Registration Dossier
Registration Dossier
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EC number: 443-870-0 | CAS number: 163520-33-0
Endpoint summary
Administrative data
Description of key information
Skin sensitisation in vivo, OECD 406, GPMT: positive for skin sensitisation
Key value for chemical safety assessment
Skin sensitisation
Link to relevant study records
- Endpoint:
- skin sensitisation: in vivo (non-LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 24 Sep - 1 Nov 1996
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 406 (Skin Sensitisation)
- Version / remarks:
- Adopted in 1992
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPP 81-6 (Skin Sensitisation)
- Version / remarks:
- Adopted in 1984
- Deviations:
- no
- GLP compliance:
- yes
- Type of study:
- guinea pig maximisation test
- Justification for non-LLNA method:
- A non-LLNA test is available that was performed prior to the current data requirements, stipulated in Regulation (EC) No 1907/2006, and before the LLNA test method was available. In accordance with the same Regulation, the data was included to avoid unnecessary testing.
- Species:
- guinea pig
- Strain:
- Dunkin-Hartley
- Sex:
- male
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Source: D. Hall, Newchurch, UK
- Age at study initiation: 7 - 8 weeks
- Body weight at study initiation: 343 - 416 g
- Housing: group housed, five animals per cage, in suspended metal cages with wire mesh floors
- Diet: vitamin C enriched guinea-pig diet FD2, ad libitum, hay was given weekly
- Water: drinking water, ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 21
- Humidity (%): 45 - 54
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 24 Sep 1996 To: 01 Nov 1996 - Route:
- intradermal and epicutaneous
- Vehicle:
- other: intradermal injection: 5% acetone in Alembicol D (coconut oil, Alembic Products, Saltney, UK) for injection site 2 and 1:1 mixture of Freund's complete adjuvant and 5% acetone in Alembicol D for injection site 3; topical application: acetone
- Concentration / amount:
- intradermal injection: 7.5% (w/v); topical application: 70% (w/v)
- Day(s)/duration:
- At study initiation, animals received 3 pairs of intradermal injections. 6 days thereafter, the injection sites were pre-treated with 10% SDS. 24 h later, a patch soaked with the test substance was placed on the pre-treated skin and fixed for 48 h.
- Adequacy of induction:
- other: non-irritant substance after topical application, skin pre-treated with 10% SDS
- No.:
- #1
- Route:
- epicutaneous, occlusive
- Vehicle:
- other: acetone
- Concentration / amount:
- 70% (w/v) in acetone (anterior site), 35% (w/v) in acetone (posterior site), 0.2 mL
- Day(s)/duration:
- 1 day
- Adequacy of challenge:
- other: Preliminary investigations indicated that the administration of the maximum technially achievable concentration of the test substance (i.e. 70% w/v) did not give rise to irritating effects.
- No. of animals per dose:
- 5 (control group), 10 (treatment group)
- Details on study design:
- RANGE FINDING TEST
In a preliminary study, animals were pre-treated with an intradermal injection of Freud's complete adjuvant (FCA), 1:1 with water, approx. 1 week prior to the start of the preliminary investigations.
The preliminary investigations indicated that the highest concentration tested, i.e. 7.5% (w/v) in 5% acetone in Alembicol D, induced erythema and oedema formation without affecting the animal adversely after intradermal injection. After topical application of the test substance, no skin irritation (erythema or oedema formation) was observed up to a concentration of 70% (w/v) in acetone. This concentration (i.e. 70%) was the maximum technically achievable concentration.
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)
- Test groups:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (v/v) FCA/water
Injection 2: 7.5% test substance in 5% acetone in Alembicol D
Injection 3: 7.5% test substance in a 1:1 mixture of FCA/5% acetone in Alembicol D
Epicutaneous: 70% test substance in acetone; pretreatment of application site with 10% (w/w) SDS in petrolatum 24 h prior to epicutaneous induction to produce irritation
- Control group:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (v/v) FCA/water
Injection 2: 5% acetone in Alembicol D
Injection 3: a 1:1 mixture (v/v) of FCA/5% acetone in Alembicol D
Epicutaneous: acetone; pretreatment of application site with 10% (w/w) SDS in petrolatum 24 h prior to epicutaneous induction to produce irritation
- Site: interscapular area (intradermal and epicutaneous)
- Frequency of applications: topical application 7 days after intradermal injections
- Duration: Days 0 - 9, dressing was removed 48 h post topical application
- Concentrations: intradermal: 7.5%, epicutaneous: 70%
B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 21 (14 days after epicutaneous induction)
- Exposure period: 24 h
- Test group: test substance
- Control group: test substance
- Site: left flank
- Concentrations: 35% posterior position, 70% anterior position
- Evaluation (h after challenge): 24 and 48 h after patch removal - Positive control substance(s):
- yes
- Remarks:
- Hexyl cinnamic aldehyde and mercaptobenzothiazole were used within 6 months prior to the conduct of the study to successfully verify the sensitivity of the test method.
