Docosanoic acid

Endpoint summary

Description of key information

Reliable studies on skin sensitisation (animal sensitisation tests) are available for the following fatty acids category members:
CAS# 124-07-2, C8 (Basketter et al., 1998);

Local Lymph Node Assay (OECD 429) - not skin sensitizing

CAS# 123-99-9, C9 (Selbie and Lea, 1995);

Guinea Pig Maximization Test (similar to OECD 406) - not skin sensitizing

CAS# 112-05-0, C9 (Celanese/Bio/dynamics, 1981);

Buehler Assay (OECD 406) - not skin sensitizing

CAS# 67701-01-3, C12-C18 (Gardner and Birnie, 1979);

Buehler Assay (similar to OECD 406) - not skin sensitizing, CAS 67701-01-3

Taken together in a weight of evidence approach members of the fatty acids category are not skin sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation, other

Remarks:

Qualitative mechanistic read-across approach

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Qualitative mechanistic read-across approach

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

QSAR Toolbox (v 2.2) prediction for skin sensitisation based on read-across (categorization approach based on organic functional groups)

GLP compliance:

no

Type of study:

other: Qualitative mechanistic read-across approach

Species:

other: not applicable

Strain:

other: not applicable

Parameter:

other: QSAR

Remarks:

(Qualitative mechanistic read-across prediction)

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

other: no skin sensitisation potential predicted

Table 1: Profiles of the analogues used for read-across

Analogue #	A1	A2	A3	A4	A5	A6
Chemical name	Octanoic acid	(2s)-2-hydroxy-2-methylbutanedioic acid	Nonanoic acid	alpha-hydroxycaprylic acid	alpha-hydroxycaproic acid	2-hydroxy-2-methylsuccinic acid
CAS No.	124-07-2	6236-09-5	112-05-0	*0-08-5	*0-09-9	*0-16-1
SMILES code	CCCCCCCC(=O)O	C(=O)(O)C{P-}(C)(O)CC(=O)O	CCCCCCCCC(=O)O	CCCCCCC(O)C(=O)O	CCCCC(O)C(=O)O	O=C(O)CC(O)(C)C(=O)O
Protein binding by OASIS	No binding	No binding	No binding	No binding	No binding	No binding
Protein binding by OECD	No binding	No binding	No binding	No binding	No binding	No binding
Protein binding potency	Not possible to classify	Not possible to classify	Not possible to classify	Not possible to classify	Not possible to classify	Not possible to classify
Organic functional groups	Alcohol; Carboxylic acid; Methyl; Methylene	Alcohol; Carboxylic acid; Methyl; Methylene	Alcohol; Carboxylic acid; Methyl; Methylene	Alcohol; Carboxylic acid; Methyl; Methylene	Alcohol; Carboxylic acid; Methyl; Methylene	Alcohol; Carboxylic acid; Methyl; Methylene
In vivo skin sensitisation	Negative	Negative	Negative	Weak sensitizer	Negative	Negative
Reference source	Unilever, 2002	Patlewicz et al, Reg Tox Pharm, 2007, 48, 225n.	Givaudan, 2007	TOPKAT database, 1986-97	TOPKAT database, 1986-97	Kern et al, Dermat, 2010, 21(1), 8n

Interpretation of results:

other: CLP criteria not met, no classification required according to Regulation (EC) No 1272/2008

Remarks:

Criteria used for interpretation of results: QSAR Toolbox prediction based on read-across

Reference 2

Endpoint:

skin sensitisation, other

Remarks:

Qualitative mechanistic QSAR model

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Qualitative mechanistic QSAR model

Justification for type of information:

QSAR prediction

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

QSAR Toolbox (v 2.2) prediction for skin sensitisation based on (Q)SAR Multicase commercial model A33 for skin sensitisation.

GLP compliance:

no

Species:

other: not applicable

Strain:

other: not applicable

Reading:

other: QSAR prediction

Group:

other: docosanoic acid

Clinical observations:

in vivo skin sensitisation: negative

Remarks on result:

other: Reading: other: QSAR prediction. Group: other: docosanoic acid. Clinical observations: in vivo skin sensitisation: negative.

The prediction of skin sensitisation for docosanoic acid is negative. The details including QMRF (QSAR Model Reporting Format) are presented in attached reports generated by OECD Toolbox.

Interpretation of results:

other: CLP criteria not met, no classification required according to Regulation (EC) No 1272/2008

Remarks:

Criteria used for interpretation of results: QSAR Toolbox prediction based on (Q)SAR model

Reference 3

Endpoint:

skin sensitisation, other

Remarks:

estimation of toxic hazard by applying a decision tree approach

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Qualitative SAR based on structural alerts

Justification for type of information:

1. SOFTWARE
Toxtree is an open source application, which is able to estimate toxic hazard by applying a decision tree approach. Toxtree was commissioned by JRC Computational Toxicology and Modelling and developed by Ideaconsult Ltd.

2. MODEL (incl. version number)
Toxtree v2.1.0

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
See “Any other information on results incl. tables”

4. ADEQUACY OF THE RESULT
The results may be used in a weight-of-evidence approach together with other information to reach a conclusion regarding the skin sensitising potential of the test substance.

