
Total no. in group:
20
Clinical observations:
Discrete/patchy erythema
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1%. No with. + reactions: 11.0. Total no. in groups: 20.0. Clinical observations: Discrete/patchy erythema.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
1%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No skin reactions
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No skin reactions.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
1%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No skin reactions
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No skin reactions.
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
1%
No. with + reactions:
1
Total no. in group:
20
Clinical observations:
Moderate/confluent erythema
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 24.0. Group: test group. Dose level: 1%. No with. + reactions: 1.0. Total no. in groups: 20.0. Clinical observations: Moderate/confluent erythema.
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
1%
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
Moderate/confluent erythema in one, discrete/patchy erythema in another animal
Remarks on result:
other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 1%. No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: Moderate/confluent erythema in one, discrete/patchy erythema in another animal.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
5%
No. with + reactions:
20
Total no. in group:
20
Clinical observations:
Discrete/patchy to moderate/confluent erythema
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 5%. No with. + reactions: 20.0. Total no. in groups: 20.0. Clinical observations: Discrete/patchy to moderate/confluent erythema.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
5%
No. with + reactions:
19
Total no. in group:
20
Clinical observations:
Discrete/patchy to moderate/confluent erythema
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 5%. No with. + reactions: 19.0. Total no. in groups: 20.0. Clinical observations: Discrete/patchy to moderate/confluent erythema.

There were no deaths during the course of the study.

No symptoms of systemic toxicity were observed in the animals.

No skin effects were observed in the first and second induction week. In the third week of induction, discrete/patchy to moderate confluent erythema (grade 1 and 2) were observed in 12/20 test animals after treatment with the test item at 5 % in purified water.

Conclusion:

According to the criteria of OECD Guideline 406 Skin Sensitisation the test substance does not have to be considered as sensitising to the skin in a non-adjuvant test system.

Interpretation of results:
not sensitising
Remarks:
Migrated information
Endpoint:
skin sensitisation, other
Remarks:
in vivo (non-LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. Reliability changed from "1" to "2" according to the ECHA guidance document “Practical guide 6: How to report read-across and categories (March 2010).
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Historical in vivo data available
Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France, L'Arbresle, France
- Age at study initiation: 1 - 2 months old
- Weight at study initiation: males 332 - 401 g; females 335 - 396 g
- Housing: individually
- Diet (e.g. ad libitum): 106 pelleted diet (SAFE, Villemoisson, Epinay-sur-Orge, France), ad libitum
- Water (e.g. ad libitum): Millipore-filtered (0.22 micron) drinking water, ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 30 - 70
- Air changes (per hr): approx. 12
- Photoperiod (hrs dark / hrs light): 12/12
Positive control results:
The sensitivity of the experimental technique is regularly assessed using a known moderate sensitizer. In a recent study performed under the experimental conditions of the testing laboratory, the strain of guinea pigs used showed a satisfactory sensitisation response in 100 % of the animals.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
1 %
No. with + reactions:
5
Total no. in group:
20
Clinical observations:
discrete erythema (grade 1), dryness of the skin in one animal
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1 %. No with. + reactions: 5.0. Total no. in groups: 20.0. Clinical observations: discrete erythema (grade 1), dryness of the skin in one animal.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
1 %
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
moderate erythema (grade 2), dryness of the skin in both animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1 %. No with. + reactions: 2.0. Total no. in groups: 20.0. Clinical observations: moderate erythema (grade 2), dryness of the skin in both animals.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
1 %
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
discrete erythema (grade 1)
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 1 %. No with. + reactions: 3.0. Total no. in groups: 10.0. Clinical observations: discrete erythema (grade 1).
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
1 %
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
discrete erythema (grade 1), dryness of the skin
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 1 %. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: discrete erythema (grade 1), dryness of the skin.

In the control group, a discrete erythema was observed on the right flank (test item application) of 3/10 animals at the 24-hour reading and persisted in one of them, associated with a dryness of the skin, at the 48-hour reading.