- Positive control results:
- The positive control substances (10% hexyl cinnamic aldehyde and 10% mercaptobenzothiazole) induced positive reactions in 10/10 animals (100%), thus meeting the reliability criteria for the guinea pig maximisation test (≥ 30% positive response).
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- negative control
- Dose level:
- not induced
- No. with + reactions:
- 0
- Total no. in group:
- 5
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- test chemical
- Dose level:
- induction: 7.5%, challenge: 35%
- No. with + reactions:
- 6
- Total no. in group:
- 10
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- test chemical
- Dose level:
- induction: 7.5%, challenge 70%
- No. with + reactions:
- 8
- Total no. in group:
- 10
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- positive control
- Dose level:
- induction: 10%, challenge: 40 and 83.33%
- No. with + reactions:
- 10
- Total no. in group:
- 10
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- negative control
- Dose level:
- not induced
- No. with + reactions:
- 0
- Total no. in group:
- 5
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- test chemical
- Dose level:
- induction: 7.5%, challenge: 35%
- No. with + reactions:
- 6
- Total no. in group:
- 10
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- test chemical
- Dose level:
- induction: 7.5%, challenge: 70%
- No. with + reactions:
- 9
- Total no. in group:
- 10
- Remarks on result:
- positive indication of skin sensitisation
- Interpretation of results:
- Category 1B (indication of skin sensitising potential) based on GHS criteria
- Conclusions:
- CLP: Skin sens Cat. 1B, H317
- Endpoint:
- skin sensitisation: in vivo (non-LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 01 Mar - 02 Apr 1999
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 406 (Skin Sensitisation)
- Version / remarks:
- Adopted in 1992
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.2600 (Skin Sensitisation)
- Version / remarks:
- Adopted in 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.6 (Skin Sensitisation)
- Version / remarks:
- Adopted in 1996
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: JMAFF, Requirements for Safety Evaluation of Agricultural Chemicals and Testing Guidelines for Toxicology Studies, Dermal Sensitization Study, 59 NohSan No. 4200, Agricultural Production Bureau
- Version / remarks:
- Adopted in 1985
- Deviations:
- no
- GLP compliance:
- yes
- Type of study:
- guinea pig maximisation test
- Justification for non-LLNA method:
- A non-LLNA test is available that was performed prior to the current data requirements, stipulated in Regulation (EC) No 1907/2006, and before the LLNA test method was available. In accordance with the same Regulation, the data was included to avoid unnecessary testing.
- Species:
- guinea pig
- Strain:
- Dunkin-Hartley
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Source: D. Hall, Newchurch, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5 - 8 weeks
- Body weight at study initiation: 325 - 397 g
- Housing: group housed, five animals per cage, in suspended metal cages with wire mesh floors
- Diet: vitamin C enriched guinea-pig diet, Harlan Teklad 9600 FD2 SQC, ad libitum; hay was given three times each week
- Water: drinking water, ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15.5 - 22.5
- Humidity (%): 35 - 58
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 01 Mar 1999 To: 02 Apr 1999 - Route:
- intradermal and epicutaneous
- Vehicle:
- coconut oil
- Remarks:
- Alembicol D, a product of coconut oil (Alembic Products, Saltney, UK)
- Concentration / amount:
- intradermal injection: 0.25% (w/v) in Alembicol D, topical application: 60% (w/v) in Alembicol D
- Day(s)/duration:
- 48 h
- Adequacy of induction:
- non-irritant substance, but skin pre-treated with 10% SDS
- No.:
- #1
- Route:
- epicutaneous, occlusive
- Vehicle:
- coconut oil
- Remarks:
- Alembicol D
- Concentration / amount:
- 60% (anterior) and 30% (posterior) (w/v) in Alembicol D, 0.2 mL
- Day(s)/duration:
- 1 day
- Adequacy of challenge:
- other: Preliminary investigations indicated that 60% (w/v) in Alembicol D was the maximum technially achievable concentration of the test substance. After topical application, it did not give rise to irritating effects.
- No. of animals per dose:
- 10 (control group), 20 (treatment group)
- Details on study design:
- RANGE FINDING TEST
In a preliminary study, animals were pre-treated with an intradermal injection of Freud's complete adjuvant (FCA), 1:1 with water, approx. 1 week prior to the start of the preliminary investigations.
The preliminary investigations revealed that concentrations ranging from 0.5% to 7.5% (v/v) in Alembicol D induced necrosis 24 and 72 h post intradermal injection. A concentration of 0.25% (v/v) test substance in Alembicol D was potent to induce erythema and oedema formation without affecting the animal adversely after intradermal injection. After topical application of the test substance, no skin irritation (erythema or oedema formation) was observed up to a concentration of 60% (v/v) in Alembicol D, which was the maximum technically achievable concentration.