Reason / purpose for cross-reference:

reference to same study

Principles of method if other than guideline:

Investigation on skin sensitisation employing Toxtree v2.1.0 (estimation of toxic hazard by applying a decision tree approach)

GLP compliance:

no

Species:

other: not applicable

Strain:

other: not applicable

Parameter:

other: QSAR

Remarks:

Toxtree v2.1.0

Remarks on result:

no indication of skin sensitisation

Remarks:

(no structural alerts)

Outcome of the prediction model:

other: no skin sensitisation potential predicted

Table 1: Results from Toxtree prediction

Properties	Docosanoic acid, C22
Alert for SNAr identified	No
Alert for SN2 identified	No
Alert for Michael Acceptor identified	No
cdk:Comment	Created from SMILES CCCCCCCCCCCCCCCCCCCCCC(=O)O
No skin sensitization alerts identified	Yes
Alert for Schiff base formation identified	No
Alert for Acyl Transfer agent identified	No

Interpretation of results:

other: CLP criteria not met, no classification required according to Regulation (EC) No 1272/2008

Remarks:

Criteria used for interpretation of results: QSAR (structural alerts)

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

1981

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

nine induction exposures

GLP compliance:

no

Type of study:

Buehler test

Justification for non-LLNA method:

The test was done before LLNA as first-choice method for in vivo testing was set into force.

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Weight at study initiation: males 302 to 396 g; females 303 to 367 g
- Housing: singly in suspended stainless steel cages
- Diet: Charles River Vitamin-C fortified Guinea pig diet, ad libitum
- Water: automatic watering system, ad libitum
- Acclimation period: 16 days


ENVIRONMENTAL CONDITIONS
- Temperature (°F): 65-75
- Photoperiod (hrs dark / hrs light): 12/12

Route:

epicutaneous, occlusive

Vehicle:

other: 80 % ethanol and acetone (induction); acetone (challenge)

Concentration / amount:

Induction: 100%

Route:

epicutaneous, occlusive

Vehicle:

other: 80 % ethanol and acetone (induction); acetone (challenge)

Concentration / amount:

Challenge: 10%
Re-challenge: 25%

No. of animals per dose:

20 (10 male, 10 females) in the definitive experiment received the test substance

Details on study design:

RANGE FINDING TESTS:
- No. of animals: 16
- Vehicle: acetone
- Concentrations: 5, 10, 25, 50, 75, and 100%
- Application of test material: each animal was dosed with two to four different concentrations, at different sites of the clipped dorsal skin.
- Application of test material: 0.2 mL of test material mixture was applied beneath a surgical gauze square, placed directly to the test site. The gauze was covered with plastic sheeting and held in place with an elastic adhesive bandage.
- Evaluation of skin reactions: observation for signs of skin irritation were made approx. 24 and 48 hours after dosing. Evaluation was made according to OECD TG 406, paragraph 23.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 9 exposures in total; 3 exposures per week
- Exposure period: 3 weeks
- Test groups: 20 animals (10 m, 10 f)

- Control group:
-- Positive control; DCNB; 12 animals (6 m, 6 f)
-- Irritation control (challenge only): nonanoic acid 8 animals; DCNB 8 animals (4m, 4f each)

- Site: dorsal skin, right side of the midline
- Frequency of applications: 3 exposures per week
- Duration: 6 hours each
- Concentrations:
-- pelargonic acid: 100% inductions 1-5; 75% from the 6th induction onwards, due to severe skin irritation
-- DCNB: 0.5 and 0.75% during inductions 1 through 8; ninth induction was omitted due to severe skin irritation


B. CHALLENGE EXPOSURE
- No. of exposures: 2
- Day(s) of challenge: 1
- Exposure period: 6 hours
- Test groups: as described above
- Control group: as described above
- Site: dorsal skin, sites left of the midline

- Concentrations:
--pelargonic acid: 10% (challenge); 25% (re-challenge, 7 days after first challenge))
-- DCNB: 0.1% at challenge and at re-challenge

- Evaluation (hr after challenge): 24 and 48 after challenge dosing (and after re-challenge dosing)


OTHER: Re-challenge was made 7 days after the challenge treatment

Challenge controls:

yes

Positive control substance(s):

yes

Remarks:

2,4-dinitrochlorbenzene

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

other: irritation controls, DCNB (or pelargonic acid) only

No. with + reactions:

0

Total no. in group:

16

Remarks on result:

other: irritation controls, DCNB (or pelargonic acid) only

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

other: irritation controls, DCNB (or pelargonic acid) only

No. with + reactions:

0

Total no. in group:

16

Remarks on result:

other: irritation controls, DCNB (or pelargonic acid) only

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10%

No. with + reactions:

0

Total no. in group:

20

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

0

Total no. in group:

20

Reading:

1st reading

Hours after challenge:

24

Group:

other: test group, re-challenge

Dose level:

25%

No. with + reactions:

3

Total no. in group:

20

Clinical observations:

barely perceptible erythema

Reading:

2nd reading

Hours after challenge:

48

Group:

other: test group, re-challenge

Dose level:

25%

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

barely perceptible erythema

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

0.1%

No. with + reactions:

10

Total no. in group:

20

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

0.1%

No. with + reactions:

10

Total no. in group:

20

Group:

negative control

Remarks on result:

not measured/tested

Nonanoic acid:

0/20 test animals challenged with 10% test substance exhibited any dermal response, 3/20 showed barely perceptible erythema (+/-) after challenge with 25% test substance at 24 h, and 1/20 at 48 h. No irritation responses were noted in the corresponding controls with the 10% and 25% solutions. Scores of +/- are considered equivocal.