In the treated group, on the right flank, a discrete erythema (grade 1) was noted in 5/20 animals at the 24-hour reading; at the 48-hour reading, a moderate erythema (grade 2) was observed in 2/20 animals. A dryness of the skin was also noted in 1/20 and 2/20 animals at the 24- and 48-hour readings, respectively.

Conclusion:

The higher number of animals demonstrating cutaneous reactions (5/20, corresponding to 25 %) in the 24-hour reading have to be ascribed to the irritant character of the test substance and must be considered as local irritation reaction. Even this number does not exceed the threshold of 30% positive reactions, demanded by the underlying OECD guideline for sensitisation tests with adjuvant, required to consider a test substance as sensitising.

After 48 hours, only 2/20 animals (10%) with cutaneous reactions remain, which have to be considered as sensitised.

Under the experimental conditions chosen, using the maximisation method of Magnusson and Kligman, the test item finally induced cutaneous reactions in 2/20 (10%) guinea pigs. According to the criteria of Council Directive 67/548/EEC (DSD) and the CLP regulation the test item does not need to be classified as sensitising to the skin.

Interpretation of results:
not sensitising
Remarks:
Migrated information
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study with acceptable restrictions. Applied doses (except the lowest) induce local skin irritation, only lowest dose acceptable for assessment, lymph nodes were pooled, therefore, no statistics possible.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Applied doses cause local skin irritation of various grades
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: Harlan Netherlands, Horst, The Netherlands
- Age at study initiation: 8 - 12 weeks (beginning of acclimatization)
- Weight at study initiation: 16 - 24 g (ordered)
- Housing: Individually
- Diet (e.g. ad libitum): Pelleted standard Kliba 3433, batch no. 94/04 mouse maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugst), ad libitum
- Water (e.g. ad libitum): Community tap water from Itingen, ad libitum
- Acclimation period: 6 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
other: ethanol/water (7/3, v/v) and undiluted, respectively
Concentration:
5, 10, 25, 50 % (w/v) and 100 % (undiluted)
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: In a non-GLP solubility pre-test, the test item was tested in different vehicles: acetone/olive oil (4/1, v/v), ethanol water (7/3, v/v), N,N-dimethylformamide (DMF), dimethylsulfoxide (DMSO) and propylene glycol. A suitable vehicle (ethanol/water) was selected and used in the main test. Detailed results were included in the report.
- Irritation: In a non-GLP animal pre-test in two mice, the test item was tested at four different concentrations: 10, 25, 50 % in ethanol/water (7/3, v/v) and 100 % (undiluted), on one ear each. One day after a single topical application no irritation effects were observed at these concentrations. The pre-test results determined that 100 % (undiluted ) was the highest technically applicable concentration.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Determination of lymphocyte proliferation by liquid scintillation counting of incorporated 3H-methyl thymidine (3HTdR)
- Criteria used to consider a positive response: 1) exposure to at least one test substance concentration resulted in an 3HTdR-incorporation of at least 3-fold or greater than that recorded in control mice; 2) the data are compatible with a conventional dose response, although allowance must be made (especially at high concentrations) for either local toxicity or immunological suppression.


TREATMENT PREPARATION AND ADMINISTRATION:
Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear lobe (left and right) with different test item concentrations of 5, 10, 25, 50 % in ethanol/water, (7/3, v/v) and 100 % (undiluted). The application volume, 25 µl, was spread over the entire dorsal surface (diameter approx. 8 mm) of each ear lobe once daily for 3 consecutive days. A further group of mice was treated with an equivalent volume of the relevant vehicle alone (control animals). A hair dryer was passed briefly over the ear's surface to prevent loss of any of the test substance applied.

3HTdR was purchased from Amersham International (Amersham product code no. TRA 310; specific activity 2Ci/mmol; concentration 1 mCi/ml). five days after the first topical application, all mice were administered with 250 µl of 77.01 µCi/ml 3HTdR (equal to 19.3 µCi 3HTdR) by intravenous injection via a tail vein.

