Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Skin sensitisation (QSAR; GPMT, read across; LLNA, read across): not sensitising

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Remarks:
in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
Refer to analogue justification provided in IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
Key result
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:
other:
Remarks:
Source: CAS 93803-87-3
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Remarks:
Source: CAS 93803-87-3
Key result
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:
other:
Remarks:
Source: CAS 93803-87-3
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Remarks:
Source: CAS 93803-87-3
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Remarks:
Source: CAS 91031-48-0
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other:
Remarks:
Source: CAS 91031-48-0
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Remarks:
Source: CAS 91031-48-0
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other:
Remarks:
Source: CAS 91031-48-0
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
Control
Remarks on result:
other:
Remarks:
Source: CAS 3687-46-5
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
25%
Remarks on result:
other:
Remarks:
Source: CAS 3687-46-5
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
50%
Remarks on result:
other:
Remarks:
Source: CAS 3687-46-5
Key result
Parameter:
SI
Value:
2.1
Test group / Remarks:
100%
Remarks on result:
other:
Remarks:
Source: CAS 3687-46-5
Cellular proliferation data / Observations:
The mean DPM values for the control, 25, 50 and 100% groups were 488, 571, 951 and 1013, respectively. The slight increase in DPM with increasing dose level was not statistically significant. (Source: CAS 3687-46-5)
Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
Conclusions:
The available data on suitable source substances did not show any skin sensitising effects. Therefore, the target substance Fatty acids, C16-18, isononyl esters is not predicted to be a skin sensitiser.
Endpoint:
skin sensitisation, other
Remarks:
QSAR
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE
The OECD QSAR Toolbox v4.1 is a Quantitative Structure-Activity Relationship model that was developed by the Laboratory of Mathematical Chemistry (http://toolbox.oasis-lmc.org).

2. MODEL (incl. version number)
OECD QSAR Toolbox version 4.1

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
The SMILES codes for the two components of the test substance were used.

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
- Predicted endpoint (OECD Principle 1 - Defined endpoint): Human Health Hazards -> Sensitisation ->Skin -> in Vivo -> GPMT LLNA Miscellaneous -> EC3 S M W N S W A N
- Calculation approach (OECD principle 2 - Unambiguous algorithm): takes the highest mode value from the nearest 5 neighbours
- Active descriptor: log Kow (calculated)
- Data usage: All values (When multiple values are available for the same chemical, all of them are taken individually in prediction calculations)
- Uncertainty of the prediction (OECD principle 4 - Uncertainty of the prediction): The prediction is based on 6 values, 4 of them (66,7%) equal to predicted value Prediction confidence is measured by the p-value: 0,1
- log Kow (calculated): 11.1 and 12.1

5. APPLICABILITY DOMAIN
- Database(s) used: Skin Sensitization
- Category boundaries (applicability domain): Active descriptor(s) range: log Kow: from 10 to 12.7 target chemical is in domain, Response range: Sensitisation: from Negative to Positive
- Profilers: Protein binding alerts for skin sensitization by OASIS (primary grouping): target chemical is in domain, no alert found
- Additional data pruning: Data inconsistency filter 12 value(s) from 9 chemical(s)

6. 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.
Principles of method if other than guideline:
- Principle of test: The OECD QSAR Toolbox v4.1 is a Quantitative Structure-Activity Relationship model that was developed by the Laboratory of Mathematical Chemistry (http://toolbox.oasis-lmc.org).
GLP compliance:
no
Key result
Parameter:
other: QSAR
Remarks on result:
no indication of skin sensitisation

The predicted skin sensitisation potential of Fatty acids, C16-18, isononyl esters was modelled in the OECD QSAR Toolbox v4.1. The test substance falls within the model applicability domain for the database 'Skin sensitisation (Danish EPA DB)'. The result of the prediction was negative.

Therefore, Fatty acids, C16-18, isononyl esters is not expected to have a skin sensitising potential.