Positive control, DCNB:
9/12 animals challenged with the positive control, 0.1% 2,4-dinitrochlorobenzene, had a score of 1 and greater. No responses for the irritation controls for this treatment were observed. This means 9/12 animals showed positive results (because of a dermal score of 1 or greater, in the absence of a dermal response in irritation control animals).

 

Materials	h	Erythema evaluation scores					Total No. of animals
		0	+/-	1	2	3
DCNB, 0.1%	24	2	1	3	6	0	12
	48	2	2	4	4	0
Irritation control	24	8	0	0	0	0	8
	48	8	0	0	0	0
nonanoic acid, 10%	24	20	0	0	0	0	20
	48	20	0	0	0	0
Re-challenge, 25%	24	17	3	0	0	0	20
	48	19	1	0	0	0
Irritation control	24	8	0	0	0	0	8
	48	8	0	0	0	0
Re-challenge irritation control	24	7	1	0	0	0	7
	48	7	1	0	0	0

 

 

Interpretation of results:

other: CLP criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

CLP: not classified

Reference 5

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

26 Apr - 25 May 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline Study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

Treatment of the control group: 1. No injection was performed on the control group. 2. After SDS treatment, the control group was not treated with the vehicle control (acetone/PEG400). 3. The exposure duration is not mentioned.

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The test was done before LLNA as first-choice method for in vivo testing was set into force.

Species:

guinea pig

Strain:

other: Albino Dunkin Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Olac
- Weight at study initiation: 318 - 347 g
- Acclimation period: 4 days

Animal husbadry was carried out according to SOP 491/05, Animal Welfare and Husbandry..

Route:

intradermal and epicutaneous

Vehicle:

other: Acetone/PEG400

Concentration / amount:

Induction application: 0.25% (intradermal) and 50.0% (epicutanous)
Challenge application: 50.0% (epicutanous)

Route:

epicutaneous, occlusive

Vehicle:

other: Acetone/PEG400

Concentration / amount:

Induction application: 0.25% (intradermal) and 50.0% (epicutanous)
Challenge application: 50.0% (epicutanous)

No. of animals per dose:

10 (test group), 5 (control)

Details on study design:

RANGE FINDING TESTS:
Intradermal injection: Preliminary irritation studies were carried out to determine concentrations of test material suitable for sensitization challenge: Four male guinea pigs weighing between 348-374 g at the start of the study were injected intradermally on the clipped flanks with 0.1 mL aliquots of 2.0, 1.0, 0.5, 0.25, 0.1, 0.05% (w/v) test material in corn oil. Approximately 24 hours later the reactions were examined for size in millimetres (length and breath), erythema and oedema.
Occluded patch: Four male guinea pigs weighing between 501-514 g were selected. For each guinea pig three eight millimetre diameter filter paper patches were saturated with the following concentrations: 1.0, 5.0, 10.0% (w/v) of test material in acetone/PEG400 and applied to the shaved and clipped flanks. The patches were left on for 24 hours and assessments carried out approximately 24 and 48 hours after the patches were removed. The study was repeated using an additional four male guinea pigs weighing between 501-608 g to find a suitably irritant concentration for induction application.

MAIN STUDY
Ten guinea pigs (five male and five female) weighing between 318-347 g were randomly selected for the test group and five guinea pigs (males) were selected to be weight matched controls for challenge. All guinea pigs were examined daily and weighed weekly as an indication of general health.

A. INDUCTION EXPOSURE
- No. of exposures: 2
On day 1, at the intradermal induction test animals were treated with 0.25% test material. Eight days later, the test and control animals were treated with 10.0% (w/w) sodium dodecyl sulphate in petrolatum by open application over the induction injection sites. Twenty four hours later the test animals were treated with 50.0% test material by induction application.


B. CHALLENGE EXPOSURE
- No. of exposures: 1
Eleven days after the induction application the test and control animals were challenged with 50.0% test material by occluded patch.

Challenge controls:

5 males

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Induction application: 0.25% (intradermal) and 50.0% (epicutanous) Challenge application: 50.0%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no effects

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Induction application: 0.25% (intradermal) and 50.0% (epicutanous) Challenge application: 50.0%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no effects

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

Challenge: 50%

No. with + reactions:

0

Total no. in group:

5

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Challenge: 50%

No. with + reactions:

0

Total no. in group:

5

Group:

positive control

Remarks on result:

not measured/tested

There was no evidence of toxicity of the test substance. Daily examination showed all guinea pigs to be in good health and no significant differences in body weights were observed between the test and control guinea pigs.

As a result of the preliminary irritation tests 0.25% was selected from the preliminary irritation test to be the highest suitable irritant concentration for the intradermal injection induction. It was not possible to establish a sufficiently irritant concentration of the test material for induction application from the preliminary work, therefore the animals were treated on day 8 of the study with 10.0% sodium dodecyl sulphate in petrolatum by open application over the induction injection sites, followed twenty four hours later by an induction application patch of 50.0%. 50.0% was selected as it was the highest non-irritant concentration for the challenge patch.

