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Administrative data

Description of key information

The substance was positive for skin sensitisation in a Guinea Pig Maximisation Test (induction - intradermic injection at 0.025% and topical application at 10%) and challenge phase conducted under occlusive dressing for 24 hours, consisted of a single topical application of the test item at 5% and diluted at 2.5% in distilled water. The substance produced positive skin sensitisation results and therefore has to be classified in category 1 (sub-category 1A) as a skin sensitizer.

In a reliable dermal sensitization study with a structurally similar substance was tested using the MAXIMISATION METHOD. The epidermal induction of sensitization was conducted for 48 hours under occlusion with the substance at 10% in PEG 300 one week after the intradermal induction. Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 1% in PEG 300 and PEG 300 alone under occlusive dressing. Ten percent of the test animals showed discrete or patchy erythema at the 24-hour scoring after the challenge treatment with test substance at 1% (w/v) in PEG 300; at the 48 h scoring the 10 animals were free of skin reactions. Based on the above mentioned findings in an adjuvant sensitization test (Magnusson Kligman maximisation test) in guinea pigs the substance does not have to be classified and labelled as a skin sensitizer.

In a reliable dermal sensitization study with a structurally similar substance, young adult Pirbright white guinea pig (10 female) were tested using the MAXIMISATION TEST method described by the US Interagency Regulatory Liaison Group (IRLG, January 1981) and Magnusson B., Kligman A. M., "The Identification of Contact Allergens by Animal Assay". The Guinea Pig Maximisation Test, Invest. Derm. 1959, 52, 268. In a small scale pilot study, necrosis occurred after intradermal application of 10, 5, 1, or 0.5% (V/V) solutions in 2/2 animals each and moderate reddening (score 2) after application of a 0.1 % (V/V) aqueous solution in 2/2 animals. The dermal application of the undiluted substance caused moderate to diffuse erythema (score 2) in 3/3 animals; 50, 25, and 10 % aqueous solutions (V/V) discrete slight erythema (score 1) in 3/3 animals each, and a 5 % (V/V) aqueous solution no skin reactions. Based on the results of this small scale pilot study, for the intradermal and epicutaneous induction procedure substance concentrations of 0.1 % and 10 % were used, respectively. The substance concentration for the challenge procedure was 5%. After intradermal induction, animals showed discrete slight erythema (score 1) to intense erythema and swelling (score 3). 48 hours after epicutaneous induction, almost all animals showed discrete slight erythema (score 1). After challenge, 9/10 and 8/10 animals of the test group exhibited discrete slight erythema (score 1) at the first and the second scoring, respectively. At both scorings all animals showed scaling. At the 48 and the 72 hours scoring 1/5 animals showed discrete slight erythema (score 1) and 3/5 and 5/5 showed scaling at these scorings, respectively. As 90 and 80 % of the test animals showed skin reactions at the first and second scoring after challenge treatment, respectively, the substance has been judged by the study author as strong sensitising agent. However, as seen in the pilot study, the substance is a strong local irritant/corrosive material especially when applied intradermally. The reaction after intradermal induction was more intense than mild to moderate (discrete slight erythema (score 1) to intense erythema and swelling (score 3) occurred). Furthermore, in the non induced negative controls skin reactions in the form of discrete slight erythema (score 1) in 1/5 animals and scaling in 5/5 animals occurred. This prompts question whether the concentrations used for intradermal induction and challenge were appropriate. In addition, as the intracutaneous pre-treatment with complete adjuvant also increases the sensibility of the test system for primary skin irritation, it is recommended to verify allergic reactions as such reactions or to separate them from the primary irritating effects by a second re-challenge with lower test concentrations. However, such a re-challenge was not included in the test procedure. Therefore, it cannot be concluded whether the reaction seen in this study after challenge treatment are really based on a sensitising activity or rather on the irritating activity of the substance.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 May 2018 - 29 June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
July 17th, 1992
Deviations:
yes
Remarks:
A relative humidity higher than 70% was registered. A temperature higher than 25°C was registered. As no effect was noted on the health of the animals, this deviation is considered as without impact on the conclusion of the study.
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
There are significant concerns raised in the toxicology literature regarding the suitability of the LLNA methodology for the assessment of surfactant substances. The literature recommends that in vivo testing of surfactants for sensitizing potential should be done using one of the available guinea pig test guidelines to insure the highest relevance of the results to humans ie either the Buehler or the GPMT. Generally the latter is considered to be the more rigorous of the two methods since it maximizes the conditions to provoke sensitization. Furthermore in this case the selection of this assay allows the results to be directly compared to the GPMT data on the analogue substances, as part of the discussion on read-across.
Specific details on test material used for the study:
Batch No.: RL 55/17
Expiry date: 31 January 2019
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
5, 6, 7 or 8 weeks old at the beginning of the test.
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Remarks:
Distilled water for topical applications.
Concentration / amount:
Intradermic injection at 0.025% and topical application at 10%
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
5% and diluted at 2.5% in distilled water
Day(s)/duration:
24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
GROUP 1 (negative control): 10 male guinea pigs.
GROUP 2 (treated): 20 male guinea pigs/dose.
Challenge controls:
Physiological saline and distilled water.
Positive control substance(s):
no
Key result
Hours after challenge:
24
Group:
test chemical
Dose level:
5%
No. with + reactions:
8
Total no. in group:
20
Clinical observations:
Moderate erythema, discrete erythema and no cutaneous reaction was noted in 11, 6 and 3 animals, respectively, 24 hours after first induction. A scab and dryness of the skin was noted in 15 and 5 animals, respectively, after the second induction.
Remarks on result:
positive indication of skin sensitisation
Key result
Hours after challenge:
48
Group:
test chemical
Dose level:
5%
No. with + reactions:
7
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Key result
Hours after challenge:
72
Group:
test chemical
Dose level:
5%
No. with + reactions:
5
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Key result
Hours after challenge:
24
Group:
test chemical
Dose level:
2.5%
No. with + reactions:
8
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Key result
Hours after challenge:
48
Group:
test chemical
Dose level:
2.5%
No. with + reactions:
6
Total no. in group:
20
Remarks on result:
positive indication of skin sensitisation
Key result
Hours after challenge:
72
Group:
test chemical
Dose level:
2.5%
No. with + reactions:
4
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Interpretation of results:
Category 1A (indication of significant skin sensitising potential) based on GHS criteria
Conclusions:
The substance produced positive skin sensitisation results and therefore has to be classified in category 1 (sub-category 1A) as a skin sensitizer.
Executive summary:

A Guinea Pig Maximisation Test was undertaken to evaluate the possible allergenic activity of the substance after intradermal and topical administration in guinea pigs. The induction phase (intradermic injection at 0.025% and topical application at 10%) was conducted with the substance to 20 Guinea pigs and a 10-day rest phase. The challenge phase conducted under occlusive dressing for 24 hours, consisted of a single topical application of the test item at 5% and diluted at 2.5% in distilled water. In the treated group (treatment dose of 5%), a discrete to intense erythema was noted in 40% (8/20), 35% (7/20) and 25% (5/20) of the animals, 24, 48 and 72 hours after the challenge phase, respectively. In the control group (associated with the treatment dose of 5%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase. In the treated group (treatment dose of 2.5%), a moderate to intense erythema was noted in 40% (8/20), 30% (6/20) and 20% (4/20) of the animals, 24, 48 and 72 hours after the challenge phase, respectively. In the control group (associated with the treatment dose of 2.5%, no macroscopic cutaneous intolerance reactions were recorded after the challenge phase. In conclusion, under these experimental conditions, the substance produced positive skin sensitisation results and therefore has to be classified in category 1 (sub-category 1A) as a skin sensitizer.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Read-across to K1 study therefore K2 is the maximum Klimisch value.
Justification for type of information:
Read-across approach - see read-across justification in section 13.
Reason / purpose for cross-reference:
read-across source
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A valid GPMT conducted according to guideline is available, which is reliable without restrictions and adequate for classification and labelling purposes. Potency estimation is not mandatory when existing guideline and GLP conforming data are available, which were conducted before the new annex of the REACH Regulation entered into force. Moreover, no indication for skin sensitisation was observed in this study, thus, no dose response information is needed. For this reason and for reasons of animal welfare no additional LLNA was conducted.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Albino Dunkin Hartley Guinea Pig, CRL:(HA)BR, SPF
- Age at study initiation: 4-6 weeks
- Weight at study initiation: pretest groups: 332 - 349 g, test and control group: 330 - 366 g
- Housing: individually in Makrolon type-4 cages with standard softwood bedding ("Lignocel", Schill AG, 4132 Muttenz, Switzerland).
- Diet: ad libitum, pelleted standard Provimi Kliba 3418 guinea pig breeding / maintenance diet batch no. 82/08, Provimi Kliba AG, 4303 Kaiseraugst, Switzerland
- Water: ad libitum, tap water as for human consumption
- Acclimation period: animals of test and control group were acclimatised, no data on acclimation length


