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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 11 April, 2013 to 31 May, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD guideline 406 and EU Method B.6, in compliance with GLP.
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
Species:
guinea pig
Strain:
other: 18 Albino Dunkin Hartley Guinea Pig, HsdPoc: DH, SPF and 2 Albino Dunkin Hartley Guinea Pig HsdDhl:DH (SPF)
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / The Netherlands
- Age at study initiation: 4 - 6 weeks
- Weight at study initiation: 284.0 - 369.6 g (for intradermal and epidermal pretests) and 291.8 - 326.3 g (for control group and test group)
- Housing: In groups of up to ten in stainless steel cages with standard softwood bedding (‘Lignocel’ J. Rettenmaier & Söhne GmbH & Co KG, 73494 Rosenberg /Germany, imported by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland).
- Diet: Teklad Global Guinea pig diet 2040C (batch no. 68/12, provided by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland), ad libitum. A haystick 4646 (batch no. 58/12, Provimi Kliba AG, 4303 Kaiseraugst / Switzerland) was provided for environmental enrichment.
- Water: Community tap-water from Itingen ad libitum in water bottles.
- Acclimation period: 24 d under laboratory conditions, after health examination for the control group and the test group. Pretest animals were treated directly upon delivery. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 - 70%,
- Air changes: 10 - 15 air changes/h
- Photoperiod: 12 h light and 12 h darkness
Route:
intradermal and epicutaneous
Vehicle:
other: polyethylene glycol 300
Concentration / amount:
Main test:
-Induction phase: 
Concentration for intradermal injection: 25%
Concentration for topical application: 10%
-Challenge phase: 
Concentration for topical application: 1%
Route:
epicutaneous, occlusive
Vehicle:
other: polyethylene glycol 300
Concentration / amount:
Main test:
-Induction phase: 
Concentration for intradermal injection: 25%
Concentration for topical application: 10%
-Challenge phase: 
Concentration for topical application: 1%
No. of animals per dose:
Main test: 15 (10 test and 5 control)
Details on study design:
RANGE FINDING TESTS:

-Intradermal pretest: The intradermal pretest was performed during the acclimatization of the main test animals. The test substance concentrations used were selected during the preliminary solubility testing and were A = 50%, B = 25% and C = 10% in PEG 300. Four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of FCA/0.9% NACl was made into the clipped and shaved neck of one guinea pig (animal no. 1). 5 d later, intradermal injections (0.1 mL/site) were performed into the clipped flank of the same guinea pig.

-Epidermal pretests: Four intradermal injections (0.1 mL/site) of a 1:1 (v/v) mixture of FCA/0.9% NaCl were made into the clipped and shaved neck of four guinea pigs (animal nos. 2; 3; 19 and 20). Five and six days later, four patches of filter paper (3 x 3 cm) were saturated with the test substance formulations of D = 100%, E = 50%, F = 25%, G = 10% in PEG 300 for the epidermal pretest 1, and H = 5%, I = 1%, J = 0.5%, K = 0.1% in PEG 300 for the epidermal pretest 2. The test substance was formulated in the vehicle or was applied undiluted. The filter paper patches were applied to the clipped and shaved flanks of the same guinea pigs. The volume of test substance preparation applied was approximately 0.2 mL.


MAIN STUDY

-Intradermal induction: The intradermal induction of the main test was performed on test day 1. The test substance concentration used for the intradermal induction was determined based on the results of the intradermal pretest. A concentration of 25% in PEG 300 well-tolerated systemically and caused discrete or patchy erythema. 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 just within the boundaries of a 4 x 6 cm area in the clipped region as follows:
Test group:
1. 1:1 (v/v) mixture of FCA/0.9% NaCl.
2. The test substance at 25% in the vehicle.
3. The test substance at 25% formulated in a 1:1 (v/v) mixture of FCA/0.9% NaCl.
Control group:
1. 1:1 (v/v) mixture of FCA/0.9% NaCl.
2. Vehicle.
3. 1:1 (w/w) mixture of the vehicle in a 1:1 (v/v) mixture of FCA/0.9% NaCl.