Interpretation of results:

other: CLP criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

CLP: not classified

Reference 6

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

not specified

Vehicle:

not specified

Concentration:

10, 25 and 50%

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Irritation: in a human 4 h patch test (Basketter et al., 1997) octanoic acid showed a high irritation potential (68% of the panel responded to octanoic acid and 58% of the panel reacted to 25% sodium lauryl sulphate (positive control))

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by β-scintillation
- Criteria used to consider a positive response: Evaluation criteria used were taken from Kimber and Basketter (1992)

TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of the test or vehicle alone was applied to the dorsum of both ears. The application was repeated on days 2 and 3. On day 6, 20 µCi 3H-methyl thymidine in 250 µL phosphate buffered saline (PBS) was injected into the tail vein. 5 h later, the draining auricular lymph nodes were excised and pooled for each experimental group. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation and washed twice in PBS. After the final wash, pellets were resuspended in 5% trichloracetic acid (TCA) and incubated at 4 °C for 18 h. Pellets were resuspended in TCA and thymidine incorporation was measured.

Positive control substance(s):

other: analogue tests with other substances are also reported

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: no data

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

10%

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

1.6

Test group / Remarks:

50%

Interpretation of results:

other: CLP criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

CLP: not classified

Reference 7

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

migrated information: read-across based on grouping of substances (category approach)

Adequacy of study:

weight of evidence

Study period:

25 Oct - 6 Dec 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions: The applied test concentration for challenge described in the materials description and the summary do not correspond (20% w/v vs 40% w/v).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

Positive control is missing. First observations after challenge were performed 26 h after removing of patches.

GLP compliance:

no

Type of study:

Buehler test

Justification for non-LLNA method:

The test was done before LLNA as first-choice method for in vivo testing was set into force.

Species:

other: albino guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Tuck, Rayleigh, Essex, UK
- Age at study initiation: 5 to 6 weeks (control and test group)
- Weight at study initiation: 291 - 353 g (males, control group), 307 - 381 (males, test group) and 327 - 361 g (females, contol group), 312 - 364 (females, test group)
- Housing: up to 10 animals of the same sex per cage in open-topped metal cages with autoclaved soft wood shavings
- Diet: Guinea-pig F.D.I, pelleted, supplemented with autoclaved hay, ad libitum
- Water: ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 2
- Humidity (%): 50 - 70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 Oct To: 6 Dec 1979

Route:

epicutaneous, occlusive

Vehicle:

other: distilled water

Concentration / amount:

Induction: 40% (w/v)

Route:

epicutaneous, occlusive

Vehicle:

other: distilled water

Concentration / amount:

Challenge: 20% (w/v)

No. of animals per dose:

20 (10 controls animals each for challange and re-challenge), 20 (in test groups)

Details on study design:

RANGE FINDING TEST
To evaluate the irritating concentration of the test substance, three animals were treated with 1, 10 and 20% (w/v) test substance in water for 6 h by occluded dermal application. 20% (w/v) of the test substance was evaluated to be the highest-non irritating concentration.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 h
- Test groups: test substance in distilled water (10m, 10f)
- Control group: untreated (5m, 5f)
- Site: left shoulder
- Frequency of applications: once a week
- Duration: 3 weeks (day 0 - 21)
- Concentrations: 40% (w/v, reflects the highest concentration to form a homogeneous solution or suspension).

B. CHALLENGE EXPOSURE
- No. of exposures: 2 (challenge and rechallenge)
- Day(s) of challenge: 13 days after the third phase of the induction period (challenge: day 27) and 8 days after primary challenge (rechallenge: day 35)
- Exposure period: 6 h
- Test groups: test substance in distilled water
- Control group: test substance in distilled water
- Site: right flank
- Concentrations: 20% (w/v)
- Evaluation (hr after challenge and rechallenge): 26 and 48 h after patch removal

APPLIED GRADING SCALE FOR THE EVALUATION OF TEST REACTIONS:
0 = no visible change
± = slight patchy erythema ( not considered as positive sensitisation response)
1 = slight, but confluent or moderate patchy erythema
2 = moderate erythema
3 = severe erythema, with or without oedema

Challenge controls:

The control group is actually a challenge control.

Positive control substance(s):

not specified

Key result

Reading:

1st reading

Hours after challenge:

26

Group:

test chemical

Dose level:

20%

No. with + reactions:

1

Total no. in group:

20

Key result

Reading:

1st reading

Hours after challenge:

26

Group:

negative control

Dose level:

20%

No. with + reactions:

0

Total no. in group:

10

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

20%

No. with + reactions:

0

Total no. in group:

20

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

20%

No. with + reactions:

0

Total no. in group:

10

Key result

Reading:

rechallenge

Hours after challenge:

26

Group:

test chemical

Dose level:

20%

No. with + reactions:

6

Total no. in group:

20

Reading:

rechallenge

Hours after challenge:

26

Group:

negative control

Dose level:

20%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 26.0. Group: negative control. Dose level: 20%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

20%

No. with + reactions:

0

Total no. in group:

20

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 20%. No with. + reactions: 0.0. Total no. in groups: 20.0.