Approximately 5 hour after treatment with 3HTdR all mice were euthanized by inhalation of CO2 (dry ice). The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechnaical disaggregation through stainless stell gauze (200 µm mesh size). After washing twice with phospahte buffered saline (approx. 10 ml) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4°C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1ml) and transferred to glass scintillation vials with 10 ml of 'Irga-Safe Plus' scintillation liquid and thoroughly mixed.

The level of 3HTdR incorporation was then measured as radioactive disintegrations per minute (pdm) on a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 ml-aliquots of 5 % trichloroacetic acid. The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporated into lymph node cells of the respective test group relative to that of the control group (Stimulation Index, S.I.). Before dpm/node values were determined, mean scintillation-background dpm was subtracted from test and control raw data.
Statistics:
The mean values and standard deviations were calculated for body weights.
A statistical analysis was conducted for assessment of the dose-repsonse relationship, and the EC3 value was calculated according to the equation EC3= (a-c)[(3-d)/(b-d)]+c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are repectively the co-ordinates of the two pair of data lying immediately below and above the S.I. value of 3 on the LLNA dose response plot.
Calculation of standard deviation for DPM was not possible as lymph nodes were directly pooled after excision.
Parameter:
SI
Remarks on result:
other: 5 %: 2.4
Parameter:
SI
Remarks on result:
other: 10 %: 6.2 (irritation)
Parameter:
SI
Remarks on result:
other: 25 %: 14.7 (irritation)
Parameter:
SI
Remarks on result:
other: 50 %: 19.0 (irritation)
Parameter:
SI
Remarks on result:
other: 100 %: 26.0 (irritation)
Parameter:
EC3
Remarks on result:
other: EC3 = 5.8 % (w/v)
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Control: 251 dpm/lymph node 5 %: 602 dpm/lymph node 10 %: 1555 dpm/lmph node 25 %: 3685 dpm/lymph node 50 %: 4764 dpm/lymph node 100 %: 6534 dpm/lymph node

No mortality occurred during the study period.

Clinical signs:

No clinical signs were observed in any animals of the control or the 5 % group. On the second application day a slight to severe ear erythema was observed at both dosing sites in all mice of the 10 % group (slight), 25 % group (moderate), 50 % group (moderate) and 100 % group (undiluted, severe), persisting for the remainder of the in-life phase of the study.

Body weights were recorded prior to first application and prior to necropsy, they were unaffected by treatment.

Conclusion:

A test substance concentration of 5.8 % (w/v) was sufficient to produce a stimulation index of 3 (EC3) under the test conditions chosen in this study.

The LLNA is based on the assessment of lymphocyte proliferation in the lymph node draining the application site during the induction phase of sensitisation. One has to keep in mind that all kinds of inflammatory reactions, among those also irritation, have the same effect on the lymph nodes draining a site of inflammation. From an immunological standpoint the LLNA has to be considered as an incomplete test, as the considered endpoint is not the elicitation of challenge-induced dermal hypersensitivity reactions. Sensitisation is characterised by the existence of memory T-cells, which mediate the secondary immune response resulting in skin erythema and edema upon challenge.

Therefore, although considered as the standard assay to assess sensitising properties by authorities over the last few years, the LLNA is not suitable for substances with irritating characteristics, which will also induce an unspecific cell proliferation in the draining lymph nodes that cannot be distinguished from a specific response. The issue has been addressed by many publications in the recent years and has also been the topic of a CESIO workshop in February 2010 in Bruxelles, where many examples for false-positive results in the LLNA with irritant substances have been demonstrated by industry, among those as the most remarkable example SLS, which has been the prime example for an irritating but not sensitising substance over the last 30 years. In such cases a guinea pig study should be considered, which adresses the appropriate endpoint, the elicitation of dermal responses by antigen-specific T-cells and more closely resembles the natural process of delyaed type hypersensitivity responses.