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30 - 70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): automatically controlled light cycle of 12 hours light and 12 hours dark


Route:
intradermal and epicutaneous
Vehicle:
polyethylene glycol
Concentration / amount:
induction: 0.5 % (V/V) in intradermal, 10 % (V/V) epicutaneous
challenge: 1 % (V/V)
Concentration selection based on a small scale dose range studies
Route:
epicutaneous, occlusive
Vehicle:
polyethylene glycol
Concentration / amount:
induction: 0.5 % (V/V) in intradermal, 10 % (V/V) epicutaneous
challenge: 1 % (V/V)
Concentration selection based on a small scale dose range studies
No. of animals per dose:
treatment group: 10
control group: 5
Details on study design:
RANGE FINDING STUDIES
Intradermal injection:
For intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of three guinea pig. Six days later intradermal injections (0.1 mL/site) were made into the clipped flank . One animal received spatially divided injections of 25, 15, and 10 % , one of 5, 2.5, and 1 % and one of 0.5 and 0.1 %. Concentrations were formulated in polyethylene glycol 300 (PEG 300). Dermal reactions were assessed 24 hours later.

Dermal application:
Four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of Freund's Complete Adjuvant/physiological saline were made into the shaved neck of two guinea pigs. Six days later, four patches of filter paper (3 x 3 cm) were saturated with the test item at 100% (technically the highest possible concentration to be applied sufficiently), 75%, 50% and 25% in PEG 300 and applied to the clipped and shaved flanks of the same guinea pigs. The amount or volume of test item preparation applied was approximately 0.2 g at 100%, 75% and 50% or 0.2 mL at 25%. The concentration causing mild (to moderate) local skin reaction and the concentration being the highest tested non-irritating concentration could not be determined after the epidermal pretest described above. Therefore, a second pretest was performed with two additional naive guinea pigs, treated in the same way as those described previously, with the concentrations of 15%, 10%, 5% and 1% in PEG 300, using an application volume of approximately 0.2 mL.

MAIN STUDY
A. INDUCTION EXPOSURE
- First stage (test day 1)
An area of dorsal skin from the scapular region (approximately 6 x 8 cm) was clipped free of hair. Three pairs of intradermal injections (0.1 mL/site) were made symmetrically in two rows on either side of the sine just within the boundaries of a 4 x 6 cm area in the clipped region.
-- Test groups:
1. 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline
2. The test item at 0.5% in PEG 300
3. The test item at 0.5% in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline
- Control group:
1. 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline
2. PEG 300
3. 1:1 (w/w) mixture of PEG 300 in a 1:1 (v/v) mixture of Freund's Complete Adjuvant and physiological saline

- Second stage - One week after the intradermal injections (test day 8), the scapular area (approximately 6 x 8 cm) was again clipped and shaved free of hair prior to the application. A 2 x 4 cm patch of filter paper was saturated with the test item at 10% in PEG 300 and placed over the injection sites of the test animals. The volume of test item preparation applied was approximately 0.3 mL. The patch was covered with aluminum foil and firmly secured by an elastic plaster wrapped around the trunk of the animal and secured with impervious adhesive tape. The occlusive dressings were left in place
for 48 hours. The epidermal application procedure described ensured intensive contact of the test item.