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epicutaneous, respectively)
- Exposure period: single injection (intradermal) and 48 h (epicutaneous)
- Test groups:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (v/v) FCA/water
Injection 2: 0.25% test substance in Alembicol D
Injection 3: 0.25% test substance in a 1:1 mixture of FCA/Alembicol D
Epicutaneous: 60% test substance in Alembicol D; pretreatment of application site with 10% (w/w) SDS in petrolatum 24 h prior to epicutaneous induction to produce irritation
- Control group:
Intradermal (3 pairs of injections):
Injection 1: a 1:1 mixture (v/v) FCA/water
Injection 2: Alembicol D
Injection 3: a 1:1 mixture (v/v) of FCA/Alembicol D
Epicutaneous: Alembicol D; pretreatment of application site with 10% (w/w) SDS in petrolatum 24 h prior to epicutaneous induction to produce irritation
- Site: interscapular area (intradermal and epicutaneous)
- Frequency of applications: topical application 7 days after intradermal injections
- Duration: Days 0 - 8 (intradermal injection Day 0, topical application Day 6, occlusive; dressing was removed 48 h post topical application)
- Concentrations: intradermal: 0.25%, epicutaneous: 60%
B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 21 (14 days after epicutaneous induction)
- Exposure period: 24 h
- Test groups: 60 and 30% (w/v) test substance in Alembicol D
- Control group: 60 and 30% (w/v) test substance in Alembicol D
- Site: left flank
- Concentrations: 60% anterior position, 30% posterior position
- Evaluation (h after challenge): 24 and 48 h after patch removal - Positive control substance(s):
- yes
- Remarks:
- The sensitivity of the guinea-pig strain and test protocol used was checked periodically at the testing laboratory with the positive control substances hexyl cinnamic aldehyde (HCA) or 2-mercaptobenzothiazole (MET).
- Positive control results:
- The positive control substance 10% hexyl cinnamic aldehyde induced positive reactions in 10/10 animals (100%), thus meeting the reliability criteria for the guinea pig maximisation test (≥ 30% positive response).
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- negative control
- Dose level:
- not induced
- No. with + reactions:
- 0
- Total no. in group:
- 10
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- test chemical
- Dose level:
- induction: 0.25%, challenge: 30%
- No. with + reactions:
- 0
- Total no. in group:
- 20
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- test chemical
- Dose level:
- induction: 0.25%, challenge: 60%
- No. with + reactions:
- 0
- Total no. in group:
- 20
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- positive control
- Dose level:
- induction intradermal: 10% HCA (v/v) in Alembicol D, induction topical: neat HCA, challenge topical: neat and 50% (v/v) in Alembicol D
- No. with + reactions:
- 10
- Total no. in group:
- 10
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- negative control
- Dose level:
- not induced
- No. with + reactions:
- 0
- Total no. in group:
- 10
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- test chemical
- Dose level:
- induction: 0.25%, challenge: 30%
- No. with + reactions:
- 0
- Total no. in group:
- 20
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- test chemical
- Dose level:
- induction: 0.25%, challenge: 60%
- No. with + reactions:
- 0
- Total no. in group:
- 20
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- positive control
- Dose level:
- induction intradermal: 10% HCA (v/v) in Alembicol D, induction topical: neat HCA, challenge topical: neat and 50% (v/v) in Alembicol D
- No. with + reactions:
- 10
- Total no. in group:
- 10
- Remarks on result:
- positive indication of skin sensitisation
- Interpretation of results:
- other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.
- Conclusions:
- CLP: not classified
- Endpoint:
- skin sensitisation: in vivo (non-LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 02 Mar - 09 Apr 1999
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 406 (Skin Sensitisation)
- Version / remarks:
- Adopted in 1992
- Deviations:
- yes
- Remarks:
- No justification for the use of Alembicol D as vehicle was provided in the study report. However, based on the water solubility and the logPow value of the test substance this vehicle is reasonable.
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.2600 (Skin Sensitisation)
- Version / remarks:
- Adopted in 1998
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.6 (Skin Sensitisation)
- Version / remarks:
- Adopted in 1996
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- other: JMAFF, Requirements for Safety Evaluation of Agricultural Chemicals and Testing Guidelines for Toxicology Studies, Dermal Sensitization Study, 59 NohSan No. 4200, Agricultural Production Bureau
- Version / remarks:
- Adopted in 1985
- Deviations:
- no
- GLP compliance:
- yes
- Type of study:
- Buehler test
- Justification for non-LLNA method:
- A non-LLNA test is available that was performed prior to the current data requirements, stipulated in Regulation (EC) No 1907/2006, and before the LLNA test method was available. In accordance with the same Regulation, the data was included to avoid unnecessary testing.