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

20%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 20%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Group:

positive control

Remarks on result:

not measured/tested

Table 1: Erythematous response after exposure

Treatment	Sex	Animal number	Erythematous response after challenge		Animal number	Erythematous response after rechallenge
			26 h	48 h		26 h	48 h
Control   (20% test solution administered at challenge only)	male	21 22 23 24 25	0 0 0 0 0	0 0 0 0 0	34 35 36 37 38	± 0 ± ± ±	± 0 0 ± 0
	female	26 27 28 29 30	0 0 0 0 0	0 0 0 0 0	39 40 41 42 43	± 0 0 ± 0	0 0 0 0 0
Test   (40% test solution administered at induction; 20% test solution administered at challenge)	male	1 2 3 4 5 6 7 8 9 10	0 0 ± 0 0 0 1 ± ± 0	0 0 ± 0 0 0 ± 0 0 ±	1 2 3 4 5 6 7 8 9 10	0 1 1 ± 0 0 ± 0 1 0	0 ± ± 0 0 0 0 0 ± 0
	Female	11 12 13 14 15 16 17 18 19 20	0 0 0 0 0 0 ± ± ± 0	0 0 0 ± 0 0 0 0 0 0	11 12 13 14 15 16 17 18 19 20	1 0 0 ± 0 0 1 ± ± 1	± 0 ± ± 0 0 0 0 0 ±

After the first challenge, only slight patchy erythema and one case of slight to moderate erythema (score 1) were observed in animals exposed to the test substance 26 and 48 h after patch removal. A rechallenge with the test substance induced confluent or moderate erythema in 6 test animals after 26 h. However, this response was reversed 48 h after challenge. In correlation to the naive control group, which showed slight patchy erythema 26 and 48 h after exposure in 6 (26 h after challenge) and 2 (48 h after challenge) animals, the observed effect in test animals is rather due to irritation than sensitization.

Therefore, the test substance is not considered to be sensitizing.

Interpretation of results:

other: CLP criteria not met, no classification required according to Regulation (EC) No 1272/2008

Conclusions:

CLP: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Human health effects on skin sensitisation are predicted from adequate and reliable data for source substances by read-across to the target substance within the group applying the group concept in accordance with Annex XI, Item 1.5, of Regulation (EC) No 1907/2006.

Fatty acids are found in all living organisms fulfilling fundamental physiological functions within the body (for details refer to IUCLID chapter 7.1). Based on this role within the body no sensitisation potential of fatty acids is expected as it could be demonstrated by animal studies with C8 fatty acid (octanoic acid), C9 fatty acids (azelaic acid, nonanoic acid), C10 fatty acid (decanoic acid), C12 fatty acid (lauric acid), C18:1 fatty acid (oleic acid), C18:3 fatty acid (linolenic acid), C12-18 fatty acids (fatty acids, C12-18), by QSAR calculations for C6 fatty acid (hexanoic acid) and C22 fatty acid (docosanoic acid) and by human data with C8 fatty acid (octanoic acid), respectively.

 

Skin sensitisation studies in animals

Octanoic acid (CAS# 124-07-2) was tested in the local lymph node assay (LLNA), which measures the induction of sensitisation as a function of primary proliferation of lymphocytes in the lymph node draining the site of topical chemical application (Basketter et al., 1998). Three experimental groups each of four CBA mice were treated once daily with octanoic acid concentrations of 10%, 25% and 50% on three consecutive days, respectively. The lymphocytes proliferation in the test animals was compared with that of sham treated controls. The stimulation indices (SI values) calculated for the substance concentrations 10%, 25% and 50% were 0.7, 1.0 and 1.6, respectively. Since octanoic acid did not elicit an SI ≥ 3 in the LLNA, it is not considered to have skin sensitizing properties.

 

Azelaic acid (CAS# 123-99-9) was examined for its skin sensitization potential in a Guinea Pig Maximisation test (GPMT) which was conducted under GLP according to OECD Guideline 406 (Selbie and Lea, 1995).10 Dunkin-Hartley guinea pigs received an intradermal injection of 0.25% azelaic acid for induction followed by the second induction 8 days later as an application of 50% azelaic acid on the same site, which had been previously treated with 10% SDS in petrolatum. The used concentrations were based on a preliminary range finding test, where the suitable concentrations for the intradermal injection and patch testing were evaluated. Eleven days after the induction application the test animals and the 5 control animals were challenged with 50% test material by occluded patch. As result, no sensitizing effects could be observed 24 and 48 hours after the challenge.

 

A dermal sensitisation study was performed with nonanoic acid (CAS# 112-05-1) according to OECD Guideline 406 (Buehler test)(Celanese/Bio/dynamics, 1981). Hartley guinea pigs were induced nine-times within three weeks with an epicutaneous occlusive application of the test substance (100% inductions 1-5; 75% inductions 6-9 due to severe dermal irritation) onto the right dorsal skin for 6 hours. 14 days after the last induction exposure, epicutaneous challenge was conducted with 10% nonanoic acid in acetone on the left flank under occlusive conditions for 6 hours. No dermal sensitisation reaction was seen at 24 and 48 hours after challenge. Following re-challenge (7 days after the first challenge) with a concentration of 25% nonanoic acid in acetone, 3 and 1 out of 20 animals exhibited barely perceptible erythema after 24 and 48 h, respectively. Since barely perceptibly erythema (score +/-) is not considered as positive sensitisation response (score ≥ 1) and since this response also reversed in two out of three animals, nonanoic acid did not elicit a skin sensitisation potential.

 

Kreiling et al. (2008) assessed the skin sensitisation potential of unsaturated C18 fatty acids (oleic acid (CAS# 112-80-1), linoleic acid (CAS# 60-33-3), linolenic acid (CAS# 463-40-1)) by the GPMT and LLNA. Additional information for oleic acid is obtained from skin sensitisation studies including LLNA and GPMT conducted by Anderson et al. (2009) and Basketter et al. (2009). 