Although demonstrated as being non-irritating up to applications of 100% (undiluted) test substance in a preliminary test, the test substance has demonstrated to induce distinct irritant responses ranging from slight to severe erythema at both ears and starting already at 10% of test substance concentration in vehicle in the main study, which remained over the whole in-life phase of the test. One can anticipate that the preliminary test failed to demonstrate irritant responses due to the use of an aqueous vehicle, which is not in accordance with the underlying guideline and might have caused run-off of the test substance, but an actual reason is not specified. This could have lead to the choice of inappropriately high concentrations for the main study.

According to OECD guideline 429, concentrations inducing excessive irritation effects should not be used for application in the LLNA. Considering the circumstances in the present test, the doses inducing distinct local skin irritation are not suitable for assessment of sensitising properties according to the OECD guideline. Therefore, only the highest non-irritating dose can be considered for assessment here. In this test only the lowest concentration of 5% did not induce local skin irritation, and at this concentration an SI of 2.4 was determined.

Due to an SI of 2.4 at the highest non-irritating dose (5%), the test substance should not be considered as sensitising according to the criteria of the DSD and the CLP regulation.

Interpretation of results:
not sensitising
Remarks:
Migrated information
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP guideline study. Reliability changed from "1" to "2" according to ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010).
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Historical in vivo data available.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Como), Italy
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 321-440g
- Housing: Groups of 5 animals in stainless steel cages
- Diet: Altromin MSK, A. Rieper S.p.A., Bolzano, Italy; ad libitum
- Water: in water bottles; ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2 September 1994 To: 3 October 1994
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
induction: 0.5% intradermal, 5% epicutaneous
challenge: 1%
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
induction: 0.5% intradermal, 5% epicutaneous
challenge: 1%
No. of animals per dose:
10
Details on study design:
RANGE FINDING TESTS:
Previously to the main study a range-finder was conducted. Intradermal injection tolerance was tested in 2 animals by injection of 0.1 mL of test substance concentrations of 1.0, 0.5, 0.2, 0.1, 0.05 and 0.01% in sterile water into the clipped region of the scapulae. Observed irritation was recorded using the Draize scoring scale. Topical application tolerance was tested in 5 animals. Each animal received two injections of 0.1 mL of emulsified Freund's complete adjuvant (FCA) into the clipped region of the scapulae. At least 7 days later the flanks were clipped free of hair before each animal was dosed with 2 concentrations of the test substance, one on either flank, applied by 20 x 20 mm gauze patches soaked with 0.2 ml of the selected test substance concentrations (100, 50, 20, 10 and 5% in sterile water), each dosed in duplicate. After application the treatment sites were occlusively covered with a strip aluminium foil and the trunk of the animals wrapped with an elastic adhesive bandage to keep the test substance in contact with the skin. After 24 hours the patches were removed, and 24 and 48 hours after removal the treated sites were assessed for signs of irritation. As irritation was observed at all concentrations investigated another 5 animals were treated in the same manner at concentrations of 1.0, 0.5, 0.1, 0.05 and 0.01% (in sterile water) to establish a suitable non-iritant level.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: On Day 1 (intradermal) and Day 8 (epicutaneous, for 48 hours)
- Test groups: Intradermal: three pairs of injections of 0.1 mL: FCA, test substance + sterile water, test substance + FCA; epicutaneous: application of a gauze patch covered with 0.4 mL: test substance + sterile water
- Control group: Intradermal: three pairs of injections of 0.1 mL: FCA, sterile water, sterile water + FCA; epicutaneous application of a gauze patch covered with 0.4 mL: sterile water
- Site: Intradermal: three pairs of injections at the edges of the clipped site in the scapular region with an area of approx. 20 x 40 mm; epicutaneous: same site
- Frequency of applications: 7 days
- Duration: Day 1 to 10
- Concentrations: 0.5% test substance intradermal, 5% test substance epicutaneous