- Species:
- guinea pig
- Strain:
- Dunkin-Hartley
- Sex:
- female
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Source: D. Hall, Newchurch, UK
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: 320 - 409 g
- Housing: group housed, five animals per cage, in suspended metal cages with wire mesh floors
- Diet: vitamin C enriched guinea-pig diet, Harlan Teklad 9600 FD2 SQC, ad libitum, hay was given three times each week
- Water: drinking water, ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 15.5 - 22.5
- Humidity (%): 35 - 60
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 02 Mar 1999 To: 09 Apr 1999 - Route:
- epicutaneous, occlusive
- Vehicle:
- coconut oil
- Remarks:
- Alembicol D, a product of coconut oil (Alembic Products, Saltney, UK)
- Concentration / amount:
- 80% (w/v) test substance in Alembicol D, 0.5 mL
- Day(s)/duration:
- 3 single treatments every 7 days within 14 days, 6 h exposure during each treatment
- Adequacy of induction:
- highest technically applicable concentration used
- No.:
- #1
- Route:
- epicutaneous, occlusive
- Vehicle:
- coconut oil
- Remarks:
- Alembicol D
- Concentration / amount:
- 80% (w/v) in Alembicol D, 0.5 mL
- Day(s)/duration:
- single treatment, 6 h exposure
- Adequacy of challenge:
- other: From preliminary investigations 80% test substance in Alembicol D was the maximum practical concentration and did not give rise to irritating effects.
- No. of animals per dose:
- 10 (control group), 20 (treatment group)
- Details on study design:
- RANGE FINDING TEST
In a preliminary study, the topical irritancy of a range of dilutions of the test substance was investigated to identify where possible (a) concentrations of the test substance that would produce irritation suitable for the induction phase of the main study and (b) a maximum non-irritant concentration for the challenge phase. For this purpose, 4 animals were treated with 10, 25, 50, and 80% test substance in Alembicol D each, by topical application. Skin reactions were scored 24 and 48 h post exposure. After topical application of the test substance, no skin irritation (neither erythema nor oedema formation) was observed at concentrations ranging from 10 to 80% in all animals for both time points. The concentration of 80% test substance in Alembicol D was the maximum practical concentration achievable.
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 h
- Test groups: test substance in Alembicol D
- Control group: Alembicol D
- Site: left shoulder region
- Frequency of applications: every 7 days
- Duration: Day 1 - 15
- Concentrations: 80%
B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day of challenge: 28
- Exposure period: 6 h
- Test groups: 0.5 mL test substance (80%) in Alembicol D
- Control group: 0.5 mL test substance (80%) in Alembicol D
- Site: right flank
- Concentrations: 80%
- Evaluation (h after challenge): 24 and 48 h after patch removal - Challenge controls:
- The control group is actually a challenge control.
- Positive control substance(s):
- yes
- Remarks:
- The sensitivity of the guinea-pig strain and the test protocol used was checked periodically at the testing laboratory with known sensitisers, i.e. hexyl cinnamic aldehyde (HCA) and 2-mercaptobenzothiazole (MET).
- Positive control results:
- The positive control substance hexyl cinnamic aldehyde (induction: neat substance, challenge: 25 and 50% in Alembicol D) induced positive reactions in 10/10 animals of the treatment group (100%), thus meeting the reliability criteria for the Buehler test (≥15% positive response). This reliability study was performed: 16 Mar - 15 Apr 1999 (study HLS/074).
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- negative control
- Dose level:
- induction: not induced, challenge: 80%
- No. with + reactions:
- 0
- Total no. in group:
- 10
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- test chemical
- Dose level:
- induction: 80%, challenge: 80%
- No. with + reactions:
- 0
- Total no. in group:
- 20
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- positive control
- Dose level:
- induction: neat substance HCA, challenge: 25 and 50% HCA
- No. with + reactions:
- 10
- Total no. in group:
- 10
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- negative control
- Dose level:
- induction: not induced, challenge: 80%
- No. with + reactions:
- 0
- Total no. in group:
- 10
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- test chemical
- Dose level:
- induction: 80%, challenge: 80%
- No. with + reactions:
- 0
- Total no. in group:
- 20
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- positive control
- Dose level:
- induction: neat substance HCA, challenge: 25 and 50% HCA
- No. with + reactions:
- 10
- Total no. in group:
- 10
- Remarks on result:
- positive indication of skin sensitisation
- Interpretation of results:
- other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.
