Registration Dossier

Administrative data

Description of key information

No information was available on the test substance, therefore read across was used to fill the endpoint. Four key studies (Klimisch 1) were available for oleic acid and linoleic acid, using the LLNA and GMPT methods. Oleic acid and linoleic acid gave clear positive results in the LLNA with Stimulation Index (SI) values in the range between 2.6 and 14.9 for oleic acid and 1.5 and 9.1 for linoleic acid. No signs of systemic toxicity were observed for any of the test substances in any of the animals in a GPMT test. A Klimisch 4 study was also available from the Cosmetics Ingredient Review conducted with undiluted glyceryl citrate/lactate/linoleate/oleate in guinea pigs and found to be non-sensitising. Human data for glyceryl linoleate and glyceryl oleate are also available and presented in the "Exposure related observations in humans" section.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
1996
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Done in conjunction with the LLNA method
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source/batch No.of test material:
Sigma-Aldrich, Munich, Germany / Aldrich No. L1376
- Purity: 99%
Species:
guinea pig
Strain:
other: SPF-Hsd Poc: DH
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Harlan Winkelmann, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
SPF
- Weight at study initiation:
300 - 500 grams
- Housing:
Terluran cages on Altromin saw fiber bedding or Lignocel bedding
- Diet (e.g. ad libitum):
Ad libitum
- Water (e.g. ad libitum):
Ad libitum
- Acclimation period:
At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route:
intradermal
Vehicle:
other: 1:1 (v/v) mixture of FCA/physiological saline
Concentration / amount:
5%
Day(s)/duration:
Day 0
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: Vaseline
Concentration / amount:
100%
Day(s)/duration:
Day 7 / 48 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: Vaseline
Concentration / amount:
50%
Day(s)/duration:
Day 20 / 24 hours
Adequacy of challenge:
not specified
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: Vaseline
Concentration / amount:
50%
Day(s)/duration:
Day 28 / 24 hours
Adequacy of challenge:
not specified
No. of animals per dose:
10 test animals and 5 control animals
Details on study design:
RANGE FINDING TESTS: Performed in a preliminary experiment assessing the irritant effects of the test compound after intradermal and topical application

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: Day 0 and Day 7 (48 hours)
- Test groups: One test group exposed to linoleic acid
- Control group: One control group exposed to vehicle only
- Frequency of applications: Once
- Duration: Day 0 to Day 20
- Concentrations: Intradermal induction - 5%; Topical induction - 100%

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 20 and 28
- Exposure period: 24 hours
- Test groups: One test group exposed to linoleic acid
- Control group: One control group exposed to vehicle only
- Concentrations: 50%
- Evaluation (hr after challenge): 24, 48 and 72 hours
Challenge controls:
Vehicle only
Positive control substance(s):
not specified
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
not determinable
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50%
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
not determinable
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
not determinable
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
50%
No. with + reactions:
4
Total no. in group:
10
Remarks on result:
not determinable
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
50%
No. with + reactions:
3
Total no. in group:
10
Remarks on result:
not determinable
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
50%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
not determinable
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100%
Remarks on result:
other: Not reported.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100%
No. with + reactions:
1
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
100%
Remarks on result:
other: Not reported.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Remarks on result:
not measured/tested
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Remarks on result:
not measured/tested
Reading:
rechallenge
Hours after challenge:
24
Group:
positive control
Remarks on result:
not measured/tested

After challenge and re-challenge with linoleic acid, skin reactions were observed in the control group as frequently or even at a higher incidence than the test group. Therefore, it was concluded that the test concentration was too high and included irritative reactions and no conclusion regarding skin sensitization could be drawn.