-Epidermal induction: The epidermal induction of the main test was performed one week after the intradermal induction on test day 8. The test substance concentration used for the epidermal induction was determined based on the results of the epidermal pretest 1. A concentration of 10% in PEG 300 was well-tolerated systemically and caused mild to moderate skin irritation. The scapular area as used in the intradermal induction (approximately 6 x 8 cm) was clipped and shaved free of hair. A 2 x 4 cm patch of filter paper was saturated with the test substance at the selected concentration and placed over the injection sites of the test group animals. The volume of test substance preparation applied was approximately 0.3 mL. The guinea pigs of the control group were treated similarly with the vehicle only. The filter paper 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 dressing was left in place for 48 ± 1 h.

-Challenge: The challenge was performed two weeks after the epidermal induction on test day 22. The test substance concentration used for the challenge was determined based on the results of the epidermal pretest 2. A concentration of 1% in PEG 300 was well-tolerated systemically and was the highest non-irritant concentration. One patch of filter paper (3 x 3 cm) was saturated with the highest non-irritating concentration of 1% test substance and applied to the right flank of the animals. Another patch of filter paper (3 x 3 cm) was saturated with the vehicle only and applied to the left flank of the animals. The patches were fastened to the animals in a similar manner as described for the epidermal induction. The volume of test substance applied was approximately 0.2 mL. The occlusive dressing was left in place for 24 ± 1 h.
Challenge controls:
Control animals received vehicle only.
Positive control substance(s):
not required
Remarks:
(data from the most recent positive control study conducted with alpha-hexylcinnamaldehyde was used)
Positive control results:
The results from the most recent positive control study (Harlan Laboratories study D65975, performed from 04-Oct-2012 to 09-Nov-2012) confirm alpha-hexylcinnamaldehyde as skin sensitizer, as it produced allergic contact dermatitis in >30% of the test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25% intradermal injection
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25% intradermal injection . No with. + reactions: 1.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25% intradermal injection
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25% intradermal injection . No with. + reactions: 1.0. Total no. in groups: 10.0.

Results and discussion:

Viability/mortality: All animals survived the scheduled observation periods.

Clinical signs: No clinical signs were recorded in any animal.

Skin reactions:

-Skin reactions in the intradermal induction: The expected and commonly observed findings after FCA injection such as erythema, oedema, necrotizing dermatitis, encrustation and exfoliation of encrustation were noted. No detailed description of the skin reactions is given in the report as these effects of FCA are well-known.

-Skin reactions in the epidermal induction: Discrete or patchy to moderate and confluent erythema was observed in all animals of the test group 24 h after treatment with 10% test substance in PEG 300. Discrete or patchy erythema was still observed in 8 of 10 test group animals 48 h after treatment with 10% test substance in PEG 300. No skin reactions were observed in the control group after treatment with the vehicle.

-Skin reactions in the challenge: No skin reactions were observed in any of the control animals (previously not exposed to the test substance) after challenge with 1% test substance in PEG 300. No skin reactions were observed in any of the ten test group animals after treatment with the vehicle, PEG 300. Discrete or patchy erythema was observed in 1 of 10 (10%) test group animals 24 h as well as 48 h after challenge with 1% test substance in PEG 300. Since this response of 10 % is still beneath the threshold of 30% in an adjuvant test for the positive control test as defined by OECD Guidelines 406, the test substance is considered not to be a sensitizer.

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

-Pathology: No unscheduled deaths occurred, hence no necropsy was performed.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the study conditions, the test substance is not considered to be a skin sensitiser in an adjuvant sensitisation test (guinea pig maximization test according to Magnusson & Kligman).
Executive summary:

The study was conducted to assess the skin sensitisation potential of C12-14 alkylmorpholine according to OECD guideline 406 and EU Method B.6 in compliance with GLP.