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 22
- Exposure period: 24 hours
- Test groups: On 20 x 20 mm gauze patches: 0.2 mL test substance (right flank); 0.2 mL sterile water (left flank)
- Control group: On 20 x 20 mm gauze patches: 0.2 mL test substance (right flank); 0.2 mL sterile water (left flank)
- Site: Clipped areas of 50 x 50 mm on each flank
- Concentrations: 1% test substance in sterile water
- Evaluation (hr after challenge): 24 and 48 after removal of the dressing
Challenge controls:
not performed
Positive control substance(s):
yes
Remarks:
Mercaptobenzothiazole
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
1%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
1%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
1%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
1%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
25%
No. with + reactions:
89
Total no. in group:
100
Clinical observations:
Animal numbers in percent from most recent reliability check at the time of the study
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 25%. No with. + reactions: 89.0. Total no. in groups: 100.0. Clinical observations: Animal numbers in percent from most recent reliability check at the time of the study.

All animals of the control group showed moderate to severe erythema (grade 3) at the injection sites of FCA or FCA in vehicle 24 hours after intradermal induction; very slight erythema (grade 1) was observed at only one vehicle injection site of a single animal, the other vehicle injection sites were without findings.

All animals of the test group showed moderate to severe erythema (grade 3) at the injection sites of FCA or test substance in FCA 24 hours after intradermal induction; injection of test substance in vehicle induced very slight (grade 1) to moderate to severe (grade 3) erythema at the respective injection sites 24 hours after intradermal induction in all animals, reflecting the irritating characteristics of the test substance.

No dermal responses were observed after epicutaneous application of a 5% test substance solution in the treated group; no responses were observed in the control group, either, which had been treated with sterile water only.

None of the control animals and none of the animals treated with the test substance demonstrated any dermal responses at 24 or 48 hours after challenge exposure.

There were no significant differences in body weight and body weight gain between treated and control group during the course of the study.

Conclusion:

According to the criteria of OECD Guideline 406 for assessment of skin sensitisation in adjuvant tests the test substance does not have to be considered as skin sensitiser. The test substance does not have to be classified as sensitising to the skin according to the criteria of EU Directive 67/548/EEC and Regulation (EC) No 1272/2008.

Interpretation of results:
not sensitising
Remarks:
Migrated information
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

There is data from five studies in animals as well as a sensitisation study in humans and medical surveillance data for occupationally exposed persons available for the substance itself and structurally closely related substances according the the criteria of Regulation (EC) No 1907/2006, Annex XI, section 1.5.

Among those animal studies there is data from two adjuvant tests (guinea pig maximisation tests) conducted according to OECD Guideline 406.

One of these adjuvant tests is a guinea pig maximisation test performed with a reaction mass consisting of 22% CAS 683-10-3 and 8% CAS 2601-33-4 in aqueous solution (Pelcot, 2006). Concentrations of 0.5% (w/w) and 25% (w/w) of the test substance were used for intradermal and epicutaneous induction, respectively, followed by an epicutaneous challenge with 1% (w/w). These concentrations had previously been determined in a preliminary range-finder test according to the criteria of the guideline. In the main study a discrete erythema was observed after the challenge in 3/10 animals of the control group at the 24-hour reading and persisted in one of them, associated with a dryness of the skin, at the 48-hour reading, reflecting the skin irritating potential of these substances.

In the treated group, a discrete erythema (grade 1) was noted in 5/20 animals at the 24-hour reading; at the 48-hour reading, a moderate erythema (grade 2) was observed in 2/20 animals. A dryness of the skin was also noted in 1/20 and 2/20 animals at the 24- and 48-hour readings, respectively. The higher number of animals demonstrating cutaneous reactions (5/20, corresponding to 25%) in the 24-hour reading have to be ascribed to the irritant character of the test substance and must be considered as local irritation reaction. Even this number does not exceed the threshold of 30% positive reactions, demanded by the underlying OECD guideline for sensitisation tests with adjuvant, required to consider a test substance as sensitising. After 48 hours, only 2/20 animals (10%) with cutaneous reactions remained, which have to be considered as sensitised. Therefore, under the experimental conditions chosen, the test item does not have to be considered as skin sensitising.