The guinea pigs of the control group were treated as described above with PEG 300 only, applied at a volume of approximately 0.3 mL.

The reaction sites were assessed 24 and 48 hours after removal of the bandage for erythema and oedema according to the method of Magnusson and Kligman.

B. CHALLENGE EXPOSURE (test day 22)
The test and control guinea pigs were challenged two weeks after the epidermal induction application and were treated in the same way.

Two patches (3 x 3 cm) of filter paper were saturated with the test item at the highest tested nonirritating concentration of 1% (applied to the left flank) and the vehicle only (PEG 300 applied to the right flank) using the same method as for the epidermal application. The volume of test item preparation and vehicle alone applied was approximately 0.2 mL. The dressings were left in place for 24 hours.

C. OBSERVATION and SCORING
The test item skin area of the animals used for the epidermal pretest and challenge was depilated approximately 21 hours after the patches have been removed, using an approved depilatory cream (VEET Cream, Reckitt & Colman AG, 4123 Allschwil, Switzerland). The depilation was performed to clean the stratum corneum from the possible staining produced by the test item and to facilitate the reading of the possible skin reaction. The depilatory cream was placed on the patch sites and surrounding areas, and left on for up to 3-5 minutes. It was then thoroughly washed off with a stream of warm, running water. The animals were then dried with a disposable towel, and returned to their cages.

The scoring system was performed by visual assessment of erythema and oedema.
0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling

Any other gross lesions not covered by this scoring system were described.

The grading method used for the pretest, induction and challenge was identical. It was performed 24 ± 2 hours after removal of the patches for the intradermal and epidermal pretest and induction and for challenge and repeated 24 ± 2 hours later (48-hour grades) for the epidermal pretest,
epidermal induction and challenge.

OTHER OBSERVATIONS
- Viability / Mortality: Daily from delivery of the animals to the termination of the test.
- Clinical Signs / Grading of Skin Response: Daily from delivery of the animals to the termination of test. Skin responses were graded during the pretest, induction and challenge periods.
- Body Weights: At delivery/acclimatization start, at the end of the pretest, at test day 1 (day of treatment) and at the termination of the study.

POSITIVE CONTROL (RELIABILITY CHECK):
- The sensitivity and reliability of the experimental technique employed was assessed by use of ALPHA-HEXYLCINNAMALDEHYDE which is recommended by the OECD 406 Guidelines and is known to have moderate skin sensitization properties in the guinea pig strain. The results from the most recent test run (Harlan Laboratories Study C16261, performed from 08-Oct-2008 to 14-Nov-2008) were included in the study report.

STATISTICAL ANALYSIS
Descriptive statistics (means and standard deviations) were calculated for body weights. No inferential statistics were used.
Positive control substance(s):
yes
Remarks:
ALPHA-HEXYLCINNAMALDEHYDE, periodically tested, most recent test run performed from 08-Oct-2008 to 14-Nov-2008
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
1 %
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
discrete or patchy erythema (score 1)
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1 %. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: discrete or patchy erythema (score 1).
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
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:
negative control
Dose level:
1 %
No. with + reactions:
0
Total no. in group:
5
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: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
1 %
No. with + reactions:
0
Total no. in group:
5
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: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
other: vehicle (PEG) control in initiated animals
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: vehicle (PEG) control in initiated animals. Dose level: 0 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: vehicle (PEG) control in initiated animals
Dose level:
0 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: vehicle (PEG) control in initiated animals. Dose level: 0 %. No with. + reactions: 0.0. Total no. in groups: 10.0.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
ALPHA-HEXYLCINNAMALDEHYDE at 1% in PEG 300
Clinical observations:
All test animals showed skin reactions after the challenge treatment with ALPHAHEXYLCINNAMALDEHYDE at 3% in PEG 300. No skin effect was observed in the animals treated with ALPHA-HEXYLCINNAMALDEHYDE at 1% in PEG 300.
Remarks on result:
positive indication of skin sensitisation

