Registration Dossier

Administrative data

Description of key information

Based on the available weight of evidence information from studies for substances representative of the main constituents, the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate' is considered to be non-sensitiser to skin.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
From May 25, 2005 to June 13, 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
Source: Jackson Laboratories
Acclimation: 5 days
Number of animals: 37 females (nulliparous and non-pregnant)
Body weight: 16 - 21g
Body weight variation was within +/- 20% of the sex mean.
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle.
Diet: Fresh PMI (Diet #5001)
Water: free access to tap water.
Vehicle:
other: Ultrapure liquid petrolatum
Concentration:
0, 2.5, 5, 10 and 25% w/w
No. of animals per dose:
5
Details on study design:
The test substance concentrations selected for the main study were based on the results of a pre-screen test. In the main study, four experimental groups of five female CBA/J mice were treated with test substance concentrations of 2.5, 5, 10 or 25% w/w for three consecutive days, by topical application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Liquid petrolatum). A positive control group with a-hexylcinnamaldehyde (HCA - 50%) was also included in the experiment. Five days after the last exposure, all animals were injected with 5-bromo-2'-deoxy-uridine (BrdU) and the draining (auricular) lymph nodes were then isolated and pooled for each animal. Cells were fixed using 70% ethanol and used for the measurement of the cell number and the BrdU determination (percentage of proliferating cells in "S" phase). Flow cytometry was conducted for analysis and stimulation index (SI) was recorded
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
SI
Positive control results:
The SI value calculated for the positive control was 9.6 and ear swelling was observed in this group.
Key result
Parameter:
SI
Value:
ca. 0.9
Variability:
+/- 0.7
Test group / Remarks:
2.5%
Key result
Parameter:
SI
Value:
ca. 0.9
Variability:
+/- 0.6
Test group / Remarks:
5%
Key result
Parameter:
SI
Value:
ca. 0.7
Variability:
+/- 0.3
Test group / Remarks:
10%
Key result
Parameter:
SI
Value:
ca. 0.3
Variability:
+/- 0.1
Test group / Remarks:
25%
Cellular proliferation data / Observations:
- SI values were similar among the control and test groups and were below 3. Three concentrations (i.e.2.5, 5 and 25%) induced ear swelling.
- No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.
Interpretation of results:
other: not classified based on EU CLP criteria
Conclusions:
Based on the results of the read across study, the test substance, is considered to be non-sensitiser to the skin.
Executive summary:

A study was conducted to determine the skin sensitisation potential of the read across substance, 'mono- and di- C16 PSE and H3PO4' (98.5%) according to OECD Guideline 429 and US EPA OPPTS 870.2600 (LLNA), in compliance with GLP. The read across substance concentrations selected for the main study were based on the results of a pre-screen test. In the main study, four experimental groups of five female CBA/J mice were treated with read across substance concentrations of 2.5, 5, 10 or 25% w/w for three consecutive days, by topical application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Liquid petrolatum). A positive control group with a-hexylcinnamaldehyde (HCA - 50%) was also included in the experiment. Five days after the last exposure, all animals were injected with 5-bromo-2'-deoxy-uridine (BrdU) and the draining (auricular) lymph nodes were then isolated and pooled for each animal. Cells were fixed using 70% ethanol and used for the measurement of the cell number and the BrdU determination (percentage of proliferating cells in "S" phase). Flow cytometry was conducted for analysis and stimulation index (SI) was recorded. Mortality/viability, body weights, clinical signs, ear size and irritation (and other local effects) were recorded as well. SI values were similar among the control and test groups and were below 3. Three concentrations (i.e.2.5, 5 and 25%) induced ear swelling. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period (MBRL, 2005). Based on the results of the read across study, the test substance, 'mono- and di- C16 PSE, K+ and C16 -OH and isostearyl isostearate' is considered to be non-sensitising to the skin.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
The test animals are initially exposed to the test substance by intradermal injection and/or epidermal application (induction exposure). Following a rest period of 10 to 14 d (induction period), during which an immune response may develop, the animals are exposed to a challenge dose. The extent and degree of skin reaction to the challenge exposure in the test animals is compared with that demonstrated by control animals which undergo sham treatment during induction and receive the challenge exposure.
GLP compliance:
not specified
Type of study:
other: modified Draize test
Justification for non-LLNA method:
Non LLNA justification
The in vivo study data were obtained in studies performed before any in vitro sensitization tests tests had been validated and accepted for regulatory purposes. Additionally, literature data demonstrates that an LLNA method is unreliable for surfactant substance, and may provide false positive results [1]. Therefore, an LLNA method is not deemed reliable for assessing the skin sensitisation of the substance.

