Registration Dossier

Administrative data

Description of key information

Skin sensitisation (OECD 406: GPMT and Buehler): not sensitising

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:
weight of evidence
Study period:
01 Feb - 03 Apr 1995
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The results of the reliability check were inconclusive.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
results of reliability check were inconclusive
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
results of reliability check were inconclusive
GLP compliance:
yes
Type of study:
Buehler test
Justification for non-LLNA method:
The Buehler Test was performed prior to the amendment of Regulation (EC) No 1907/2006 in which the Local Lymph Node Assay is given as the first-choice in vivo study.
Species:
guinea pig
Strain:
other: Dunkin Hartley Crl:(HA)BR
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River, Kisslegg, Germany
- Age at study initiation: approximately 5 weeks
- Weight at study initiation: approximately 380 g
- Housing: in groups of 2-3 animals in Makrolon Type IV cages (EBECO) with standard softwood bedding (ARWI-Center, Essen, Germany); bedding chaged twice weekly
- Diet: pelleted Altromin Maintenance Diet 3022 (Fa. Altromin, Lage, Germany), ad libitum. Carrots optionally added.
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 45-70
- Air changes (per hr): 100 m³
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: 7 Feb - 3 Apr 1995
Route:
epicutaneous, occlusive
Vehicle:
peanut oil
Concentration / amount:
70%
Route:
epicutaneous, occlusive
Vehicle:
peanut oil
Concentration / amount:
60%
No. of animals per dose:
10 (control group), 20 (treatment group)
Details on study design:
RANGE FINDING TESTS:
Irritation test: 0.5 mL of the test substance was applied topically to the shaved skin of 3 guinea pigs, successively. A concentration of 12.5, 25, 50 and 100 % was applied to the left flank and 30, 50 and 70% concentration to the right flank. The exposure was terminated after 6 hours by removing the plaster and cleaning the skin with 20% propylene glycol. The severity of erythema and oedema was assessed 24 and 48 h after exposure. The undiluted substance caused weak, confluent erythema in 1/3 animals at the 24 and 48 hour reading time point. The 70% solution was the lowest concentration to induce minimal irritation; causing weak, confluent erythema in 1/3 animals 24 hours after exposure. This effect had cleared within 48 hours.
Challenge test: the maximum non-irritating concentration was tested one week before the challenge in the main study by applying 0.5 mL of 30, 50 and 70% solution to the right flank of 5 animals in the control group under occlusive conditions. After 6 hours, the plaster was removed and the site cleaned with 20% propylene glycol. 24 and 48 hours after the exposure ended, the skin irritating effects were assessed. None of the concentrations caused skin irritation. 60% was selected to be the challenge dose.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 3
- Exposure period: 6 h
- Test groups: test substance in peanut oil
- Control group: peanut oil
- Site: left cranial shaved flanks
- Frequency of applications: once weekly
- Duration: Day 1-14
- Concentrations: 70%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 28
- Exposure period: 6 h
- Test groups: test substance in peanut oil
- Control group: test substance in peanut oil
- Site: left and right caudal flanks
- Concentrations: 60%
- Evaluation (hr after challenge): 24, 48 and 72 h
Positive control substance(s):
yes
Remarks:
alpha-hexyl cinnamic aldehyde
Positive control results:
A reliability check is carried out at regular intervals with alpha-hexyl cinnamic aldehyde to check the sensitivity of the test system and the reliability of the experimental methods used by the test laboratory. An independent study was performed in October-December 1994 (report No. R 9400844), according to the Buehler method. During the first challenge with 25% alpha-hexyl cinnamic aldehyde, a sensitisation reaction was induced in 25% (5/20) of the Dunkin Hartley guinea pigs, while the second challenge did not lead to conclusive sensitisation reactions. In the negative control group, the first challenge induced sensitisation in 20% (2/10) of the animals, and the second challenge did not caused any sensitisation reactions. The 50% solution in peanut oil that was used for the topical inductions, caused weak to moderate skin irritation. A 25% solution was applied in the first challenge, while a 25% solution was applied to the left flank and 15, 20 and 25% solutions were applied to the right flank during the second challenge. As 20% of the animals in the negative control group reacted, the results of the reliability check are inconclusive.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
60%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
1/10 animals showed slight, patchy erythema on the right flank only
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
60%
No. with + reactions:
0
Total no. in group:
20
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
60%
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
1/10 animals showed slight, patchy erythema on the left flank only
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
60%
No. with + reactions:
2
Total no. in group:
20
Clinical observations:
2/20 animals showed slight, patchy erythema on the left flank only
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
negative control
Dose level:
60%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test group
Dose level:
60%
No. with + reactions:
0
Total no. in group:
20

Skin rections, induction phases

The 3rd induction with a 70% solution caused slight skin irritation in 2/20 treatment animals.