Interpretation of results:
other: Data inconclusive
Conclusions:
Due to non-specific irritative skin reactions observed, it was concluded that the challenge test concentration was too high, and no conclusion in regard to skin sensitization could be made.
Executive summary:

Female SPF-Hsd Poc: DH guinea pigs, weighing between 300 and 500 grams, were obtained for this guinea pig maximisation test (GPMT). The guinea pigs were divided into 2 groups, a negative control group containing 5 guinea pigs, and a test group containing 10 guinea pigs. The animals recieved two induction treatments, an intradermal injection (day 0) and a topical application (day 7). The test concentrations used for the two induction tests were 5% linoleic acid for the intradermal induction and 100% linoleic acid for the topical induction. The animals also recieved two topical challenge treatments, one on day 20 and one on day 28. The test concentration used for the two challenge tests was 50% linoleic acid. Isotonic saline was used as the vehicle for the intradermal injection and vaseline was used as the vehicle for the topical exposures, and control animals were dosed with the vehicle alone. After challenge and re-challenge with linoleic acid, skin reactions were observed in the control group as frequently or even at a higher incidence than the test group. Therefore, it was concluded that the test concentration was too high and included irritative reactions and therefore no conclusion regarding skin sensitization could be drawn.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
1996
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Done in conjunction with the LLNA method
Species:
guinea pig
Strain:
other: SPF-Hsd Poc: DH
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Weight at study initiation: 300 - 500 grams
- Housing: Terluran cages on Altromin saw fiber bedding or Lignocel bedding
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route:
intradermal
Vehicle:
other: 1:1 (v/v) mixture of FCA/physiological saline
Concentration / amount:
5%
Day(s)/duration:
Day 0
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: Vaseline
Concentration / amount:
50%
Day(s)/duration:
Day 7 / 48 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: Vaseline
Concentration / amount:
25%
Day(s)/duration:
Day 20 / 24 hours
Adequacy of challenge:
not specified
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: Vaseline
Concentration / amount:
25%
Day(s)/duration:
Day 28 / 24 hours
Adequacy of challenge:
not specified
No. of animals per dose:
10 test animals and 5 control animals
Details on study design:
RANGE FINDING TESTS: Performed in a preliminary experiment assessing the irritant effects of the test compound after intradermal and topical application

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: Day 0 and Day 7 (48 hours)
- Test groups: One test group exposed to oleic acid
- Control group: One control group exposed to vehicle only
- Frequency of applications: Once
- Duration: Day 0 to Day 20
- Concentrations: Intradermal induction - 5%; Topical induction - 50%

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 20 and 28
- Exposure period: 24 hours
- Test groups: One test group exposed to oleic acid
- Control group: One control group exposed to vehicle only
- Concentrations: 25%
- Evaluation (hr after challenge): 24, 48 and 72 hours
Challenge controls:
Vehicle only
Positive control substance(s):
not specified
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25%
No. with + reactions:
1
Total no. in group:
10
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
25%
No. with + reactions:
4
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
25%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
25%
No. with + reactions:
3
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
25%
No. with + reactions:
3
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100%
Remarks on result:
other: Not reported
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100%
No. with + reactions:
1
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
100%
Remarks on result:
other: Not reported.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Remarks on result:
not measured/tested
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Remarks on result:
not measured/tested
Reading:
rechallenge
Hours after challenge:
24
Group:
positive control
Remarks on result:
not measured/tested

For oleic acid, one animal showed a skin reaction at 48 and 72 hours and an additional 3 animals showed skin reactions only at the 72 hours reading. Upon re-challenge, a skin reaction was reproduced only in two of the four animals. The skin sensitization was confirmed in two animals.

Interpretation of results:
study cannot be used for classification
Conclusions:
For oleic acid, the skin sensitization potential was confirmed in two animals out of ten.
Executive summary:

Female SPF-Hsd Poc: DH guinea pigs, weighing between 300 and 500 grams, were obtained for this guinea pig maximisation test (GPMT). The guinea pigs were divided into 2 groups, a negative control group containing 5 guinea pigs, and a test group containing 10 guinea pigs. The animals recieved two induction treatments, an intradermal injection (day 0) and a topical application (day 7). The test concentrations used for the two induction tests were 5% oleic acid for the intradermal induction and 50% oleic acid for the topical induction. The animals also recieved two topical challenge treatments, one on day 20 and one on day 28. The test concentration used for the two challenge tests was 25% oleic acid. Isotonic saline was used as the vehicle for the intradermal injection and vaseline was used as the vehicle for the topical exposures, and control animals were dosed with the vehicle alone. For oleic acid, one animal showed a skin reaction at 48 and 72 hours and an additional 3 animals showed skin reactions only at the 72 hours reading. Upon re-challenge, a skin reaction was reproduced only in two of the four animals. The skin sensitization was confirmed in two animals.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
Reason / purpose:
read-across: supporting information
Qualifier:
no guideline followed
GLP compliance:
not specified
Type of study:
Buehler test
Species:
guinea pig
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
Not specified.
Route:
epicutaneous, open
Vehicle:
unchanged (no vehicle)
Day(s)/duration:
54 hours
Adequacy of induction:
not specified
Route:
epicutaneous, open
Vehicle:
unchanged (no vehicle)
Day(s)/duration:
54 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Day(s)/duration:
54 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
20 test animals and 10 control animals
Positive control substance(s):
not specified
Reading:
1st reading
Hours after challenge:
30
Group:
test group
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
54
Group:
test group
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Conclusions:
Glyceryl citrate/lactate/linoleate/oleate did not induce sensitisation in guinea pigs.
Executive summary:

The skin sensitisation potential of glyceryl citrate/lactate/linoleate/oleate was assessed using guinea pigs. Thirty guinea pigs were used for this study, 20 serving as test guinea pigs and 10 serving as controls. The test item was undiluted as the undiluted test item did not induce skin irritation in a preliminary test. The test item was applied via dermal application during induction phases I, II and III and the reactions were assessed 30 and 54 hours after initiation of treatment. A trigger concentration was also determined in week 4, as the highest non-irritating concentration. No skin reactions (erythema or oedema) were observed in induction phases I, II or III, or with the "trigger" concentration. Therefore, it was concluded that glyceryl citrate/lactate/linoleate/oleate did not induce sensitisation in guinea pigs.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2004
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: SPF-CBA/Ca01aHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Females nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: SPF
- Age at study initiation: 6 to 12 weeks
- Housing: Macrolon cages on Altromin saw fiber bedding
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least 5 days
- Indication of any skin lesions: Not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Vehicle:
other: 3 + 1 (v/v) acetone/olive oil
Concentration:
0, 10%, 25% and 50%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Systemic toxicity: Animals observed for systemic effects and local irritant effects
- Irritancy: Local irritant effects expressed as ear swelling were assessed by measuring ear thickness.
- Ear thickness measurements: Ear thickness measured in all groups using a mechanical calliper on day 1 and day 5
- Other measurements: Lymph node weight index (LNWI) were calculated as the arithmetic mean lymph node weight values of the test group and the negative control group

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: SI > 3

TREATMENT PREPARATION AND ADMINISTRATION: Treated by topical applications of test or control solution to the entire dorsal surface of each ear. Treatments performed once daily over three consecutive days.
Positive control substance(s):
other: p-phenylenediamine
Statistics:
A one-sided t-test was used to identify a statistically significant increase in lymph node weights in treated groups vs. the vehicle control group
Positive control results:
Not reported
Parameter:
SI
Value:
1.5
Variability:
0.7
Test group / Remarks:
10%
Parameter:
SI
Value:
7
Variability:
1.2
Test group / Remarks:
25%
Parameter:
SI
Value:
9.1
Variability:
2.4
Test group / Remarks:
50%
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION : For each dose group, the stimulation index was calculated as the ratio of the arithmetic mean DPM (disintegrations per minute)/node values of the test group and the negative control group.

CLINICAL OBSERVATIONS: No systemic effects were observed for any of the treatments.

Increased lymph node weight indices (LNWI) were observed at 25% (p<0.01) and 50% (p<0.01).