- Conclusions:
- CLP: not classified
- Endpoint:
- skin sensitisation: in vivo (non-LLNA)
- Type of information:
- experimental study
- Adequacy of study:
- weight of evidence
- Study period:
- 18 Oct - 25 Nov 1999
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 406 (Skin Sensitisation)
- Version / remarks:
- Adopted in 1992
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EU Method B.6 (Skin Sensitisation)
- Version / remarks:
- Guideline in place during study conduct: adopted in 1996
- Deviations:
- no
- Qualifier:
- according to guideline
- Guideline:
- EPA OPPTS 870.2600 (Skin Sensitisation)
- Version / remarks:
- Adopted in 1998
- Deviations:
- no
- GLP compliance:
- yes
- Type of study:
- Buehler test
- Justification for non-LLNA method:
- A non-LLNA test is available that was performed prior to the current data requirements, stipulated in Regulation (EC) No 1907/2006, and before the LLNA test method was available. In accordance with the same Regulation, the data was included to avoid unnecessary testing.
- Species:
- guinea pig
- Strain:
- Dunkin-Hartley
- Sex:
- male
- Details on test animals and environmental conditions:
- TEST ANIMALS
- Source: D. Hall, Newchurch, UK
- Age at study initiation: 5 - 8 weeks
- Body weight range at study initiation: 351 - 441 g
- Housing: group housed, 5 animals per cage, in suspended metal cages with wire mesh floors
- Diet: vitamin C enriched guinea-pig diet, Harlan Teklad 9600 FD2 SQC, ad libitum; hay was given 3 times each week
- Water: drinking water, ad libitum
- Acclimation period: 5 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 - 20
- Humidity (%): 26 - 62
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 18 Oct 1999 To: 25 Nov 1999 - Route:
- epicutaneous, occlusive
- Vehicle:
- coconut oil
- Remarks:
- Alembicol D, a product of coconut oil (Alembic Products, Saltney, UK)
- Concentration / amount:
- 80% (w/v) in Alembicol D, 0.5 mL
- Day(s)/duration:
- 3 single treatments, one every 7 days within 14 days (Day 1, 8, and 15), 6 h exposure during each treatment
- Adequacy of induction:
- highest technically applicable concentration used
- No.:
- #1
- Route:
- epicutaneous, occlusive
- Vehicle:
- coconut oil
- Remarks:
- Alembicol D
- Concentration / amount:
- 80% (w/v) in Alembicol D, 0.5 mL
- Day(s)/duration:
- single treatment 2 weeks after the final induction application, 6 h exposure
- Adequacy of challenge:
- other: From preliminary investigations 80% test substance in Alembicol D was the maximum practical concentration and did not give rise to irritating effects.
- No. of animals per dose:
- 10 (control group), 20 (treatment group)
- Details on study design:
- RANGE FINDING TEST
The topical irritancy of a range of dilutions of the test substance was investigated to identify where possible (a) concentrations of the test substance that would produce irritation suitable for the induction phase of the main study and (b) a maximum non-irritant concentration for the challenge phase.
Approximately 0.5 mL of a range of concentrations (20 to 80% (v/v)) of the test substance in sterile water was applied to patches of surgical gauze (20 x 20 mm). These were placed on the clipped and shaved flanks of each of 4 guinea pigs. The patches were covered by a strip of an impermeable plastic adhesive tape ("Blenderm") and firmly secured by an elastic adhesive bandage ("Elastoplast") wound round the trunk of the animals and fixed with "Sleek" impervious plastic adhesive tape. After an exposure period of 6 h the dressings were removed and the test sites were assessed for erythema and oedema. Further examination of the sites was carried out 24 and 48 h after removal of the dressings.
None of the tested concentrations induced erythema or oedema in any of the animals.
Based on these results the following concentrations of the test substance were selected for the main study:
Induction topical application: 80% (w/v) in Alembicol D
Topical challenge: 80% (w/v) in Alembicol D
From preliminary investigations 80% (w/v) in Alembicol D was the maximum practical concentration.
MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 h
- Test groups: 80% test substance in Alembicol D
- Control group: Alembicol D
- Site: left shoulder region
- Frequency of applications: every 7 days (Days 1, 8 and 15)
- Duration: Days 1 - 15
- Concentrations: 80% test substance in Alembicol D
B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: two weeks after the final induction application
- Exposure period: 6 h
- Test groups: 80% test substance (0.5 mL) in Alembicol D
- Control group: 80% test substance (0.5 mL) in Alembicol D
- Site: right flank
- Concentrations: 80% test substance in Alembicol D
- Evaluation (h after challenge): 24 and 48 h after patch removal - Challenge controls:
- The control group is actually a challenge control.
- Positive control substance(s):
- yes
- Remarks:
- hexyl cinnamic aldehyde (HCA) (induction topical applications: HCA, as supplied (neat); challenge application: HCA, as supplied and 50% (v/v) in Alembicol D)
- Positive control results:
- The sensitivity of the guinea pig strain used was checked periodically at the testing laboratory with known sensitisers hexyl cinnamic aldehyde (HCA) and 2-mercaptobenzothiazole (MET).
A reliability test with the positive substance HCA was routinely performed 1 week prior to the present study (study HLS/075).