Skin reactions, challenge phase

At the first reading, 1/10 control animals had slight, patchy erythema on the challenge site on the left flank. 1/10 in the control group had slight, patchy erythema on the right flank at the second reading, while 2/20 treatment animals had slight, patchy erythema on the left flank at the second reading. All skin irritation effects had cleared within 72 hours after exposure ended.

Table 1: Skin reactions 24, 48 and 72 hours after challenge

 

24 hours

48 hours

72 hours

group

(# of animals)

flank

control

(10)

l/r

treatment

(20)

l/r

control

(10)

l/r

treatment

(20)

l/r

control

(10)

l/r

treatment

(20)

l/r

none

10/9

20/20

9/10

18/20

10/10

20/20

slight

0/1

0/0

1/0

2/0

0/0

0/0

weak

0/0

0/0

0/0

0/0

0/0

0/0

moderate

0/0

0/0

0/0

0/0

0/0

0/0

strong

0/0

0/0

0/0

0/0

0/0

0/0

l: left flank, induction site and challenge site

r: right flank, challenge site

Mortality and body weight

There was no mortality during the study period and the animals in the treatment group showed a similar gain in body weight compared with the control group.

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
24 Mar - 24 Apr 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The epicutaneous induction and challenge were performed under semi-occlusive conditions.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
epicutaneous induction and challenge performed under semi-occlusive conditions
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
epicutaneous induction and challenge performed under semi-occlusive conditions
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The Guinea Pig Maximisation Test was performed prior to the amendment of Regulation (EC) No 1907/2006 in which the Local Lymph Node Assay is given as the first-choice in vivo study.
Species:
guinea pig
Strain:
Himalayan
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BRL Ltd., Basel, Switzerland
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 416 ± 23 g (mean ± SD, control group); 434 ± 24 g (mean ± SD, treatment group)
- Housing: animals were housed in groups of 5 in labelled metal cages with wire-mesh floors (ITL, Bergen, the Netherlands)
- Diet: standard guinea pig diet (LC 23-B, pellet diameter 4mm, including ascorbic acid (1600 mg/kg); Hope farms, Woerden, the Netherlands), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 24 March 1998 To: 24 April 1998
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Route:
epicutaneous, semiocclusive
Vehicle:
corn oil
No. of animals per dose:
10 (treatment group)
5 (control group)
Details on study design:
RANGE FINDING TESTS:
Prior to the start of the main study, the intradermal and epidermal irritancy of the test substance was investigated to select suitable concentrations for the induction and challenge phase of the main study. The selection was based on the absence of toxicity and on the following criteria for each route and/or study phase:
Induction (intradermal and epidermal): The highest possible concentration that produced moderate irritation (the intradermal reactions may include slight necrosis (< 3 mm in diameter)).
Challenge: The maximum non-irritant concentration.

A series of test substance concentrations were tested. The first and subsequent concentrations were selected from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1% and if needed, further lower concentrations using the same steps. The test system and procedures were identical to those used during the main study, unless otherwise specified. The four animals were 5-9 weeks old. The body weights were determined prior to treatment (results not shown).

Intradermal Injections:
A series of four test substance concentrations was used; the highest concentration was the maximum concentration that could technically be
injected (undiluted). One animal received 50 and 100% (undiluted), and the second animal received 10 and 20%, respectively, in duplicate (0.1 mL/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment. Slight erythema was observed at the injection site of the undiluted test substance 48 h after exposure. The udiluted test substance was selected for the induction phase in the main study.

Epidermal application:
A series of four test substance concentrations (10, 20, 50 and 100%) was used. All concentrations could technically be applied. Two different concentrations were applied (0.5 mL each) per animal to the clipped flank, using Metalline patches (2x3 cm) mounted on Medical tape, which were held in place with Micropore tape and subsequently Coban elastic bandage (semi-occlusive covering). The animals receiving intradermal injections were treated with the lowest concentrations (10 and 20%) and two further animals with the highest concentrations (50 and 100%). After 24 hours, the dressing was removed and the skin cleaned of residual test substance. The treated skin areas were assessed for irritation 24 and 48 hours after exposure. No skin irritation was observed at any of the treated sites at any of the reading time points. As the epidermal induction using the test substance did not cause any skin irritation, the test site of all animals was treated with 10% SDS approximately 24 hours before the epidermal induction in the main study, to provoke a mild inflammatory reaction. The udiluted test substance was selected for the challenge phase in the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2, intradermal and epicutaneous
- Exposure period: single injection (epidermal) and 48 h (epicutaneous)
- Test groups:
Intradermal, Day 1 (3 pairs of injections, 0.1 mL/site):
Injection 1: a 1:1 mixture (w/w) Freunds Complete Adjuvant (FCA)/water for injection
Injection 2: undiluted test substance
Injection 3: undiluted test substance in a 1:1 mixture (w/w) with FCA (final concentration is 50% test substance)
48 h after intradermal injection (Day 3), the degree of erythema and edema was evaluated.