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Linoleic acid resulted in SI values above 3 and so is considered a skin sensitizer according to OECD TG 429.
Executive summary:

Female SPF-CBA/Ca01aHsd mice, between 6 and 12 weeks old, were obtained for this local lymph node assay (LLNA). The mice were divided into 5 groups, a negative control, a vehicle control and three treatment groups, each containing 5 mice. The test substance, linoleic acid, was administered via topical application to the entire dorsal surface of each ear at concentrations of 0 (controls), 10, 25 and 50% once daily for three consecutive days. The vehicle used was 3 + 1 (v/v) acetone/olive oil. For each treatment group a stimulation index (SI) was calculated. The stimulation indexes for linoleic acid were 1.5, 7.0 and 9.1 for the 10%, 25% and 50% groups, respectively. Increased lymph node weight indexes were also observed at 25% (p<0.01) and 50% (p<0.01). An SI value > 3 indicated a positive result, therefore concentrations of 25% and 50% were considered positive as skin sensitizing. Linoleic acid was therefore considered a skin sensitizer according to the OECD TG 429.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2004
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: SPF-CBA/Ca01aHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Females nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: SPF
- Age at study initiation: 6 to 12 weeks
- Housing: Macrolon cages on Altromin saw fiber bedding
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least 5 days
- Indication of any skin lesions: Not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Vehicle:
other: 3 + 1 (v/v) acetone/olive oil
Concentration:
0, 10%, 25% and 50%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Systemic toxicity: Animals observed for systemic effects and local irritant effects
- Irritancy: Local irritant effects expressed as ear swelling were assessed by measuring ear thickness.
- Ear thickness measurements: Ear thickness measured in all groups using a mechanical calliper on day 1 and day 5
- Other measurements: Lymph node weight index (LNWI) were calculated as the arithmetic mean lymph node weight values of the test group and the negative control group

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: SI > 3

TREATMENT PREPARATION AND ADMINISTRATION: Treated by topical applications of test or control solution to the entire dorsal surface of each ear. Treatments performed once daily over three consecutive days.
Positive control substance(s):
other: p-phenylenediamine
Statistics:
A one-sided t-test was used to identify a statistically significant increase in lymph node weights in treated groups vs. the vehicle control group
Positive control results:
Not reported
Parameter:
SI
Value:
2.6
Variability:
1.6
Test group / Remarks:
10%
Parameter:
SI
Value:
14.9
Variability:
6.8
Test group / Remarks:
25%
Parameter:
SI
Value:
6.9
Variability:
3.6
Test group / Remarks:
50%
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION : For each dose group, the stimulation index was calculated as the ratio of the arithmetic mean DPM (disintegrations per minute)/node values of the test group and the negative control group.

CLINICAL OBSERVATIONS: No systemic effects were observed for any of the treatments.

Increased lymph node weight indices (LNWI) were observed at 25% (p<0.01) and 50% (p<0.01).

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Oleic acid resulted in SI values above 3 and so is considered a skin sensitizer according to OECD TG 429.
Executive summary:

Female SPF-CBA/Ca01aHsd mice, between 6 and 12 weeks old, were obtained for this local lymph node assay (LLNA). The mice were divided into 5 groups, a negative control, a vehicle control and three treatment groups, each containing 5 mice. The test substance, oleic acid, was administered via topical application to the entire dorsal surface of each ear at concentrations of 0 (controls), 10, 25 and 50% once daily for three consecutive days. The vehicle used was 3 + 1 (v/v) acetone/olive oil. For each treatment group a stimulation index (SI) was calculated. The stimulation indexes for oleic acid were 2.6, 14.9 and 6.9 for the 10%, 25% and 50% groups, respectively. Increased lymph node weight indexes were also observed at 25% (p<0.01) and 50% (p<0.01). An SI value > 3 indicated a positive result, therefore concentrations of 25% and 50% were considered positive as skin sensitizing. The results showed a bell-shaped dose response curve as the SI value was lower at 50% than at 25%. Oleic acid was therefore considered a skin sensitizer according to the OECD TG 429.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
1996
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Done in conjunction with the LLNA method
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source/batch No.of test material:
Sigma-Aldrich, Munich, Germany / Aldrich No. L1376
- Purity: 99%
Species:
guinea pig
Strain:
other: SPF-Hsd Poc: DH
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source:
Harlan Winkelmann, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known:
SPF
- Weight at study initiation:
300 - 500 grams
- Housing:
Terluran cages on Altromin saw fiber bedding or Lignocel bedding
- Diet (e.g. ad libitum):
Ad libitum
- Water (e.g. ad libitum):
Ad libitum
- Acclimation period:
At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route:
intradermal
Vehicle:
other: 1:1 (v/v) mixture of FCA/physiological saline
Concentration / amount:
5%
Day(s)/duration:
Day 0
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: Vaseline
Concentration / amount:
100%
Day(s)/duration:
Day 7 / 48 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: Vaseline
Concentration / amount:
50%
Day(s)/duration:
Day 20 / 24 hours
Adequacy of challenge:
not specified
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: Vaseline
Concentration / amount:
50%
Day(s)/duration:
Day 28 / 24 hours
Adequacy of challenge:
not specified
No. of animals per dose:
10 test animals and 5 control animals
Details on study design:
RANGE FINDING TESTS: Performed in a preliminary experiment assessing the irritant effects of the test compound after intradermal and topical application