The positive control substance (HCA, as supplied and 50% (v/v) in Alembicol D) induced positive reactions in 7/10 animals (70%), thus meeting the reliability criteria for the Buehler test (≥15% positive response). - Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- negative control
- Dose level:
- induction: not induced, challenge: 80%
- No. with + reactions:
- 0
- Total no. in group:
- 10
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- test chemical
- Dose level:
- induction: 80%, challenge: 80%
- No. with + reactions:
- 0
- Total no. in group:
- 20
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 1st reading
- Hours after challenge:
- 24
- Group:
- positive control
- Dose level:
- induction: neat, challenge: neat and 50%
- No. with + reactions:
- 7
- Total no. in group:
- 10
- Remarks on result:
- positive indication of skin sensitisation
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- negative control
- Dose level:
- induction: not induced, challenge: 80%
- No. with + reactions:
- 0
- Total no. in group:
- 10
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- test chemical
- Dose level:
- induction: 80%, challenge: 80%
- No. with + reactions:
- 0
- Total no. in group:
- 20
- Remarks on result:
- no indication of skin sensitisation
- Key result
- Reading:
- 2nd reading
- Hours after challenge:
- 48
- Group:
- positive control
- Dose level:
- induction: neat, challenge: neat and 50%
- No. with + reactions:
- 7
- Total no. in group:
- 10
- Remarks on result:
- positive indication of skin sensitisation
- Interpretation of results:
- other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No. 1272/2008.
- Conclusions:
- CLP: not classified
Referenceopen allclose all
Induction
Intradermal injections: Necrosis was recorded at sites receiving Freund's Complete Adjuvant in test and control animals. Slight irritation was seen in test animals at sites receiving 7.5% (w/v) test substance in 5% acetone in Alembicol D and slight irritation was observed in control animals receiving 5% acetone in Alembicol D alone. Necrosis was observed in animals receiving 7.5% (w/v) test substance in a 1:1 mixture of FCA and 5% acetone in Alembicol D.
Topical application: Slight erythema was observed in test animals following topical application of 70% (w/v) test substance in acetone. Slight erythema was also seen in the control guinea pigs.
Clinical signs
No signs of toxicity were recorded.
Body weight
No effect on body weight and body weight gain was observed.
Table 1: Dermal reactions observed after each induction
| Site | Intradermal injection | Topical application | ||
| Test animals | Control animals | Test animals | Control animals | |
| 1 | Necrosis | Necrosis | Slight erythema | Slight erythema |
| 2 | Slight irritiation | Slight irritiation | ||
| 3 | Necrosis | Necrosis | ||
Injection site 1: Freund's complete adjuvant : water, 1:1
Injection site 2: 7.5% test substance in 5% acetone in Alembicol D
Injection site 3: 7.5% test substance in a 1:1 mixture of Freund's complete adjuvant and 5% acetone in Alembicol D
Table 2: Dermal reactions observed after the challenge application with the test substance
| Animal number | Skin reaction | Score | Results Positive (+) Negative (-) |
|||
| 24 h | 48 h | |||||
| Anterior | Posterior | Anterior | Posterior | |||
| control - 1 | Erythema | 0 | 0 | 0 | 0 | - |
| Oedema | 0 | 0 | 0 | 0 | ||
| control - 2 | Erythema | 0 | 0 | 0 | 0 | - |
| Oedema | 0 | 0 | 0 | 0 | ||
| control - 3 | Erythema | 0 | 0 | 0 | 0 | - |
| Oedema | 0 | 0 | 0 | 0 | ||
| control - 4 | Erythema | 0 | 0 | 0 | 0 | - |
| Oedema | 0 | 0 | 0 | 0 | ||
| control - 5 | Erythema | 0 | 0 | 0 | 0 | - |
| Oedema | 0 | 0 | 0 | 0 | ||
| test animal - 1 | Erythema | 1 | 1 | NE 1 | # 1 | + |
| Oedema | 1 | 1 | # 2 | 2 | ||
| test animal - 2 | Erythema | # 1 | 2 | NP 3 | # 1 | + |
| Oedema | 2 | 1 | # 2 | 1 | ||
| test animal - 3 | Erythema | 2 | 1 | # 2 | # 1 | + |
| Oedema | 1 | 0 | 1 | 1 | ||
| test animal - 4 | Erythema | # 2 | 1 | # 2 | # 1 | + |
| Oedema | 2 | 1 | 2 | 1 | ||
| test animal - 5 | Erythema | 1 | 0 | 1 | 0 | + |
| Oedema | 0 | 0 | * 0 | 0 | ||
| test animal - 6 | Erythema | 2 | 0 | # 2 | 0 | + |
| Oedema | 1 | 0 | 2 | * 0 | ||
| test animal - 7 | Erythema | 0 | 0 | 0 | 0 | - |
| Oedema | 0 | 0 | 0 | 0 | ||
| test animal - 8 | Erythema | 0 | 1 | 1 | 1 | + |
| Oedema | 0 | * 0 | * 0 | * 0 | ||
| test animal - 9 | Erythema | # 2 | 1 | # 2 | # 2 | + |
| Oedema | 2 | 2 | 2 | 2 | ||
| test animal - 10 | Erythema | L 1 | 0 | # 1 | 0 | + |
| Oedema | * 0 | * 0 | 1 | * 0 | ||
Score: grading from 0 (no erythema/oedema) to maximum 4 (severe erythema/oedema)
Anterior: anterior site exposed to 70% test substance in acetone
Posterior: posterior site exposed to 35% test substance in acetone
Control: control animals were treated with Freund's complete adjuvant
NE: necrotic edge
NP: necrotic patch
#: thickening, dryness and sloughing of the epidermis
*: dryness and sloughing of the epidermis
L: localised dermal reaction
INDUCTION
Intradermal injections: Necrosis was recorded at sites receiving Freund's Complete Adjuvant in all test and control animals. Slight irritation was seen in all test animals at sites receiving 0.25% test substance in Alembicol D. Slight irritation was also observed in all control animals receiving Alembicol D only. At the third injection site, necrosis was observed in animals receiving 0.25% (w/v) test substance in a 1:1 mixture of FCA and Alembicol D.