RESULTS

Range finding studies:

Intradermal injection: All tested concentrations caused skin reactions:

- 25 and 10 %: moderate and confluent erythema (score 2) with swelling

- 10, 5, 2.5 and 1 %: moderate and confluent erythema (score 2)

- 0.5 %: discrete or patchy erythema (score 1)

- 0.1 %: discrete or patchy erythema (score 1) with blanching

Dermal application: The test item at a concentration of 1 % caused no skin reactions. Concentration depended skin reactions after application of 5 to 100 %:

- 100 and 75 %: at 24 and 48 h scoring intense erythema and swelling (score 3), at 48 h with scaling and necrosis

- 50 %: at 24 and 48 h scoring moderate and confluent erythema (score 2), at 24 with swelling, at 48 h with scaling and necrosis

- 25 %: at 24 h scoring in both animals moderate and confluent erythema (score 2), at 48 h scoring one animals with discrete or patchy erythema (score 1) with scaling and one animals with moderate and confluent erythema (score 2) with scaling and necrosis

- 15, 10 and 5 %: at 24 and 48 h scoring discrete or patchy erythema (score 1), at 48 h with scaling

- 1 %: at 24 and 48 h scoring no visible changes

Main study:

- Induction:

-- intradermal: The expected and common findings were observed in the control and test group after the different applications using FCA intradermally. These findings consisted of erythema, oedema, necrotizing dermatitis, encrustation and exfoliation of encrustation.

-- epicutaneous: No erythematous or oedematous reaction was observed in the animals treated with PEG 300 only. Discrete or patchy (score 1) to moderate and confluent (score 2) erythema was observed in all animals at the 24- and 48 -hour scorings after treatment with the test item at 10% in PEG 300; in detail at 24 h scoring 8/10 animals with discrete or patchy erythema (score 1), 2/10 animals with moderate and confluent erythema (score 2), at 48 h scoring 9/10 animals with discrete or patchy erythema (score 1), 1/10 animals with moderate and confluent erythema (score 2). Scaling was observed in 70% of the test animals at the 48-hour reading.

- Challenge

-- test group: Discrete or patchy erythema (score 1) was observed in 1/10 of the test animals at the 24-hour scoring after treated with the test item at 1% in PEG 300. No local skin reaction was observed at the 48-hour scoring.

No skin reactions were observed in the test animals when treated with PEG 300 only.

-- control group: No skin reactions were observed in the animals when treated with PEG 300 alone or when treated with the test item at 1% in PEG 300.

OTHER OBSERVATIONS

- Viability / Mortality: There were no deaths during the course of the study, hence no necropsies were performed.

- Clinical Signs: No signs of systemic toxicity were observed in the animals.

- Body Weights: The body weight of the animals was within the range commonly recorded for animals of this strain and age.

Reliability check (positive control):

The test was performed from 08-Oct-2008 to 14-Nov-2008. All test animals showed skin reactions after the challenge treatment with ALPHAHEXYLCINNAMALDEHYDE at 3% in PEG 300. No skin effect was observed in the animals treated with ALPHA-HEXYLCINNAMALDEHYDE at 1% in PEG 300. No skin effect was observed in the control group.

Interpretation of results:
GHS criteria not met
Conclusions:
The substance is not a sensitiser.
Executive summary:

In a dermal sensitization study with the partially unsaturated IQAC, DMS quaternised (NO SOLVENT, 100 % pure) in PEG 300, 15 Dunkin Hartley guinea pigs (10 in treatment group, 5 in control group), were tested using the MAXIMISATION METHOD described by OECD Guideline No. 406 and the Commission Regulation (EC) No 440/2008, B.6. The sensitivity of the test system was assessed by use of the positive control substance ALPHA-HEXYLCINNAMALDEHYDE.