In the studies performed by Kreiling et al. (2008) unsaturated C18 fatty acids (oleic acid, linoleic acid and linolenic acid were assessed for their skin sensitisation potential in the GPMT according to OECD Guideline 406 and in the LLNA according to OECD Guideline 429 both under GLP.

In the GPMT, first a range-finding test was performed to assess the irritation effects of the test substances after intradermal and topical application. 10 SPF-Hsd Poc:DH guinea pigs each received an intradermal induction of 5% oleic acid, linoleic acid and linolenic acid (day 0). 7 days later the second induction treatment was performed by applying a gauze patch with 50% oleic acid and 100% linoleic and linolenic acid, respectively to the clipped skin under occlusive conditions for 48 h. No sodium lauryl sulphate was applied 24 h before the topical induction, since the three unsaturated fatty acids itself caused a clear skin irritation at the topical induction concentration used. On day 20 and day 28 the test animals and 5 control animals were challenged and re-challenged by occluded patch with 25% oleic acid and 50% linoleic acid and linolenic acid, respectively. 24, 48 and 72 h after removal of the challenge patches, skin reactions were recorded. For oleic acid skin sensitisation was considered to be present in two animals. For linolenic acid two test animals showed a skin reaction at the 24 h reading, but not after 48 h and 72 h, indicating a non-specific rather than an allergic reaction, since also one control animal showed grade 1 skin reaction at the 48 h reading. In another test animal a skin reaction was observed at 48 and 72 h. Since the response of at least 30% of the animals is not achieved in the GPMT for oleic acid (2/10 animals with skin sensitisation reaction) and linolenic acid (no animal with skin sensitisation reaction), they are considered not to be skin sensitiser. Linoleic acid caused skin reactions in the control group as frequently or even at a higher incidence than in the test group after challenge and re-challenge. Thus it was assumed, that the test concentrations for challenge were too high and induced non-specific, irritation skin reactions. As a consequence, no conclusion with regard to skin sensitisation could be drawn for linoleic acid in the GPMT.

In parallel to the GPMT the LLNA was performed in SPF-CBA/Ca01aHsd mice. The highest tolerable exposure concentration was determined in preliminary experiments. 10, 25 and 50% of the unsaturated C18 fatty acids in acetone/olive oil (3+1 (v/v)) and the vehicle alone were applied daily to the ears of 5 animals each for three consecutive days. Local irritant effects expressed as ear swelling were assessed by measuring the ear thickness. Oleic acid, linoleic acid and linolenic acid gave clear positive results in the LLNA with SI values ≥ 3. For 10, 25 and 50% oleic acid the SI values were 2.6, 14.9 and 6.9, for 10, 25 and 50% linoleic acid 1.5, 7.0 and 9.1 and for linolenic acid the SI values were 3.1, 9.3 and 10.3, respectively. There was a good agreement between SI values > 3 and statistically significant lymph node weight increase. No clinical signs of local irritation were noted and no significant ear swelling (data not shown) was measured according to the authors. However, these positive results are in discrepancy to the negative results of the GPMT tests.

 

Furthermore, Basketter et al. (2009) evaluated the sensitising potential of oleic acid including a GPMT and LLNA. In the GPMT assay according to OECD 406, 10 guinea pigs were induced via intradermal injection of 5% oleic acid followed by epicutaneous induction with undiluted oleic acid. For challenge and re-challenge, 1% oleic acid was applied topically. No positive skin reaction was observed in the test animals. The validity of the test was confirmed by a positive control group treated with 10% hexyl cinnamic aldehyde, in which 40% of the animals showed a positive sensitisation reaction. The LLNA was conducted in compliance with OECD 429 with the deviation that no pre-screening test was performed to determine the concentration level, that does not induce systemic toxicity and/or excessive local skin irritation. Topical application of oleic acid induced a dose-dependent lymphoproliferative response as shown by stimulation indices of 3.4, 5.7 and 6.5 for 25, 50 and 100% oleic acid, respectively. Thus, the results from the two test systems are also in conflict as the GPMT revealed a negative and the LLNA a positive test result, as already shown by Kreiling et al. (2008).

 

However, the positive LLNA result for oleic acid is considered a false positive based on a LLNA performed by Anderson et al. (2009), in which an excessive skin irritation was noted after application of 25 and 50% oleic acid as indicated by ear swelling measurements. Beside increases in the stimulation indices (SI: 1.6, 2.4 and 5.6 for 10, 25 and 50% oleic acid, respectively, resulting in an EC3 value of 29.7), oleic acid induced a dose-dependent increase in ear thickness: control animals revealed no ear swelling (-1.8 ± 2%) whereas oleic acid exposed animals showed ear swellings of 11.2 ± 2.6%, 29.2 ± 3.9% and 51.6 ± 6.2% for 10, 25 and 50%, respectively, with statistical significance for the high-dose group. In compliance with OECD 429, an increase in ear thickness of ≥ 25% has to be interpreted as excessive skin irritation. Thus, positive results after treatment with more than 10% oleic acid have to be considered as ambiguous due to the induction of excessive skin irritation. Furthermore, 10% oleic acid, which seems not to induce excessive skin irritation, did not reveal a stimulation index above 3. Taken together, oleic acid seems to induce false positive results in the LLNA when concentrations of ≥ 30% (EC3 value) are applied to the skin due to skin irritation properties.