Topical application: Slight to well-defined erythema was observed in 19/20 test animals following topical application of 60% test substance in Alembicol D. Slight erythema was also seen in 6/10 control guinea-pigs receiving Alembicol D alone.
CHALLENGE
The erythema and oedema scores for 20/20 test animals were 0 for both reading time points. Dryness/sloughing of epidermis was noted in 2/20 test animals.
CLINICAL SIGNS
No signs of toxicity were recorded.
BODY WEIGHT
No effect on body weight and body weight gain was observed.
INDUCTION
Isolated incidences of slight erythema (grade 1 of 4) were noted for some test animals throughout the induction period. No dermal reactions were observed for the control animals.
CHALLENGE
No skin irritation reactions were observed in 20/20 test animals at both reading time points.
CLINICAL SIGNS
No signs of toxicity were observed.
BODY WEIGHT
No effect on body weight and body weight gain was observed.
CLINICAL SIGNS
No clinical signs of toxicity were observed.
BODY WEIGHT
Body weight increases were recorded for all guinea-pigs over the period of the study. The treatment group values were comparable to control group values.
INDUCTION
There were no dermal reactions seen in any of the test or control animals.
CHALLENGE
There were no dermal reactions seen in any of the test or control animals, therefore all 20 test animals gave negative responses.
Endpoint conclusion
- Endpoint conclusion:
- adverse effect observed (sensitising)
- Additional information:
The skin sensitising characteristics of the test substance were assessed in a guinea pig maximisation test (GPMT) performed according to OECD test guideline (TG) 406 and GLP (M-141891-01-1, 1997). In this study, 10 male Dunkin-Hartley guinea pigs were induced intradermally on Day 0 with 7.5% test substance (w/v) in 5% acetone in Alembicol D (a product of coconut oil) and 7.5% test substance (w/v) in a 1:1 mixture of Freund’s complete adjuvant (FCA) and 5% acetone in Alembicol D. Intradermal induction was followed on Day 7 by an epicutaneous induction for 48 h under occlusive conditions with 70% test substance (w/v, in 5% acetone). The highest concentrations technically possible were used for the induction phases. A control group with 5 animals was induced with the vehicle only according to the same protocol as the treatment group.
After the intradermal injections, necrosis was recorded at sites injected with FCA in all test and control animals. Slight irritation was seen in test animals at sites treated with 7.5% (w/v) test substance in 5% acetone in Alembicol D and slight irritation was observed in control animals injected with 5% acetone in Alembicol D alone. Necrosis was observed in animals treated with 7.5% (w/v) test substance in a 1:1 mixture of FCA and 5% acetone in Alembicol D.
After topical application, slight erythema was observed in test animals following application of 70% (w/v) test substance in acetone. Slight erythema was also seen in the control guinea pigs.
Two weeks later, on Day 27 the challenge was performed with 70% and 35% test substance (w/v, in 5% acetone), which was applied to the right flank under occlusive conditions for 24 h. 24 and 48 h after patch removal the application site was assessed for signs of local irritation. Dermal reactions were observed in 9/10 test animals challenged with 70% test substance, compared to 0/5 in the controls. No dermal reactions were noted in the remaining test animal. The sensitisation response exceeded 30% of the relative number of the test animals. The test substance was a skin sensitizer under the conditions of the test. Positive control tests were performed periodically with hexyl cinnamic aldehyde and mercaptobenzothiazole, to confirm the validity of the test protocol.