The intradermal induction of sensitization in the test group was performed in the nuchal region with a 0.5% dilution of the test item in PEG 300 and in an emulsion of Freund's Complete Adjuvant (FCA)/physiological saline. The epidermal induction of sensitization was conducted for 48 hours under occlusion with the substance at 10% in PEG 300 one week after the intradermal induction. The animals of the control group were intradermally induced with PEG 300 and FCA/physiological saline and epidermally induced with PEG 300 under occlusion. Two weeks after epidermal induction the control and test animals were challenged by epidermal application of the test item at 1% in PEG 300 and PEG 300 alone under occlusive dressing.

Ten percent of the test animals showed discrete or patchy erythema at the 24-hour scoring after the challenge treatment with test substance at 1% (w/v) in PEG 300; at the 48 h scoring the 10 animals were free of skin reactions. None of the 5 control animals showed skin reactions at challenge.

Based on the above mentioned findings in an adjuvant sensitization test (Magnusson Kligman maximisation test) in guinea pigs the partially unsaturated IQAC, DMS quaternised does not have to be classified and labelled as a skin sensitizer.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
Read-across to K2 study therefore K2 is the maximum Klimisch value.
Justification for type of information:
Read-across approach - see read-across justification in section 13.
Reason / purpose for cross-reference:
read-across source
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A valid GPMT conducted according to guideline is available, which is reliable without restrictions and adequate for classification and labelling purposes. Potency estimation is not mandatory when existing guideline and GLP conforming data are available, which were conducted before the new annex of the REACH Regulation entered into force. For this reason and for reasons of animal welfare no additional LLNA was conducted.
Species:
guinea pig
Strain:
other: Pirbright-white
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Züchter Siegfried Knopp, Osnabrück, Germany
- Age at study initiation: 4 to 6 weeks
- Weight at study initiation: 345-485 g
- Housing: collective housing up to a maximum of 5 animals per cage (Macrolon type)
- Diet: ad libitum, standard laboratory guinea pig diet Ssniff-G
- Water: ad libitum, drinking water as for human consumption
- Acclimation period: 16 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 40 -70
- Air changes (per hr): 10
- Photoperiod: 12 hours light, 12 hours dark
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
induction: 0.1 % (V/V) intradermal, 10 % (V/V) epicutaneous
challenge: 5 % (V/V)
Concentration selection based on a small scale dose range study
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
induction: 0.1 % (V/V) intradermal, 10 % (V/V) epicutaneous
challenge: 5 % (V/V)
Concentration selection based on a small scale dose range study
No. of animals per dose:
treatment group: 10
control group: 5
Details on study design:
RANGE FINDING TESTS:
- Intradermal Injection:
2 animals were treated at 5 treatment sites each with test item concentrations of 10, 5 , 1, 0.5, or 0.1 % V/V (solutions in aqua destillata).
Skin reactions were recorded 24 h after treatment.

- Dermal Application:
3 animals were treated at 5 treatment sites each with test item concentrations of 100 % (as delivered by the sponsor) and 50, 25, 10, or 5 % V/V (solutions in aqua destillata). A closed patch (saturated fiter paper pieces (Whatman No. 3 MM, 1 x 1 cm)) exposure was effected by means of an occlusive bandage. Exposure period was 24 hours. Skin reactions were recorded 3 h after patch removal.

MAIN STUDY
A. INDUCTION EXPOSURE
- First stage - an area of 4 x 6 cm over the shoulders was clipped short with electric clippers. Three pairs of intradermal injections were then made symmetrically in two rows on either side of the spine:
-- Test group:
1. 0.1 ml FCA (50 % V/V in aqua ad inject.)
2. 0.1 ml test article (0.1 % V/V in aqua destillata)
3. 0.1 ml test article (0.1 % V/V in destillata emulsified in FCA (50 %))
-- Control group:
1. 0.1 ml FCA (50 % V/V in aqua ad inject.)
2. 0.1 ml aqua destillata
3. 0.1 ml FCA (50 % V/V in aqua destillata)

- Second stage - One week after intradermal injection the clipped area was reclipped. A filter paper (Whatman No. 3 MM (2 x 4 cm)) was saturated with the test substance (10 % in aqua destillata), applied to clipped area and covered for 48 h.