The second adjuvant test was a guinea pig maximisation test performed with Dehyton AB 30, i.e. coco betaine (CAS 68424-94-2) marketed in the form of a 30% aqueous solution (Haynes, 1994). Concentrations of 0.5% and 5% of the test substance were used for intradermal and epicutaneous induction, respectively, followed by an epicutaneous challenge with a concentration of 1%. These concentrations had been determined in a preliminary range-finder study according to the criteria of the guideline, comparable to the study by Pelcot. The main study was conducted with 10 animals in each the treatment and the negative control group. No positive control was included in the actual study, but the results of a concurrent reliability check with mercaptobenzothiazole were reported, demonstrating the validity of the test system. In the main study all animals of both the treated and the control group demonstrated moderate to severe erythema (grade 3) at the injection sites of Friend's complete adjuvant (FCA), FCA in vehicle and test substance in FCA 24 hours after intradermal induction. However, injection of test substance in vehicle without FCA also induced very slight (grade 1) to moderate to severe (grade 3) erythema at the respective injection sites of all animals of the test group 24 hours after intradermal induction, again reflecting the irritant characteristics of the betaines in general and of the respective test substance in particular. No dermal reactions were observed after epicutaneous induction with 5% test substance solution in the treated group; the control group had received only water in the epicutaneous induction, anyway. There were no dermal responses observed either 24 or 48 hours after epicutaneous challenge with a 1% solution of the test substance, neither in the treatment nor in the control group. Therefore, no dermal sensitisation had occurred under the experimental conditions chosen here, confirming the conclusion from the previously described adjuvant test by Pelcot. According to the criteria of the maximisation method of Magnusson and Kligman, the test item does not have to be considered as skin sensitising.

Furthermore, two non-adjuvant studies in guinea pigs are available. The first one with higher validity was performed according to OECD Guideline 406 with a test material consisting of 30% CAS 66455-29-6 (with which Betaines, C12-14 (even numbered)-alkyldimethyl was previously referred to) in aqueous solution (Ott, 2003). A concentration of 5% of the test substance was used for occlusive epicutaneous induction over a period of 3 weeks, followed by an epicutaneous challenge with 1% of test substance. These concentrations had been determined in a preliminary range-finder study according to the criteria of the guideline and demonstrated to be suitable for the induction of a hypersensitivity response and the elicitation of a secondary response, respectively. In the main study no skin effects were observed in the first and second induction week. In the third induction week, discrete/patchy to moderate confluent erythema (grade 1 and 2) were observed in 12/20 test animals after treatment with the test item, again reflecting the skin irritating characteristics of the alkyl dimethyl betaines. Challenge exposure was performed after a treatment-free period of 2 weeks, and readings were performed 24 and 48 hours later. In both readings 11/20 animals demonstrated discrete/patchy erythema comparable to the observations in the induction period. Due to the irritant effects of the test material it was not possible to correctly distinguish between erythema induced by irritation or by sensitisation. Therefore, the animals were rechallenged after a second treatment-free period of 14 days. If those animals had indeed been sensitised then the reactions should have been comparable to the first challenge with the test substance due to the mechanism of delayed type hypersensitivity, which is characterised by the presence of antigen-specific memory T-cells. These T-cells facilitate the elicitation of allergic reactions upon renewed contact with antigen and are responsible for a faster and stronger response. In contrast, after rechallenge only 1/20 and 2/20 (corresponding to 10%) animals demonstrated a reaction at the 24- and 48-hour reading, respectively, characterised by discrete/patchy to moderate/confluent erythema. Therefore, the high number of positive reactions observed after the first challenge has indeed to be considered as local irritation due to the known irritant characteristics of the test substance, also reflected by a classification as skin irritant. Taking into consideration the results from both challenges, the response of 10% positive reactions does not exceed the threshold for non-adjuvant tests; the test substance does not have to be regarded as skin sensitising according to this non-adjuvant test, either.