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: Day 0 and Day 7 (48 hours)
- Test groups: One test group exposed to linoleic acid
- Control group: One control group exposed to vehicle only
- Frequency of applications: Once
- Duration: Day 0 to Day 20
- Concentrations: Intradermal induction - 5%; Topical induction - 100%

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 20 and 28
- Exposure period: 24 hours
- Test groups: One test group exposed to linoleic acid
- Control group: One control group exposed to vehicle only
- Concentrations: 50%
- Evaluation (hr after challenge): 24, 48 and 72 hours
Challenge controls:
Vehicle only
Positive control substance(s):
not specified
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
50%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
not determinable
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50%
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
not determinable
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
not determinable
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
50%
No. with + reactions:
4
Total no. in group:
10
Remarks on result:
not determinable
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
50%
No. with + reactions:
3
Total no. in group:
10
Remarks on result:
not determinable
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
50%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
not determinable
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100%
Remarks on result:
other: Not reported.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100%
No. with + reactions:
1
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
100%
Remarks on result:
other: Not reported.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Remarks on result:
not measured/tested
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Remarks on result:
not measured/tested
Reading:
rechallenge
Hours after challenge:
24
Group:
positive control
Remarks on result:
not measured/tested

After challenge and re-challenge with linoleic acid, skin reactions were observed in the control group as frequently or even at a higher incidence than the test group. Therefore, it was concluded that the test concentration was too high and included irritative reactions and no conclusion regarding skin sensitization could be drawn.

Interpretation of results:
other: Data inconclusive
Conclusions:
Due to non-specific irritative skin reactions observed, it was concluded that the challenge test concentration was too high, and no conclusion in regard to skin sensitization could be made.
Executive summary:

Female SPF-Hsd Poc: DH guinea pigs, weighing between 300 and 500 grams, were obtained for this guinea pig maximisation test (GPMT). The guinea pigs were divided into 2 groups, a negative control group containing 5 guinea pigs, and a test group containing 10 guinea pigs. The animals recieved two induction treatments, an intradermal injection (day 0) and a topical application (day 7). The test concentrations used for the two induction tests were 5% linoleic acid for the intradermal induction and 100% linoleic acid for the topical induction. The animals also recieved two topical challenge treatments, one on day 20 and one on day 28. The test concentration used for the two challenge tests was 50% linoleic acid. Isotonic saline was used as the vehicle for the intradermal injection and vaseline was used as the vehicle for the topical exposures, and control animals were dosed with the vehicle alone. After challenge and re-challenge with linoleic acid, skin reactions were observed in the control group as frequently or even at a higher incidence than the test group. Therefore, it was concluded that the test concentration was too high and included irritative reactions and therefore no conclusion regarding skin sensitization could be drawn.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
1996
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Done in conjunction with the LLNA method
Species:
guinea pig
Strain:
other: SPF-Hsd Poc: DH
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Weight at study initiation: 300 - 500 grams
- Housing: Terluran cages on Altromin saw fiber bedding or Lignocel bedding
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Route:
intradermal
Vehicle:
other: 1:1 (v/v) mixture of FCA/physiological saline
Concentration / amount:
5%
Day(s)/duration:
Day 0
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
other: Vaseline
Concentration / amount:
50%
Day(s)/duration:
Day 7 / 48 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: Vaseline
Concentration / amount:
25%
Day(s)/duration:
Day 20 / 24 hours
Adequacy of challenge:
not specified
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: Vaseline
Concentration / amount:
25%
Day(s)/duration:
Day 28 / 24 hours
Adequacy of challenge:
not specified
No. of animals per dose:
10 test animals and 5 control animals
Details on study design:
RANGE FINDING TESTS: Performed in a preliminary experiment assessing the irritant effects of the test compound after intradermal and topical application