Based on the results of a preliminary test, 25 and 10% were selected as intradermal and topical induction doses respectively and 1% as topical challenge dose for the main test. The test was performed in 15 (10 test and 5 control) female albino Dunkin Hartley guinea pigs. The intradermal induction of sensitisation in the test group was performed in the nuchal region by injection of 25% of the test substance in polyethylene glycol 300 (PEG 300) and an emulsion of Freund's Complete Adjuvant and physiological saline (FCA/0.9% NaCl). One week after the intradermal induction, the epidermal induction of sensitisation was performed by topical application of 10% of the test substance for 48 h under occlusion. The animals of the control group were intradermally induced with PEG 300 and FCA/0.9% NaCl and epidermally induced with PEG 300 under occlusion. Two weeks after epidermal induction the test and control animals were challenged by epidermal application of 1% test substance in PEG 300 on the right flank and PEG 300 was applied on the left flank. Skin reactions were evaluated at 24 and 48 h after removal of the dressing.

Discrete or patchy to moderate and confluent erythema was observed in all animals of the test group 24 h after treatment with 10% test substance in PEG 300. Discrete or patchy erythema was still observed in 8 of 10 test group animals 48 h after treatment with 10% test substance in PEG 300. No skin reactions were observed in the control group after treatment with the vehicle. No skin reactions were observed in any of the control animals (previously not exposed to the test substance) after challenge with 1% test substance in PEG 300. Discrete or patchy erythema was observed in 1 of 10 test group animals 24 h as well as 48 h after treatment with 1% test substance in PEG 300. It can be concluded that the test substance is not considered to be a skin sensitiser.

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

A GLP compliant study was conducted to assess the skin sensitisation potential of C12 -14 alkylmorpholine according to OECD guideline 406 and EU Method B.6. Based on the results of a preliminary test, 25 and 10% were selected as the intradermal and topical induction doses respectively and 1% as the topical challenge dose for the main test. The test was performed in 15 (10 test and 5 control) female albino Dunkin Hartley guinea pigs. Induction: Skin reactions were evaluated at 24 and 48 h after removal of the dressing. Discrete or patchy to moderate and confluent erythema was observed in all animals of the test group 24 h after treatment with 10% test substance in PEG 300. Discrete or patchy erythema was still observed in 8 of 10 test group animals 48 h after treatment with 10% test substance in PEG 300. No skin reactions were observed in the control group after treatment with the vehicle.

Challenge: No skin reactions were observed in any of the control animals (previously not exposed to the test substance) after challenge with 1% test substance in PEG 300. Discrete or patchy erythema was observed in 1 of 10 test group animals 24 h as well as 48 h after treatment with 1% test substance in PEG 300. It can be concluded that the test substance is not considered to be a skin sensitiser (Harlan, D71623, 2013).


Migrated from Short description of key information:
In vivo data suggests that C12-14 alkylmorpholine is not a skin sensitiser.

Justification for selection of skin sensitisation endpoint:
One reliable guideline-compliant study is available.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

As chemical respiratory sensitisers also elicit positive results in predictive tests for contact sensitisation, the negative outcome for dermal sensitisation is also predictive for non-respiratory sensitisation of the substance. The likelihood for exposure via inhalation is low, based on the high boiling point (> 300 °C) and low vapour pressure (0.18 Pa at 20°C).


Migrated from Short description of key information:
The negative outcome for dermal sensitisation is also predictive for non- sensitising properties of the substance via inhalation.

Justification for classification or non-classification

Skin sensitisation: The results of the available adjuvant sensitisation test (guinea pig maximization test according to Magnusson & Kligman), the test substance does not need to be classified for skin sensitisation in accordance with EU CLP Regulation (EC) No. 1272/2008.

Respiratory sensitisation: As indicated in REACH guidance R.7.3.5 it can be concluded that chemicals that are negative in dermal sensitisation studies and are considered as not being skin sensitisers, can also be regarded as lacking the potential to cause allergic sensitisation of the respiratory tract.

Furthermore, C12-14 alkylmorpholine has low vapour pressure so that normal processing and use conditions will not generate inhalation exposure.