Interpretation of results:
other: The prediction results was negative for skin sensitising potential, based on QSAR prediction (OECD QSAR Toolbox v4.1, Danish EPA database). The results may only be used for classification purposes together with other data in a weight-of-evidence approach.
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
26 May - 23 Jun 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2002
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: 22-25 g (range)
- Housing: animals were housed individually in labelled Makrolon cages (MI type, height 12.5 cm) containing sterilised sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and paper as cage-enrichment (Enviro-dri, Wm. Lillico & Son (Wonham Mill Ltd.), Surrey, UK). The paper was removed on Day 1 prior to dosing and was supplied again after scoring of the ears on the third day 3. During the acclimation period the animals were housed in groups in Macrolon cages (MIII type, height 18 cm).
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.5-23.1
- Humidity (%): 41-68
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 26 May 2010 To: 23 Jun 2010
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50 and 100% (w/w)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Irritation: A preliminary irritation study was performed according to the procedure of the main study. Two mice were treated daily for 3 consecutive days with either a 50% solution or 100% test substance. 3-4 hours after the last exposure, the irritation severity on the treated skin site was assessed. The body weight was recorded on Day 1 and 3. Very slight erythema (grade 1 of 4) was observed on both the treated sites in the animal exposed to 100% concentration. None of the animals exhibited signs of toxicity during the study period and the body weight was not affected by the treatment. Based on these results, the highest test substance concentration selected for the main study was 100%.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by beta-scintillation counting
- Criteria used to consider a positive response: DMP values will be measured for each animal and for each dose group. The stimulation Index (SI) will be calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥3, the test substance may be regarded as a skin sensitiser, based on the test guidelines and the recommendations done by ICCVAM.

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
During the induction phase (Day 1-3), the dorsal surface of both ears was epidermally treated (25µL/ear) with the vehicle control or 25, 50 and 100% test substance, at approximately the same time each day for 3 consecutive days. On Day 6, all the animals were injected via the tail vein with 0.25 mL sterile phosphate buffered saline (PBS), containing 20 µCi 3H-methyl thymidine. After approximately 5 hours, all the animals were sacrificed by intraperitoneal injection of Euthasol 20% and the draining (auricular) lymph node of both ears was excised. The relative size of the nodes (as compared to normal) was estimated by visual examination, and abnormalities of the nodes and surrounding area were recorded. The nodes were pooled for each animal in approximately 3 mL PBS. A single cell suspension of lymph node cells (LNC) was prepared the same day in PBS by gentle separation through a stainless steel gauze (diameter 125 µm).
A single cell suspension of LNCs was prepared in PBS by gentle separation through stainless steel gauze (diameter 125 µm). To precipitate the DNA, the LNCs were exposed to 5% trichloroacetic acid (TCA) and stored in the refrigerator until the next day.
Radioactivity measurements were performed on Day 7, using Ultima Gold cocktail as the scintillation fluid. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).
Positive control substance(s):
other: A reliability check was performed with alpha-hexylcinnamaldehyde every 6 months to demonstrate that the LLNA test system is reliable and sufficiently sensitive
Positive control results:
A reliability check with a positive control substance was performed every 6 months to demonstrate that the LLNA test system, as used by the test laboratory, is reliable and sufficiently sensitive. In the test performed in April 2010 (project No. 494039), 5, 10 and 25% alpha-hexylcinnamaldehyde, technical grade in acetone/olive oil (4:1 v/v) was used as the positive control. The SI values calculated for the substance concentrations 5, 10 and 25% were 1.7, 2.7 and 8.8 respectively. The SI-value for the vehicle control was 1.0. An EC3 value of 10.7% was calculated using linear interpolation. The calculated EC3 value was found to be in the acceptable range of 2 and 20%. The results of the 6 -monthly HCA reliability checks in CBA/J female mice of the recent years were 14.1, 13.8, 13.9, 16.0, 11.9 and 16.9%. Based on the results, it was concluded that the Local Lymph Node Assay in the mouse as supplied by Janvier performed at NOTOX is an appropriate model for testing for contact hypersensitivity.
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
control
Key result
Parameter:
SI
Value:
1.2
Test group / Remarks:
25%
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
50%
Key result
Parameter:
SI
Value:
2.1
Test group / Remarks:
100%
Cellular proliferation data / Observations:
The mean DPM values for the control, 25, 50 and 100% groups were 488, 571, 951 and 1013, respectively (see Table 1). The slight increase in DPM with increasing dose level was not statistically significant.

Effects on the lymph nodes:

The right auricular node was enlarged in 1/5 mice in the highest dose group. As this was a single case, it is not considered to be a sensitivity reaction. No macroscopic abnormalitites were observed in the surrounding area.

Table 1: Individual values for radioactivity measurements (disintegrations per minute) and mean stimulation indices

Group

Animal No.

Concentration (% w/w)

DPM/animal

Stimulation index (mean± SEM)

1

1

0

480

-

 

2

0

629

-

 

3

0

367

-

 

4

0

447

-

 

5

0

515

-

Mean ± SEM

 

 

488 ± 43

1.0 ± 0.1

 

 

 

 

 

2

6

25

514

-

 

7

25

516

-

 

8

25

519

-

 

9

25

817

-

 

10

25

491

-

Mean ± SEM

 

 

571 ± 62

1.2 ± 0.2

 

 

 

 

 

3

11

50

928

-

 

12

50

778

-

 

13

50

589

-

 

14

50

1084

-

 

15

50

1376

-

Mean ± SEM

 

 

951 ± 134

1.0 ± 0.3

 

 

 

 

 

4

16

100

637

-

 

17

100

796

-

 

18

100

1137

-

 

19

100

1013

-

 

20

100

1483

-

Mean ± SEM

 

 

1013 ± 146

1.1 ± 0.1

DPM = disintegrations per minute

SEM = standard error of the mean

Skin irritation effects:

Slight erythema was observed at the test site on both ears in 5/5 mice exposed to 100% test substance. This was not considered to have affected the activity of the lymph nodes.