[1]: Evaluating the sensitization potential of surfactants: Integrating data from the local lymph node assay, guinea pig maximization test, and in vitro methods in a weight-of-evidence approach. Ball et al. Regulatory Toxicology and Pharmacology 60 (2011) 389–400
Species:
guinea pig
Strain:
Hartley
Sex:
not specified
Details on test animals and environmental conditions:
Test animals:
- Source: not reported
- Weight at study initiation: ca 350 g
- Number of animals: 10
- Controls: only at rechallenge

Route:
intradermal
Vehicle:
not specified
Concentration / amount:
0.25%
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
other: Topical
Vehicle:
not specified
Concentration / amount:
10%
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
intradermal
Vehicle:
not specified
Concentration / amount:
0.1%
Day(s)/duration:
14 d after induction each animal received an intradermal injection in one flank and a topical application on the other
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
other: Topical
Vehicle:
not specified
Concentration / amount:
10%
Day(s)/duration:
14 d after induction each animal received an intradermal injection in one flank and a topical application on the other
Adequacy of challenge:
highest non-irritant concentration
Details on study design:
Administration / Exposure
- Study type: Non-adjuvant
- Preparation of test substance for induction: not reported, 0.1 mL of the test solution was administered.
- Induction schedule: 4 intradermal injections at one time point over the 2 auxillary and 2 inguinal lymph nodes.
- Concentrations used for induction: Based on a primary irritation screen the concentration used was 2.5 times the injection challenge concentration (the concentration giving slight barely perceptible irritation with no oedema).
- Challenge schedule: 14 days after induction each animal received an intradermal injection in one flank and a topical application on the other.
- Concentrations used for challenge: 0.1% intradermally ad 10% topically
- Rechallenge: Where materials test negative at challenge a repeat set of induction applications was carried out followed by challenge at 14 days and rechallenge (with controls) 7 days later.
- Positive control: not reported
- Pilot study: A preliminary irritation study was undertaken to determine the injection challenge concentration (the concentration giving slight barely perceptible irritation with no oedema) and the application challenge concentration (the highest concentration producing no irritation).

Examinations
- Grading system: A colour matching lighting unit was used to examine the skin reactions. Each injection reaction was scored based on size, erythema and oedema and considered positive if the total score was greater than the total average of the control scores. Application reactions were scored on a scale of 0 to +++ and considered positive if individual reactions were => + and there was no erythema in the controls.
Positive control substance(s):
not specified
Positive control results:
Results of the pilot study:
- 0.25%, 0.1% and 10% solutions were chosen for the intradermal induction, intradermal challenge and topical challenge respectively.

Results of test:
- Sensitization reaction: No sensitisation reactions at challenge or rechallenge following a second induction procedure. The result was reported as non-sensitising, individual animal data were not presented.
- Clinical signs: None
- Rechallenge: No sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.1% intradermal
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No sensitisation reactions
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
test group
Dose level:
10% topical
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No sensitisation reactions
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Group:
positive control
Remarks on result:
other: not reported
Key result
Reading:
1st reading
Group:
negative control
Remarks on result:
other: not reported
Interpretation of results:
other: not classified based on EU CLP criteria
Conclusions:
Under the study conditions, the test substance was determined to be non-sensitiser to the skin.
Executive summary:

A study was conducted to determine the skin sensitisation potential of the test substance, hexadecan-1-ol (purity:>95%), according to modified draize test in guinea pigs. Based on a primary irritation screening, 0.25%, 0.1% and 10% test substance solutions (the concentration giving slight barely perceptible irritation with no oedema) were chosen for the intradermal induction, intradermal challenge and topical challenge respectively. For induction phase, 0.1 mL of 0.25% intradermal and 10% topical application were performed. For challenge phase, 14 d after induction, each animal received  0.1% intradermal and 10% topical application of the test substance. Where materials test negative at challenge a repeat set of induction applications was carried out followed by challenge at 14 d and rechallenge (with controls) 7 d later. No positive control were reported. A colour matching lighting unit was used to examine the skin reactions. Each injection reaction was scored based on size, erythema and oedema and considered positive if the total score was greater than the total average of the control scores. Application reactions were scored on a scale of 0 to 3 and considered positive if individual reactions were ≥1 and there was no erythema in the controls. No sensitisation reactions at challenge or rechallenge following a second induction procedure were observed. There were no clinical signs were noted in all the animals. The study author had reported the test substance as non-sensitiser, whereas, the individual animal data were not presented. Under the study conditions, the test substance was determined to be non-sensitiser to the skin (OECD SIDS, 2006).