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: Day 0 and Day 7 (48 hours)
- Test groups: One test group exposed to oleic acid
- Control group: One control group exposed to vehicle only
- Frequency of applications: Once
- Duration: Day 0 to Day 20
- Concentrations: Intradermal induction - 5%; Topical induction - 50%

B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 20 and 28
- Exposure period: 24 hours
- Test groups: One test group exposed to oleic acid
- Control group: One control group exposed to vehicle only
- Concentrations: 25%
- Evaluation (hr after challenge): 24, 48 and 72 hours
Challenge controls:
Vehicle only
Positive control substance(s):
not specified
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
25%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25%
No. with + reactions:
1
Total no. in group:
10
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
25%
No. with + reactions:
4
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
25%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
25%
No. with + reactions:
3
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
72
Group:
test group
Dose level:
25%
No. with + reactions:
3
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100%
Remarks on result:
other: Not reported
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100%
No. with + reactions:
1
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
100%
Remarks on result:
other: Not reported.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Remarks on result:
not measured/tested
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Remarks on result:
not measured/tested
Reading:
rechallenge
Hours after challenge:
24
Group:
positive control
Remarks on result:
not measured/tested

For oleic acid, one animal showed a skin reaction at 48 and 72 hours and an additional 3 animals showed skin reactions only at the 72 hours reading. Upon re-challenge, a skin reaction was reproduced only in two of the four animals. The skin sensitization was confirmed in two animals.

Interpretation of results:
study cannot be used for classification
Conclusions:
For oleic acid, the skin sensitization potential was confirmed in two animals out of ten.
Executive summary:

Female SPF-Hsd Poc: DH guinea pigs, weighing between 300 and 500 grams, were obtained for this guinea pig maximisation test (GPMT). The guinea pigs were divided into 2 groups, a negative control group containing 5 guinea pigs, and a test group containing 10 guinea pigs. The animals recieved two induction treatments, an intradermal injection (day 0) and a topical application (day 7). The test concentrations used for the two induction tests were 5% oleic acid for the intradermal induction and 50% oleic acid for the topical induction. The animals also recieved two topical challenge treatments, one on day 20 and one on day 28. The test concentration used for the two challenge tests was 25% oleic acid. Isotonic saline was used as the vehicle for the intradermal injection and vaseline was used as the vehicle for the topical exposures, and control animals were dosed with the vehicle alone. For oleic acid, one animal showed a skin reaction at 48 and 72 hours and an additional 3 animals showed skin reactions only at the 72 hours reading. Upon re-challenge, a skin reaction was reproduced only in two of the four animals. The skin sensitization was confirmed in two animals.

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:
4 (not assignable)
Rationale for reliability incl. deficiencies:
secondary literature
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
Reason / purpose:
read-across source
Qualifier:
no guideline followed
GLP compliance:
not specified
Type of study:
Buehler test
Species:
guinea pig
Strain:
not specified
Sex:
not specified
Details on test animals and environmental conditions:
Not specified.
Route:
epicutaneous, open
Vehicle:
unchanged (no vehicle)
Day(s)/duration:
54 hours
Adequacy of induction:
not specified
Route:
epicutaneous, open
Vehicle:
unchanged (no vehicle)
Day(s)/duration:
54 hours
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Day(s)/duration:
54 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
20 test animals and 10 control animals
Positive control substance(s):
not specified
Reading:
1st reading
Hours after challenge:
30
Group:
test group
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
54
Group:
test group
No. with + reactions:
0
Total no. in group:
20
Remarks on result:
no indication of skin sensitisation
Conclusions:
Glyceryl citrate/lactate/linoleate/oleate did not induce sensitisation in guinea pigs.
Executive summary:

The skin sensitisation potential of glyceryl citrate/lactate/linoleate/oleate was assessed using guinea pigs. Thirty guinea pigs were used for this study, 20 serving as test guinea pigs and 10 serving as controls. The test item was undiluted as the undiluted test item did not induce skin irritation in a preliminary test. The test item was applied via dermal application during induction phases I, II and III and the reactions were assessed 30 and 54 hours after initiation of treatment. A trigger concentration was also determined in week 4, as the highest non-irritating concentration. No skin reactions (erythema or oedema) were observed in induction phases I, II or III, or with the "trigger" concentration. Therefore, it was concluded that glyceryl citrate/lactate/linoleate/oleate did not induce sensitisation in guinea pigs.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2004
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: SPF-CBA/Ca01aHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Females nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: SPF
- Age at study initiation: 6 to 12 weeks
- Housing: Macrolon cages on Altromin saw fiber bedding
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least 5 days
- Indication of any skin lesions: Not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Vehicle:
other: 3 + 1 (v/v) acetone/olive oil
Concentration:
0, 10%, 25% and 50%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Systemic toxicity: Animals observed for systemic effects and local irritant effects
- Irritancy: Local irritant effects expressed as ear swelling were assessed by measuring ear thickness.
- Ear thickness measurements: Ear thickness measured in all groups using a mechanical calliper on day 1 and day 5
- Other measurements: Lymph node weight index (LNWI) were calculated as the arithmetic mean lymph node weight values of the test group and the negative control group

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: SI > 3

TREATMENT PREPARATION AND ADMINISTRATION: Treated by topical applications of test or control solution to the entire dorsal surface of each ear. Treatments performed once daily over three consecutive days.
Positive control substance(s):
other: p-phenylenediamine
Statistics:
A one-sided t-test was used to identify a statistically significant increase in lymph node weights in treated groups vs. the vehicle control group
Positive control results:
Not reported
Parameter:
SI
Value:
1.5
Variability:
0.7
Test group / Remarks:
10%
Parameter:
SI
Value:
7
Variability:
1.2
Test group / Remarks:
25%
Parameter:
SI
Value:
9.1
Variability:
2.4
Test group / Remarks:
50%
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION : For each dose group, the stimulation index was calculated as the ratio of the arithmetic mean DPM (disintegrations per minute)/node values of the test group and the negative control group.

CLINICAL OBSERVATIONS: No systemic effects were observed for any of the treatments.

Increased lymph node weight indices (LNWI) were observed at 25% (p<0.01) and 50% (p<0.01).

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Linoleic acid resulted in SI values above 3 and so is considered a skin sensitizer according to OECD TG 429.
Executive summary:

Female SPF-CBA/Ca01aHsd mice, between 6 and 12 weeks old, were obtained for this local lymph node assay (LLNA). The mice were divided into 5 groups, a negative control, a vehicle control and three treatment groups, each containing 5 mice. The test substance, linoleic acid, was administered via topical application to the entire dorsal surface of each ear at concentrations of 0 (controls), 10, 25 and 50% once daily for three consecutive days. The vehicle used was 3 + 1 (v/v) acetone/olive oil. For each treatment group a stimulation index (SI) was calculated. The stimulation indexes for linoleic acid were 1.5, 7.0 and 9.1 for the 10%, 25% and 50% groups, respectively. Increased lymph node weight indexes were also observed at 25% (p<0.01) and 50% (p<0.01). An SI value > 3 indicated a positive result, therefore concentrations of 25% and 50% were considered positive as skin sensitizing. Linoleic acid was therefore considered a skin sensitizer according to the OECD TG 429.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included under 'Attached justification' in IUCLID section 13 and 'Cross-reference'.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2004
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: SPF-CBA/Ca01aHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Females nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: SPF
- Age at study initiation: 6 to 12 weeks
- Housing: Macrolon cages on Altromin saw fiber bedding
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: At least 5 days
- Indication of any skin lesions: Not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 / 12
Vehicle:
other: 3 + 1 (v/v) acetone/olive oil
Concentration:
0, 10%, 25% and 50%
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Systemic toxicity: Animals observed for systemic effects and local irritant effects
- Irritancy: Local irritant effects expressed as ear swelling were assessed by measuring ear thickness.
- Ear thickness measurements: Ear thickness measured in all groups using a mechanical calliper on day 1 and day 5
- Other measurements: Lymph node weight index (LNWI) were calculated as the arithmetic mean lymph node weight values of the test group and the negative control group