Control animals received a patch loaded with the vehicle.

B. CHALLENGE EXPOSURE
Both control and test animals were subjected to a challenge exposure two weeks after the second stage of induction. The challenge test was performed on a 5 x 5 cm clipped area of the flank. The test substance (5 % in aqua destillata) was applied by a 2 x 2 cm filter paper to the left flank of both test and control animals. In each case the duration of exposure was 24 h under an occlusive dressing.
dressing. 24 and 48 h after patch removal (= 48 and 72 hours after challenge initiation), allergic responses were evaluated on a numerical scale.

C. SCORING SYSTEM:
No skin reaction: score 0
Discrete slight erythema: score 1
Moderate to diffuse erythema: score 2
Intense erythema and swelling: score 3

D. SENSITIZATION CLASSIFICATION SYSTEM used by STUDY AUTHOR:
Sensitization rate 0%: nonsensitizer
Sensitization rate 10-20%: weak sensitizer
Sensitization rate 30-60%: moderate sensitizer
Sensitization rate 70-100%: strong sensitizer
Positive control substance(s):
not specified
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5 %
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
discrete slight erythema (score 1), scaling
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5 %. No with. + reactions: 9.0. Total no. in groups: 10.0. Clinical observations: discrete slight erythema (score 1), scaling.
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
5 %
No. with + reactions:
1
Total no. in group:
5
Clinical observations:
one animal with discrete slight erythema (score 1), this animal and two further animals with scaling
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 5 %. No with. + reactions: 1.0. Total no. in groups: 5.0. Clinical observations: one animal with discrete slight erythema (score 1), this animal and two further animals with scaling.
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
5 %
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
discrete slight erythema (score 1), scaling
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 5 %. No with. + reactions: 8.0. Total no. in groups: 10.0. Clinical observations: discrete slight erythema (score 1), scaling.
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
5 %
No. with + reactions:
1
Total no. in group:
5
Clinical observations:
one animal with discrete slight erythema (score 1), all animals with scaling
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 5 %. No with. + reactions: 1.0. Total no. in groups: 5.0. Clinical observations: one animal with discrete slight erythema (score 1), all animals with scaling.
Key result
Group:
positive control
Remarks on result:
other: positive control was not specified

RESULTS

Pilot study (range finding):

- Intradermal: necrosis after application of 10, 5, 1, or 0.5% (v/v) solutions in 2/2 animals each; moderate reddening (score 2) after application of 0.1 % (v/v) solution in 2/2 animals

- Epicutaneous:

-- 100 % (v/v): moderate to diffuse erythema (score 2) in 3/3 animals

-- 50, 25, and 10 % (v/v): discrete slight erythema (score 1) in 3/3 animals each

-- 5 % (v/v): No skin reactions were observed.

Main study:

- Induction:

-- intradermal: after intradermal induction, animals showed discrete slight erythema (score 1) to intense erythema and swelling (score 3)

-- epicutaneous: 48 hours after epicutaneous induction, almost all animals showed discrete slight erythema (score 1)

- Challenge:

-- test group: at the 48 and 72 hours scoring 9/10 and 8/10 animals exhibited discrete slight erythema (score 1), at both scorings all animals showed scaling

-- negative control group: at the 48 and the 72 hours scoring 1/5 animals showed discrete slight erythema (score 1) and 3/5 and 5/5 showed scaling at these scorings, respectively.

Discussion:

The test item has been judged by the study author as strong sensitising agent. However, as seen in the pilot study, the test substance is a strong local irritant/corrosive material especially when applied intradermally.