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
From March 24, 1998 to April 24, 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
KL2 due to RA
Justification for type of information:
Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.
Reason / purpose:
read-across source
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:
A valid GPMT study was available before REACH came into force, therefore no additional LLNA study was conducted.
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 d

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:
unchanged (no vehicle)
Concentration / amount:
Day 1:
Intradermal: undiluted and 50% in 1:1 mixture (w/w) with FCA with water for injection

Epicutaenous:
Day 8: undiluted test substance
Day(s)/duration:
Day 1 and Day 8
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
Intradermal:
Day 1 (3 pairs of injections, 0.1 mL/site): 1:1 mixture (w/w) FCA/water, corn oil, corn oil (w/v) in a 1:1 mixture (w/w) FCA (final concentration is 50% corn oil)

Epicutenous
Day 8: 0.5 mL corn oil
Day(s)/duration:
Day 1 and Day 8
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
Day 1:
Intradermal: 5% solution of positive control substance

Day 8:
Epicutaneous: undiluted positive control substance
Day(s)/duration:
Day 1 and Day 8
Route:
epicutaneous, semiocclusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
Test substance undiluted
Day(s)/duration:
Day 21
Adequacy of challenge:
highest non-irritant concentration
Route:
epicutaneous, semiocclusive
Vehicle:
corn oil
Concentration / amount:
Control substance: Corn oil
Day(s)/duration:
Day 21
Route:
epicutaneous, semiocclusive
Vehicle:
corn oil
Concentration / amount:
5 and 10% positive control substance solution
Day(s)/duration:
Day 21
No. of animals per dose:
10 (treatment group)
5 (control group)
Details on study design:
Range finding test:
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 h after treatment. Slight erythema was observed at the injection site of the undiluted test substance 48 h after exposure. The undiluted 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 h, the dressing was removed and the skin cleaned of residual test substance. The treated skin areas were assessed for irritation 24 and 48 h 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 h before the epidermal induction in the main study, to provoke a mild inflammatory reaction. The undiluted 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 h
- 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 (h after challenge): 24 and 48 h 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.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
undiluted
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:
negative control
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10% solution
No. with + reactions:
8
Total no. in group:
10
Clinical observations:
Sensitisation
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
24
Group:
positive control
Dose level:
5% solution
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
Sensitisation
Remarks on result:
positive indication of skin sensitisation

48 h 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 h 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: not classified based on EU CLP criteria
Conclusions:
Based on the results of the read across study, the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate' was determined to be non-sensitiser to the skin.
Executive summary:

A study was conducted to determine the skin sensitisation potential of the read across substance, octyldodecyl isooctadecanoate (purity: assumed to be 100%), using guinea pig maximisation test (GPMT), according to OECD Guideline 406 and EU Method B.6, in compliance with GLP. The test was performed in 15 (10 test and 5 control) female albino Himalayan guinea pigs. Based on the preliminary study, undiluted substance (100%) was considered as induction and challenge phase concentration. In the main test, one treated group of ten females received 3 pairs of intradermal injections (0.1 mL/site) [i.e., Injection 1: a 1:1 mixture (w/w) Freud’s 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)] on Day 1. 48 h after intradermal injection (i.e., on 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 (SDS) in Vaseline using a spatula. This concentration of SDS provoked a mild inflammatory reaction. On Day 8, 0.5 mL undiluted read across substance was applied to the SDS-treated skin area under semi-occlusive conditions for 48 h. The degree of erythema and edema was evaluated directly after cleaning the skin area with water (Day 10). Similar procedure was followed for control substance (corn oil). For challenge phase, on Day 21, 0.5 mL of undiluted read across substance or control substance was applied to approximately 20 mm x 30 mm, on one flank of the animals and evaluation was made 24 and 48 h after the challenge ended. The animals were observed for mortality twice daily and for signs of toxicity at least once daily. The body weight of the animals was determined on Day 1 (prior to exposure) and at the end of the study period (Day 24). A reliability check was carried out by the lab at regular intervals with alpha-hexylcinnamic aldehyde to check the sensitivity of the test system and the reliability of the experimental methods. 48 h after intradermal induction of the test substance, 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. 48 and 72 h 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. Under the study conditions, the read across substance was determined to be non-sensitiser to the skin (Notox, 1998). Based on the results of the read across study, similar non-sensitising behaviour can be expected for the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearly isostearate'.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Reason / purpose:
data waiving: supporting information
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

In absence of skin sensitisation study with the test substance, the endpoint has been assessed based on studies for substances representative of the main constituents, which can be categorised as phosphate esters (PSE i.e., mono- and di C16 PSE, K+), alcohol (i.e., hexadecanol) and isoalkylester (i.e., isostearyl isostearate). The results are presented below:

Constituent PSE - read across study:

A study was conducted to determine the skin sensitisation potential of the read across substance, 'mono- and di- C16 PSE and H3PO4' (98.5%) according to OECD Guideline 429 and US EPA OPPTS 870.2600 (LLNA), in compliance with GLP. The read across substance concentrations selected for the main study were based on the results of a pre-screen test. In the main study, four experimental groups of five female CBA/J mice were treated with read across substance concentrations of 2.5, 5, 10 or 25% w/w for three consecutive days, by topical application on the ears. Five vehicle control animals were similarly treated, but with the vehicle alone (Liquid petrolatum). A positive control group with a-hexylcinnamaldehyde (HCA - 50%) was also included in the experiment. Five days after the last exposure, all animals were injected with 5-bromo-2'-deoxy-uridine (BrdU) and the draining (auricular) lymph nodes were then isolated and pooled for each animal. Cells were fixed using 70% ethanol and used for the measurement of the cell number and the BrdU determination (percentage of proliferating cells in "S" phase). Flow cytometry was conducted for analysis and stimulation index (SI) was recorded. Mortality/viability, body weights, clinical signs, ear size and irritation (and other local effects) were recorded as well. SI values were similar among the control and test groups and were below 3. Three concentrations (i.e.2.5, 5 and 25%) induced ear swelling. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period (MBRL, 2005).

Constituent alcohol:

A study was conducted to determine the skin sensitisation potential of hexadecan-1-ol (purity:>95%), according to modified draize test in guinea pigs. Based on a primary irritation screening, 0.25%, 0.1% and 10% test substance solutions (the concentration giving slight barely perceptible irritation with no oedema) were chosen for the intradermal induction, intradermal challenge and topical challenge respectively. For induction phase, 0.1 mL of 0.25% intradermal and 10% topical application were performed. For challenge phase, 14 d after induction, each animal received  0.1% intradermal and 10% topical application of the test substance. Where materials test negative at challenge a repeat set of induction applications was carried out followed by challenge at 14 d and rechallenge (with controls) 7 d later. No positive control were reported. A colour matching lighting unit was used to examine the skin reactions. Each injection reaction was scored based on size, erythema and oedema and considered positive if the total score was greater than the total average of the control scores. Application reactions were scored on a scale of 0 to 3 and considered positive if individual reactions were ≥1 and there was no erythema in the controls. No sensitisation reactions at challenge or rechallenge following a second induction procedure were observed. There were no clinical signs were noted in all the animals. The study author had reported the test substance as non-sensitiser, whereas, the individual animal data were not presented. Under the study conditions, the test substance was determined to be non-sensitiser to the skin (OECD SIDS, 2006).

Constituent isoalkylester:

A study was conducted to determine the skin sensitisation potential of the read across substance, octyldodecyl isooctadecanoate (purity: assumed to be 100%), using guinea pig maximisation test (GPMT), according to OECD Guideline 406 and EU Method B.6, in compliance with GLP. The test was performed in 15 (10 test and 5 control) female albino Himalayan guinea pigs. Based on the preliminary study, undiluted substance (100%) was considered as induction and challenge phase concentration. In the main test, one treated group of ten females received 3 pairs of intradermal injections (0.1 mL/site) [i.e., Injection 1: a 1:1 mixture (w/w) Freud’s 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)] on Day 1. 48 h after intradermal injection (i.e., on 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 (SDS) in Vaseline using a spatula. This concentration of SDS provoked a mild inflammatory reaction. On Day 8, 0.5 mL undiluted read across substance was applied to the SDS-treated skin area under semi-occlusive conditions for 48 h. The degree of erythema and edema was evaluated directly after cleaning the skin area with water (Day 10). Similar procedure was followed for control substance (corn oil). For challenge phase, on Day 21, 0.5 mL of undiluted read across substance or control substance was applied to approximately 20 mm x 30 mm, on one flank of the animals and evaluation was made 24 and 48 h after the challenge ended. The animals were observed for mortality twice daily and for signs of toxicity at least once daily. The body weight of the animals was determined on Day 1 (prior to exposure) and at the end of the study period (Day 24).A reliability check was carried out by the lab at regular intervals with alpha-hexylcinnamic aldehyde to check the sensitivity of the test system and the reliability of the experimental methods.48 h after intradermal induction of the test substance, 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. 48 and 72 h 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. Under the study conditions, the read across substance was determined to be non-sensitiser to the skin (Notox, 1998).

Overall, based on the available weight of evidence information from studies for substances representative of the main constituents, the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate' is considered to be non-sensitising to skin.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available weight of evidence information from studies for substances representative of the main constituents, the test substance, 'mono- and di- C16 PSE, K+ and C16-OH and isostearyl isostearate' does not warrant a classification for skin sensitisation according to EU CLP criteria (Regulation 1272/2008/EC).