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: SI > 3

TREATMENT PREPARATION AND ADMINISTRATION: Treated by topical applications of test or control solution to the entire dorsal surface of each ear. Treatments performed once daily over three consecutive days.
Positive control substance(s):
other: p-phenylenediamine
Statistics:
A one-sided t-test was used to identify a statistically significant increase in lymph node weights in treated groups vs. the vehicle control group
Positive control results:
Not reported
Parameter:
SI
Value:
2.6
Variability:
1.6
Test group / Remarks:
10%
Parameter:
SI
Value:
14.9
Variability:
6.8
Test group / Remarks:
25%
Parameter:
SI
Value:
6.9
Variability:
3.6
Test group / Remarks:
50%
Cellular proliferation data / Observations:
DETAILS ON STIMULATION INDEX CALCULATION : For each dose group, the stimulation index was calculated as the ratio of the arithmetic mean DPM (disintegrations per minute)/node values of the test group and the negative control group.

CLINICAL OBSERVATIONS: No systemic effects were observed for any of the treatments.

Increased lymph node weight indices (LNWI) were observed at 25% (p<0.01) and 50% (p<0.01).

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Oleic acid resulted in SI values above 3 and so is considered a skin sensitizer according to OECD TG 429.
Executive summary:

Female SPF-CBA/Ca01aHsd mice, between 6 and 12 weeks old, were obtained for this local lymph node assay (LLNA). The mice were divided into 5 groups, a negative control, a vehicle control and three treatment groups, each containing 5 mice. The test substance, oleic acid, was administered via topical application to the entire dorsal surface of each ear at concentrations of 0 (controls), 10, 25 and 50% once daily for three consecutive days. The vehicle used was 3 + 1 (v/v) acetone/olive oil. For each treatment group a stimulation index (SI) was calculated. The stimulation indexes for oleic acid were 2.6, 14.9 and 6.9 for the 10%, 25% and 50% groups, respectively. Increased lymph node weight indexes were also observed at 25% (p<0.01) and 50% (p<0.01). An SI value > 3 indicated a positive result, therefore concentrations of 25% and 50% were considered positive as skin sensitizing. The results showed a bell-shaped dose response curve as the SI value was lower at 50% than at 25%. Oleic acid was therefore considered a skin sensitizer according to the OECD TG 429.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Four key studies (Klimisch 1) were available with oleic acid and linoleic acid giving clear positive results in the LLNA test, but not in the GMPT test. An additional Klimisch 4 GMPT study was also available in which glyceryl citrate/lactate/linoleate/oleate was found to be non-sensitising. The review of LLNA and GMPT tests on unsaturated compounds by Kreiling (2008) discussed various scenarios which could lead to potential false positives in the LLNAs and suggests that formal classification and labelling for such compounds based on LLNA alone would be inappropriate. Also, although oleic acid and linoleic acid gave clear positive results in the LLNA test, there is human data available on glyceryl linoleate and oleate indicating that these substances are not skin sensitizers (see section on Exposure-related observations in humans). Based on the human data, triglycerides C16 and 18 (unsaturated) are not expected to be a skin sensitizing substances. Therefore, according to CLP Regulation (EC) No 1272/2008, Guidance on the Application of the CLP Criteria" (ECHA 2017) and the 8th Adaptation to Technical Progress to CLP Regulation, the test item is not classified as a skin sensitiser.