The second non-adjuvant test in guinea pigs was performed with CAS 66455-29-6 according to the principles of Draize (1959 in FDA handbook) in 1974 before the recent OECD Guideline 406 was established (Albright & Wilson Limited, 1974). The test was performed with only 6 animals. Induction exposures were performed for even 10 times with intradermal injections of 0.1% aqueous solution of the test article, followed by an intradermal challenge exposure with 0.1% of the test article after a treatment-free period of 12 days. None of the animals showed skin reactions at the readings 4 and 24 hours after the challenge that were more severe than those produced by any of the previous injections, therefore, the test substance was not considered to be sensitising. Due to the deviations from the current guideline this test alone would not be sufficient for regulatory purposes, but it supports the chain of evidence already suggested by the other guinea pig tests.

The fifth available test is a local lymph node assay performed according to OECD Guideline 429 with a test substance consisting of the reaction mass of CAS 683-10-3 and CAS 2601-33-4 (29.53% active) in aqueous solution (Wang-Fan, 2005). In deviation to the guideline an aqueous vehicle consisting of 70% ethanol was used, which had been justified by preliminary solubility tests. Test concentrations used in the main study had been shown in a preliminary range finder study to produce no irritation effects, and, therefore, concentrations of 5, 10, 25, 50% in vehicle and 100% (undiluted) were applied.

No clinical signs were observed in any animals of the control or the 5% group. On the second application day a slight to severe ear erythema was observed at both dosing sites in all mice of the 10% group (slight), 25% group (moderate), 50% group (moderate) and 100% group (undiluted, severe), persisting for the remainder of the in-life phase of the study. After excision the draining lymph nodes were pooled; therefore, no statistics on individual cell numbers and disintegrations per minute could be performed. The different concentrations produced stimulation indices of 2.4 (5%), 6.2 (10%), 14.7 (25%), 19.0 (50%) and 26.0 (100%); under the conditions of the study a test substance concentration of 5.8% was sufficient to produce a stimulation index of 3 (EC3).

Although at first glance it seems that the test substance has to be considered as sensitising in the LLNA, one has to keep in mind that, except for the lowest concentration of 5%, all test concentrations produced distinct irritation responses. The responses ranged from slight to severe erythema at both ears and started already in the 10% group in the main study. These effects remained over the whole in-life phase of the test, although the concentrations used were demonstrated to be non-irritating up to applications of 100% (undiluted) in the preliminary test.

One can anticipate that the preliminary test failed to demonstrate such irritant responses due to the use of an aqueous vehicle. Being not in accordance with the underlying guideline, it might have caused a run-off of the test substance and therewith prevented the induction of irritation. Nevertheless, this is only speculative since an actual reason for the discrepancy between the preliminary and the main test was not specified. Moreover, this could have lead to the inappropriately high concentrations chosen for the main study.

However, considering the nature of the LLNA, which is based on the assessment of lymphocyte proliferation in the lymph node draining the application site during the induction phase of sensitisation, results obtained under irritation conditions cannot be used for assessment. Cell proliferation is not an exclusive hallmark of sensitisation and can have various reasons. All kinds of inflammatory reactions, among those also irritation, have the same effect on the lymph nodes draining a site of inflammation, which is recruitment of cells and proliferation. This is reflected in OECD Guideline 429, as well, according to which concentrations inducing excessive irritation effects should not be used for application in the LLNA.

Considering the circumstances in the present test, those doses inducing distinct local skin irritation are not suitable for assessment of sensitising properties according to the guideline. Therefore, only the highest non-irritating dose can be considered for assessment here. In this test only the lowest concentration of 5% did not induce local skin irritation, and at this concentration an SI of 2.4 was determined, which is below the threshold of 3. Only those stimulation indices greater than 3 in the absence of local skin irritation have to be regarded as proof for a positive sensitisation reaction. Therefore, according to the criteria of the OECD Guideline 429, the test substance does not have to be regarded as sensitising to the skin under the conditions of the study.