On Day 7, the scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium dodecyl sulfate in vaseline using a spatula. This concentration of SDS provoked a mild inflammatory reaction.

Epicutaneous, Day 8:
0.5 mL undiluted test substance was applied to the SDS-treated skin area. The semi-occlusive dressing was kept in place for 48 h. The degree of erythema and edema was evaluated directly after cleaning the skin area with water (Day 10).

- Control group:
Intradermal, day 1 (3 pairs of injections, 0.1 mL/site):
Injection 1: a 1:1 mixture (w/w) FCA/water
Injection 2: corn oil
Injection 3: corn oil (w/v) in a 1:1 mixture (w/w) FCA (final concentration is 50% corn oil)

On Day 7, the scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium dodecyl sulfate (SDS, Boom, Meppel, the Netherlands) in vaseline using a spatula. This concentration of SDS provoked a mild inflammatory reaction.

Epicutaneous, Day 8: 0.5 mL corn oil

- Site: the shoulder region
- Frequency of applications: once (intradermal on Day 1 and epicutaneous on Day 8)
- Duration: Day 1 (intradermal), Day 8-10 (epicutaneous)
- Concentrations: undiluted (intradermal and epicutaneous)

B. CHALLENGE EXPOSURE
- No. of exposures: 1 (challenge)
- Day(s) of challenge: 21
- Exposure period: 24 hours
- Test groups: 0.5 mL test substance
- Control group: 0.5 mL test substance
- Site: approximately 20 mm x 30 mm, on one flank of the animals
- Concentration: undiluted
- Evaluation (hr after challenge): 24 and 48 hours after the challenge ended
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamicaldehyde, tech. 85%
Positive control results:
A reliability check is carried out at regular intervals with alpha-hexylcinnamic aldehyde to check the sensitivity of the test system and the reliability of the experimental methods used by the test laboratory. In an independent study performed in 1998 (report No. 217812), alpha-hexylcinnamic aldehyde induced sensitisation in 80% (8/10) of the Himalayan guinea pigs challenged with a 10% solution, and in 70% (7/10) of the guinea pigs challenged with a 5% solution. A 5% solution was used for intradermal induction and undiluted test substance was used for topical induction.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
10

48 hours after intradermal induction, slight to severe erythema was noted at all of the sites injected with FCA/water and FCA/test substance in 10/10 treated and 5/5 control animals. 4/5 control animals also exhibited necrosis at the FCA/test substance injection site. In 3/10 treated animals slight to well-defined erythema was observed at the injection site of the test substance. Following the topical induction, severe erythema and scabs were observed at the test site in 3/10 treated animals. A further 4/10 (in total 7/10) treated and 4/5 control animals exhibited only scabs. No edema was observed (see Table 1).

48 and 72 hours after the challenge, no sensitisation was observed in the treated animals.

There was no mortality, no signs of toxicity and no treatment-related effects on body weight.

Table 1: skin irritation effects of intradermal and epidermal induction

 

Group/

animal No.

Intradermal induction (Day 3), undiluted test substance

Epidermal induction (Day 10), undiluted test substance

Control

A

B

C

Erythema

Edema

16

E2

NA

E3

0p

0

17

E2

NA

N2

0a

0

18

E3

NA

N3

0a

0

19

E3

NA

N3

0

0

20

E2

NA

N3

0a

0

Experimental

 

 

 

 

 

21

E4

NA

E2

0a

0

22

E1

NA

E1

0

0

23

E2

E2

E2

0a

0

24

E2

NA

E1

0a

0

25

E1

NA

E1

4k

0

26

E1

NA

E1

0

0

27

E3

E1

E2

0a

0

28

E2

E1

E2

4s

0

29

E2

NA

E2

4k

0

30

E3

NA

E2

0

0

A. 1:1 mixture of FCA and water for injection

B. undiluted test substance (experimental group) or vehicle (control group)

C. 1:1 mixture of FCA and undiluted test substance (experimental group) or vehicle (control group)

a. small scabs

k. scabs

p. scaliness

s. eschar formation

 

Skin effect intradermal injections:

NA. No abnormalities

E. erythema

N. signs of necrosis (mm in diameter)

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Justification for read-across

There are no data on the skin sensitisation potential of Fatty acids, C16 and C18-22 - unsatd., C16-18 and C18 unsatd. alkyl ester (CAS 90990-29-7). The assessment was therefore based on studies conducted with analogue (source) substances as part of a read across approach, which is in accordance with Regulation (EC) No. 1907/2006, Annex XI, 1.5. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13).