The reaction after intradermal induction was more intense than mild to moderate (discrete slight erythema (score 1) to intense erythema and swelling (score 3) occurred). Furthermore, in the non induced negative controls skin reactions in the form of discrete slight erythema (score 1) in 1/5 animals and scaling in 5/5 animals occurred. This prompts question whether the concentrations used for intradermal induction and challenge were appropriate.

In addition, as the intracutaneous pre-treatment with complete adjuvant also increases the sensibility of the test system for primary skin irritation, it is recommended to verify allergic reactions as such reactions or to separate them from the primary irritating effects by a second re-challenge with lower test concentrations. However, such a re-challenge was not included in the test procedure. Therefore, it can not be decided whether the reaction seen after challenge treatment are really based on a sensitising activity or rather on the irritating activity of the test item.

.

Interpretation of results:
study cannot be used for classification
Remarks:
Migrated information
Conclusions:
It cannot be decided whether the reaction seen in this study after challenge treatment are really based on a sensitising activity or rather on the irritating activity of the test item since verification of a sensitizing reaction by a second challenge treatment was not performed.
Executive summary:

In a dermal sensitization study with the oleic-acid based IQAC, DMS quaternised, young adult Pirbright white guinea pig (10 female) were tested using the MAXIMISATION TEST method described by the US Interagency Regulatory Liaison Group (IRLG, January 1981) and Magnusson B., Kligman A. M., "The Identification of Contact Allergens by Animal Assay". The Guinea Pig Maximisation Test, Invest. Derm. 1959, 52, 268.The negative control group comprised 5 animals.

In a small scale pilot study, necrosis occurred after intradermal application of 10, 5, 1, or 0.5% (V/V) solutions in 2/2 animals each and moderate reddening (score 2) after application of a 0.1 % (V/V) aqueous solution in 2/2 animals. The dermal application of the undiluted test item caused moderate to diffuse erythema (score 2) in 3/3 animals; 50, 25, and 10 % aqueous solutions (V/V) discrete slight erythema (score 1) in 3/3 animals each, and a 5 % (V/V) aqueous solution no skin reactions.

Based on the results of this small scale pilot study, for the intradermal and epicutaneous induction procedure test substance concentrations of 0.1 % and 10 % were used, respectively. The test article concentration for the challenge procedure was 5 %.

After intradermal induction, animals showed discrete slight erythema (score 1) to intense erythema and swelling (score 3). 48 hours after epicutaneous induction, almost all animals showed discrete slight erythema (score 1).

After challenge, 9/10 and 8/10 animals of the test group exhibited discrete slight erythema (score 1) at the first and the second scoring, respectively. At both scorings all animals showed scaling.

Skin effects were also seen in the negative control after challenge. At the 48 and the 72 hours scoring 1/5 animals showed discrete slight erythema (score 1) and 3/5 and 5/5 showed scaling at these scorings, respectively.

As 90 and 80 % of the test animals showed skin reactions at the first and second scoring after challenge treatment, respectively, the test item has been judged by the study author as strong sensitising agent. However, as seen in the pilot study, the test substance is a strong local irritant/corrosive material especially when applied intradermally.

The reaction after intradermal induction was more intense than mild to moderate (discrete slight erythema (score 1) to intense erythema and swelling (score 3) occurred). Furthermore, in the non induced negative controls skin reactions in the form of discrete slight erythema (score 1) in 1/5 animals and scaling in 5/5 animals occurred. This prompts question whether the concentrations used for intradermal induction and challenge were appropriate.

In addition, as the intracutaneous pre-treatment with complete adjuvant also increases the sensibility of the test system for primary skin irritation, it is recommended to verify allergic reactions as such reactions or to separate them from the primary irritating effects by a second re-challenge with lower test concentrations. However, such a re-challenge was not included in the test procedure.

Therefore, it cannot be decided whether the reaction seen in this study after challenge treatment are really based on a sensitising activity or rather on the irritating activity of the test item.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the findings of a reliable Guinea Pig Maximisation Test the substance should be classified in category 1 (sub-category 1A) as a skin sensitizer.