From an immunological standpoint the LLNA has to be regarded as an incomplete test, as the considered endpoint is cell proliferation and not the elicitation of challenge-induced dermal hypersensitivity reactions. Delayed type hypersensitivity is characterised by the existence of antigen-specific memory T-cells, which mediate the secondary immune response resulting in skin erythema and oedema upon challenge with their specific antigen. Therefore, although considered as the standard assay to assess sensitising properties by authorities over the last few years, the LLNA is not suitable for substances with irritating characteristics, which will also induce an unspecific cell proliferation in the draining lymph nodes that cannot be distinguished from a specific response. The issue has been addressed by many publications in the recent years and has also been the topic of a CESIO workshop in February 2010 in Brussels, where many examples for false-positive results in the LLNA with irritant substances have been demonstrated by industry, among those, as the most remarkable example, SLS, which has been the prime example for an irritating but not sensitising substance over the last 30 years. In such cases a guinea pig study should be conducted, which addresses the appropriate endpoint, the elicitation of dermal responses by antigen-specific T-cells, and more closely resembles the natural process of delayed type hypersensitivity responses. This is also valid for surfactants, which are known for their irritating characteristics.

The results from the in this case more appropriate guinea pig tests are supported by a human patch test carried out with a solution containing 0.1% (active ingredient) of CAS 683-10-3 in 20 human volunteers from the general population (Haskell Laboratory, 1963). The volunteers were exposed epicutaneously under occlusive conditions over 6 days for induction and were epicutaneously challenged for 24 hours after a treatment-free period of 10 days. After the induction period observations of local irritation were reported in two individuals, ranging from mild to strong. Directly after the challenge no reactions were observed, effects arising during the next 4 days in 4 individuals ranged from mild to strong, but according to their nature they were considered as primary irritation and not sensitisation. Although suffering from limited documentation this report supports the view that the test substance is not sensitising in humans, either.

 

This view is further supported by industrial medical surveillance data provided for 25 workers of a production plant for CAS 683-10-3 (InfraServ GmbH & Co. KG, 2009). This routine medical check up is performed every 3 years, and the last one was carried out in 2009. The workers were examined for disorders like irritation of the respiratory tract, the eyes, skin, and allergic disorders or sensitisation related to contact with the test substance. In these examinations no observations were made that were related to the test substance.

 

According to the ECETOC Monograph No. 32 (ISSN-0773-6347-32, Brussels, 2002) on the Use of Human Data in Hazard Classification for Irritation and Sensitisation, "no classification with R43 is necessary, where a significant number of individuals (e.g. 100,000) have frequent (daily) skin exposure for at least one year and there is a system in place to pick up complaints and adverse reaction reports, and where no or only a few isolated cases of allergic contact dermatitis are observed".

Based on the fact that betaines have been frequently used in cleaning products like detergents, washing powders, shampoos, etc. for more than 30 years, and an eminent number of persons have daily contact with the cleaning products mentioned above, without the occurrence of clinical case reports on the sensitisation potential of these substances available at the same time, a sensitisation potential appears very unlikely. 

In conclusion, neither the appropriate experimental animal nor exposure-related human data indicate the need for a classification as sensitiser.

Migrated from Short description of key information:

GPMT (OECD 406): not sensitising

Non-adjuvant test (OECD 406, Buehler): not sensitising

LLNA (OECD 429): not sensitising

Justification for selection of skin sensitisation endpoint:

Hazard assessment is conducted by a weight of evidence approach including studies either for the registration substance itself or read-across from structural analogues.  All available studies are adequate and reliable based on the identified similarities in structure and intrinsic properties, applied methods and overall assessment of quality and dose descriptor level (refer to the endpoint discussion for further details).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data for Betaines, C12-14 (even numbered)-alkyldimethyl itself and structurally closely related substances according to Regulation (EC) No 1907/2006, Annex XI, section 1.5 the substance does not have to be classified for sensitisation according to the criteria of EU Directive 67/548/EEC and Regulation (EC) No 1272/2008.