 

Skin sensitisation

In vivo

CAS 93803-87-3

A Guinea pig maximisation test was performed with 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) under GLP conditions and using a protocol similar to OECD guideline 406 (WoE, 1998). 10 test and 5 control Himalayan guinea pigs were induced intradermally with undiluted test substance on both sides of the spine with and without Freud's complete adjuvant. On Day 7, the animals were treated with 10% sodium dodecyl sulfate to induce mild skin irritation. On Day 8, a 48-hour epicutaneous induction treatment with the undiluted test substance was performed under semi-occlusive conditions. On Day 22, the challenge treatment was performed by topical application of the test substance at 100% (right flank) and a blank patch (left flank) to all animals for 24 hours, under semi-occlusive conditions. Skin reactions were evaluated 24 and 48 hours after the challenge application. During the study, no test substance-related clinical signs and no effects on body weight gain were observed. 8 hours after intradermal induction, slight to severe erythema was noted at all of the sites injected with FCA/water and FCA/test substance in 10/10 treated and 5/5 control animals. 4/5 control animals also exhibited necrosis at the FCA/test substance injection site. In 3/10 treated animals slight to well-defined erythema was observed at the injection site of the test substance. Following the topical induction, severe erythema and scabs were observed at the test site in 3/10 treated animals. A further 4/10 (in total 7/10) treated and 4/5 control animals exhibited only scabs. No skin reactions were observed after the challenge treatment in any of the animals of the test and control groups. The results of the reliability check carried out at regular intervals were positive, confirming the reliability of the assay. Based on the results, the test substance showed no skin sensitising effect in guinea pigs.

CAS 17673-56-2

The skin sensitising potential of (Z)-octadec-9-enyl (Z)-docos-13-enoate (CAS 17673-56-2) was evaluated in a Buehler test performed according to a protocol similar to OECD 406 and under GLP conditions (WoE, 1995). The induction treatments were performed on 20 Dunkin Hartley guinea pigs on Day 0, 7 and 14. A 70% solution of the test substance in peanut oil was applied to the shaved skin on the flank of the animals, and covered with an occlusive dressing for 6 hours. On Day 28, all the animals were challenged with a 60% solution of the test substance via topical application on the flank for 6 hours, using an occlusive dressing. 10 guinea pigs in the control group were treated according to the same protocol with the vehicle as the control substance. During the first reading, 1/20 treated animals and 1/10 negative control animals had a positive reaction, respectively. In the second reading, 2/20 treated animals and 1/10 negative control animals had a positive reaction, respectively. The slight, patchy erythema was limited to the left flank, where both the induction and challenge doses were applied. It is possible that the reaction was skin irritation, rather than a sensitisation reaction. All skin irritation effects had cleared within 72 hours after the challenge exposure ended. The result of the reliability check was inconclusive. During the first challenge application with 25% alpha-hexyl cinnamic aldehyde, a sensitisation reaction was induced in 25% (5/20) of the Dunkin Hartley guinea pigs, while the second challenge application did not cause sensitisation reactions. In the negative control group, the first challenge application induced sensitisation in 20% (2/10) of the animals, and the second challenge application did not cause sensitisation reactions. The results of the study are still considered to be valid as the first results could not be reproduced. Under the conditions of this study, the test substance is considered to be not skin sensitising.

Conclusion

A weight-of-evidence approach was applied to assess the skin sensitising potential of the target substance fatty acids, C16 and C18-22 - unsatd., C16-18 and C18 unsatd. alkyl ester (CAS 90990-29-7). In vivo studies (GPMT and Buehler test) performed with 2 source substances both gave negative results. Therefore, Fatty acids, C16 and C18-22 - unsatd., C16-18 and C18 unsatd. alkyl ester (CAS 90990-29-7) is not expected to be skin sensitising. 

Justification for classification or non-classification

According to Article 13 of Regulation (EC) No. 1907/2006 "General Requirements for Generation of Information on Intrinsic Properties of substances", information on intrinsic properties of substances may be generated by means other than tests e.g. from information from structurally related substances (grouping or read-across), provided that conditions set out in Annex XI are met. Annex XI, "General rules for adaptation of this standard testing regime set out in Annexes VII to X” states that “substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or ‘category’ of substances. This avoids the need to test every substance for every endpoint". Since the analogue concept is applied to Fatty acids, C16 and C18-22 - unsatd., C16-18 and C18 unsatd. alkyl ester (CAS 90990-29-7), data will be generated from data for reference source substance(s) to avoid unnecessary animal testing. Additionally, once the analogue read-across concept is applied, substances will be classified and labelled on this basis.

Therefore, based on the analogue read-across approach, the available data on skin sensitisation do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.