The known limitation of the test system to produce false positive findings with certain skin irritants as indicated in OECD 429 supports the hypothesis that the effects observed in the LLNA performed by Anderson et al. (2009) and thus in the LLNAs performed by Kreiling et al. (2008) and Basketter et al. (2009) are secondary effects due to irritation and cannot clearly be attributed to a sensitisation potential of the test substance. Kreiling et al. (2008) measured the ear swelling in the LLNA, but the data was not presented or described in the result part. It was only stated, that no significant increases in ear thickness in the LLNA studies were measured for any of the test substance concentrations. However, statistically non-significant increases can be obtained even when ear thickness is greater than 25% (e.g. Andersons et al. measured at a test concentration of 25% oleic acid, a statistical non-significant ear swelling of 29.2 ± 3.9%). Thus, a biological significant increase in ear thickness cannot be excluded.. Further, regarding the study performed by Basketter et al. (2009) no information about the induction of skin irritation in the applied oleic acid concentrations is available, which might be the reason for the discrepancy between the two test models GPMT and LLNA. Thus it is not comprehensible, if local irritant effects have been considered adequately in the studies by Kreiling et al. and Basketter et al.. For linoleic acid and linolenic acid also skin irritation potential was noted during the GPMT assay by Kreiling et al. (2008). Thus, the design of the LLNA studies should be regarded as not suitable to evaluate the sensitisation potential for the tested unsaturated C18 fatty acids, since local irritation cannot be excluded definitely.

 

The basic principle underlying the LLNA is that sensitiser induce proliferation of lymphocytes in the lymph nodes draining the sited of test substance application (OECD 429). Kreiling et al. (2008) speculated, that unsaturated fatty acids may cause cell activation by a number of several specific mechanisms (e.g. by induction of intracellular Ca signals, activation of protein kinase C, inhibition GTPase activity, induction of interleukin release etc.) leading to the activation of epidermal cells. “This stimulation could have activated Langerhans cells to migrate out of the skin to the auricular lymph node where they induced a hapten-unrelated cell proliferation of lymph node cells. The gathered information on possible activation of cells in the skin by mechanisms different from a hapten-specific activation suggests that, perhaps, at least part of the lymph node reactions observed after application of oleic acid, linoleic acid and linolenic acid has been caused in an unspecific way” (Kreiling et al., 2008). Furthermore, regarding the fact that the LLNA assesses the induction/sensitization phase of allergic responses only via 3H-thymidine incorporation in the auricular draining lymphe node whereas the GPMT assess the induction/sensitisation period in combination with the elicitation phase of an allergic response via scoring of skin reactions after intradermal induction including a powerful adjuvant which enhances the induction of the immune response (Basketter et al., 2009), the result of the GPMT is considered to be of stronger biological significance than the LLNA.

 

In conclusion, taken into account (1) the discussions about possible false positive findings in the LLNA due to irritating effects and non-specific cell activation, (2) the fact, that unsaturated C18 fatty acids occur in fatty acid constituents of foods and as natural building block in membrane phospholipids and triglycerides, (3) the absence of reports on human cases of allergic reactions, (4) negative QSAR predictions and (5) the negative result in the GPMT for oleic acid and linolenic acid, the unsaturated C18 fatty acids do not fulfill the criteria for skin classification.

 

An UVCB substance with C12-18 fatty acids (0.1% C8, 2.6% C10, 55.9% C12, 19.1% C14, 9.8% C16, 0.1% C17, 11.1% C18 and 0.3% C20) was examined in a dermal sensitization study (Buehler test) (Gardner and Birnie, 1979). 20 Dunkin-Hartley guinea pigs were induced with 40% of the test substance (CAS# 67701-01-3) in distilled water by applying an occlusive patch on the left shoulder once a week for three consecutive weeks. This induction concentration represented the highest concentration to form a homogenous suspension and causing some irritation reactions at the dermal sites of induction. 13 days after the last induction, the test animals and 10 control animals were challenged with 20% test substance under occlusive conditions for 6 hours. Only one animal showed slight, but confluent to moderate erythema at the 26 h reading after patch removal. No positive sensitizing reactions were observed at the 48 h reading after challenge. 8 days after challenge, a re-challenge was carried out including a new naïve control group with ten 10 animals. In 6 animals confluent to moderate erythema was noted at the 26 h reading after re-challenge. However, this response reversed within 22 h in all animals and is therefore not considered as sensitizing effect.

 

Skin sensitisation tests have also been performed with decanoic acid (CAS# 334-48-5) and lauric acid (CAS# 143-07-7). Since only short abstracts are available, these studies have been judged with reliability 4 "not assignable". However, the results of these studies confirm the non-sensitising properties of fatty acids. The skin sensitization potential of decanoic acid was tested in a Buehler test, where 20 guinea pigs were induced with an epicutanous application of 5% decanoic acid in 40% ethanol under occlusion for 6 hours once a week for three consecutive weeks (Sauter and Ritz, 1975). Two weeks after the last induction, the animals were challenged epicutanously under occlusion with a concentration of 5% decanoic acid in acetone for 6 hours. The readings 24 and 48 hours after removal of the patches revealed occasional very slight degree of irritation in the dose and control groups, respectively. However, no signs of a sensitization reaction were noted.