The skin sensitising potential of the test substance was assessed in a second GPMT performed according to OECD TG 406 and GLP (M-192535-01-1, 1999). In this study, 20 female Dunkin-Hartley guinea pigs were induced intradermally on Day 0 with 0.25% test substance (w/v) in Alembicol D and 0.25% test substance in a 1:1 mixture of FCA/Alembicol D. Intradermal induction was followed on Day 7 by an epicutaneous induction for 48 h under occlusive conditions with 60% test substance (w/v) in Alembicol D. The highest concentrations technically possible were used for the induction phases. A control group with 10 animals was induced with the vehicle only according to the same protocol as the treatment group.
After the intradermal injections, necrosis was recorded at sites injected with FCA in all test and control animals. Slight irritation was seen in all test animals at sites treated with 0.25% test substance in Alembicol D. Slight irritation was also observed in all control animals injected with Alembicol D only. At the third injection site, necrosis was observed in animals treated with 0.25% (w/v) test substance in a 1:1 mixture of FCA and Alembicol D.
After topical application, slight to well-defined erythema was observed in 19/20 test animals following application of 60% test substance in Alembicol D. Slight irritation was also seen in 6/10 control guinea-pigs receiving Alembicol D alone.
Two weeks later, on Day 27, the challenge was performed with 60% and 30% test substance (w/v) in Alembicol D, which was applied to the right flank under occlusive conditions for 24 h (anterior site and posterior site, respectively). 24 and 48 h after patch removal the application site was assessed for signs of local irritation.
No dermal reactions were noted in animals challenged with 60% and 30% test substance and the control animals. The sensitisation response did not exceed 30% of the relative number of the test animals. The test substance was not a skin sensitizer under the conditions of the test. Positive control tests were performed periodically with hexyl cinnamic aldehyde and mercaptobenzothiazole to confirm the validity of the test protocol.
The skin sensitising potential of the test substance was assessed in a Buehler test performed according to OECD TG 406 and GLP (M-192538-01-1, 1999). In this study, 20 female Dunkin-Hartley guinea pigs were induced topically with 80% test substance (w/v) in Alembicol D. During 3 single treatments performed once every 7 days within 14 days, animals were treated with 0.5 mL test substance solution for 6 h under occlusive conditions during each treatment. A control group with 10 animals was induced with the vehicle only according to the same protocol as the treatment group.
After topical application of the highest technically applicable concentration of the test substance (80%), isolated incidences of slight erythema (grade 1 of 4) were noted: in 1/20 treated animals following the first induction and in 10/20 test animals following the last induction treatment. No dermal reactions were observed for the control animals receiving Alembicol D only.
Two weeks after the last induction, animals were challenged by epicutaneous occlusive challenge exposure of the right flank for 6 h to 80% test substance (w/v) in Alembicol D. 24 and 48 h after patch removal the application site was assessed for signs of local irritation.
No dermal reactions were noted in induced and control animals challenged with 80% test substance. The sensitisation response did not exceed 15% of the relative number of test animals and the test substance was not a skin sensitizer under the conditions of the test. Positive control tests were performed periodically with hexyl cinnamic aldehyde and mercaptobenzothiazole. In study HLS/074, performed in Mar - Apr 1999, 10/10 guinea pigs responded to the skin sensitizer, confirming the validity of the test protocol.
The skin sensitising potential of the test substance was assessed in a second Buehler test performed according to OECD TG 406 and GLP (M-197213-01-1, 2000). In this study, 20 male Dunkin-Hartley guinea pigs were induced topically with 80% test substance (w/v) in Alembicol D. During 3 single treatments performed once every 7 days within 14 days, animals were treated with 0.5 mL test substance solution for 6 h under occlusive conditions during each treatment. A control group with 10 animals was induced with the vehicle only according to the same protocol as the treatment group.
After topical application of the highest technically applicable concentration of the test substance (80%) or Alembicol D only, no dermal reactions were seen in any of the test or control animals, respectively.
Two weeks after the last induction, animals were challenged by epicutaneous occlusive challenge exposure of the right flank for 6 h to 80% test substance (w/v) in Alembicol D. 24 and 48 h after patch removal the application site was assessed for signs of local irritation.
No dermal reactions were noted in induced and control animals challenged with 80% test substance. The sensitisation response did not exceed 15% of the relative number of test animals. The test substance was not a skin sensitizer under the conditions of the test. Positive control tests were performed periodically with hexyl cinnamic aldehyde and mercaptobenzothiazole. In study HLS/075, performed in Oct - Nov 1999, 7/10 guinea pigs responded to the skin sensitizer, confirming the validity of the test protocol.
Respiratory sensitisation
Endpoint conclusion
- Endpoint conclusion:
- no study available
Justification for classification or non-classification
The available data on sensitisation of the test substance meet the criteria for classification as Skin Sens Cat. 1B (H317) according to Regulation (EC) No. 1272/2008.
The substance is listed in Annex VI of Regulation (EC) No. 1272/2008 with the harmonised classification Skin Sens Cat. 1.
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