The same negative result was obtained for lauric acid tested in a study according to the method described by Magnusson and Kligman (Gloxhuber and Potokar, 1979). 20 female Pirbright-white guinea pigs received an induction by intradermal injection and were challenged with a concentration of 2.5% epicutanously under occlusion for 24 hours. The readings of the skin sites 24 and 48 hours later did not reveal any reaction so that lauric acid is regarded as not sensitizing to skin.

 

Skin sensitisation predictions using Toxtree and OECD Toolbox

The skin sensitisation potential of hexanoic acid (CAS# 142-62-1) and docosanoic acid (CAS# 112-85-6) was estimated using QSAR calculations (KNOELL CONSULT, 2012). These two substances are representing the fatty acids with the shortest (C6 fatty acid) and the longest carbon chain length (C22 fatty acid) within the fatty acids category.

Structural alerts of hexanoic acid and docosanoic acid using Toxtree (Estimation of Toxic Hazard – A Decision Tree Approach v.2.1.0) were examined. No structural alerts were detected for the test substances.

In addition a read-across approach based on organic functional groups was conducted using the OECD Toolbox databases. For 5 read-across substances which contain the same functional groups, skin sensitisation data could be found in OECD Toolbox databases. The experimental results of the surrogate substance were negative.

With the help of the OECD Toolbox (v 2.2) the sensitisation potential for hexanoic acid and docosanoic acid was also predicted based on the (Q)SAR Multicase commercial model A33 also referred to as Danish EPA QSAR Database. The prediction for both substances C6 and C22 are negative.

 

Skin sensitisation studies in humans

Octanoic was not found to be a skin sensitizer in a published study with 25 human subjects, who received an application of 0.3 g octanoic acid (CAS# 124-07-2) at 5% concentration under occlusion for induction onto the forearm 5 times for 24 hours (Opdyke, 1981). The challenge with a concentration of 1% did not result in any positive reaction when scored 72 and 96 hours later.

Conclusion

Taken into account all available data on skin sensitisation testing in animals and humans and by QSAR applications, fatty acids do not fulfil the criteria for classification as skin sensitiser.

References:

Anderson, SE. et al. (2009). Evaluation of irritancy and sensitization potential of metalworking fluid mixtures and components. Journal of Immuntoxicology 6(1):19 - 29.

Auletta, C. S. (1981). A dermal sensitization study in guinea pigs. Testing laboratory: Bio/dynamics Inc., East Millstone, NJ, USA. Report no.: 6752-81. Owner company: OXEA Group. Study number: T00875. Report date: 1981-12-31.

Basketter, D.A. et al. (1998).Strategies for Identifying False Positive Responses in Predictive Skin Sensitization Tests. Food and Chemical Toxicology 36(4):327 – 33.

Basketter, D. et al. (2009). Application of a weight of evidence approach to assessing discordant sensitisation datasets: Implications for REACH. Regulatory Toxicology and Pharmacology 55:90 - 96.

Gardner, J. R. and Birnie, J. V. (1979). DELAYED CONTACT HYPERSENSITIVITY IN GUINEA-PIGS (BUEHLER TEST): ECM BTS 579 E9226. Testing laboratory: Life Science Research, Stock, Essex, UK. Report no.: 79/PGN427/567. Owner company: Protcter & Gamble (NTC) Limited, Newcastle-upon-Tyne, UK.Study number: 25596.Report date: 1979-12-31.

Gloxhuber and Potokar (1979). Cetiol A und seine Ausgangsprodukte Laurinsäure und Hexanol, Prüfungen auf allergieauslösende Eigenschaften.Testing Laboratory: Hauptabteilung Toxikologie, Henkel KGaA.Report no. 252. Owner Company: Henkel KGaA, Düsseldorf, Germany. Company study no. TBD790129. Report data: 1979-06-08.

Kreiling, R. et al. (2008). Comparison of the skin sensitizing potential of unsaturated compounds as assessed by the murine local lymphnode assay (LLNA) and the guinea pig maximization test (GPMT).Food and Chemical Toxicology 46:1896 - 1904.

Opdyke, D.L.J. (1981). Monographs on Fragrance Raw Materials. Fd Cosmet. Toxicol. Vol. 19: 237-254.

Sauter and Ritz (1975) Guinea Pig Closed Patch Test (SL: U300-117). Testing Laboratory: Procter & Gamble. Company. Owner Company: Procter & Gamble Company. Company study no.: 13003. Report date: 1975-05-05.

Selbie, L. and Lea, L. (1995). Azelaic Acid: Skin Sensitization Study in Guinea Pigs. Testing laboratory: Environmental safety Laboratory, Bedford, England. Report no.: SM950173. Owner Company: Unilever Research, Colworth House, Sharnbrook, Bedford MK44 1LQ, England.Report date: 1995-06-27.

Szymoszek, A. (2012). Combined QSAR & read-across approach for fatty acids C6 (hexanoic acid) and C22 (docosanoic acid) - skin sensitisation. Testing laboratory: DR. KNOELL CONSULT GmbH, Mannheim, Germany. Owner company: FATAC Ltd., Gloucestershire, Great Britain.Report date: 2012-01-20.

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

Study not required according to Annex VII-X of Regulation (EC) No 1907/2006.

Justification for classification or non-classification

All available data on skin sensitisation of the members of the fatty acids category do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.