Systemic effects:

There was no mortality. No clinical signs were observed during the study period and there were no effects on body weight.

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:
6 Aug - 30 Aug 1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Analytical purity of test substance not specified, no positive control group
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
adopted in 1981
Deviations:
yes
Remarks:
analytical purity of test substance not specified, no positive control group
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
A non-LLNA test is available that was performed prior to the current data requirements, stipulated in Regulation (EC) No 1907/2006. In accordance with the same Regulation, the data was included to avoid unnecessary testing.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Lebeau breeding centre, Gambais, France
- Age at study initiation: approx. 5 weeks
- Weight at study initiation: male mean 436 g, female mean 423 g
- Housing: individual housing in polycarbonate cages
- Diet: guinea pigs sustenance ref. 106 (U.A.R., Villemoisson-sur-Orge, France), ad libitum
- Water: tap water, ad libitum
- Acclimation period: for a minimal period of 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Photoperiod (hrs dark / hrs light): 12 / 12
Route:
intradermal
Vehicle:
paraffin oil
Concentration / amount:
25%
Day(s)/duration:
day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%


Day(s)/duration:
day 8
Adequacy of induction:
highest technically applicable concentration used
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
50%
Day(s)/duration:
day 22
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Treatment group: 20 (10 males and 10 females)
Control group: 10 (5 males and 5 females)
Details on study design:
RANGE FINDING TESTS:
Based on the results of a preliminary study a 25% dilution of the test substance in paraffin oil was used for intradermal induction and the undiluted (100%) substance was used for the epidermal induction exposure. The undiluted test substance was found to be slightly irritating upon dermal application.
A 50% test substance concentration was selected for the challenge phase as the maximum non-irritant dose upon dermal exposure for 24 h under occlusive dressing.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: Intradermal induction on day 1. On day 7 a local irritation was induced using 0.5 mL of a 10 % sodium laurylsulphate in vaseline. On day 8 a epidermal induction was performed with 0.5 mL vehicle or 0.5 mL test substance. The dermal applications were held in place for 48 hours under occlusive dressing.
- Test groups: 20 animals treated with test substance
- Control group: 10 animals treated with vehicle only
- Site: the scapular region of both sides
- Concentrations: 0.1 mL of 25% dilution of the test substance in paraffin oil in the presence of Freund' adjuvant was used for intradermal induction and 0.5 mL of the undiluted (100%) test substance was used for epidermal induction.
- Duration: 10 days

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 14 days after the last induction treatment
- Exposure period: 24 h under occlusive dressing
- Test groups: 20 animals treated with test substance
- Control group: 10 animals treated analogous to the test groups
- Site: the right flank was treated with the test substance; the left flank was treated with vehicle.
- Concentrations: 0.5 mL of a 50% solution in paraffin oil
- Evaluation (hr after challenge): 24 and 48 h after removal of the dressing
Positive control substance(s):
no
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Key result
Reading:
other:
Group:
positive control
Remarks on result:
not measured/tested

Table 1: Challenge readings

Group

Sex

Animals

24 h scoring period

48 h scoring period

Erythema

Edema

Erythema

Edema

LF

RF

LF

RF

LF

RF

LF

RF

Control

Males

1

0

0

0

0

0

0

0

0

2

0

0

0

0

0

0

0

0

3

0

0

0

0

0

0

0

0

4

0

0

0

0

0

0

0

0

5

0

0

0

0

0

0

0

0

Females

16

0

0

0

0

0

0

0

0

17

0

0

0

0

0

0

0

0

18

0

0

0

0

0

0

0

0

19

0

0

0

0

0

0

0

0

20

0

0

0

0

0

0

0

0

Treated

Males

6

0

0

0

0

0

0

0

0

7

0

0

0

0

0

0

0

0

8

0

0

0

0

0

0

0

0

9

0

0

0

0

0

0

0

0

10

0

0

0

0

0

0

0

0

11

0

0

0

0

0

0

0

0

12

0

0

0

0

0

0

0

0

13

0

0

0

0

0

0

0

0

14

0

0

0

0

0

0

0

0

15

0

0

0

0

0

0

0

0

Females

21

0

0

0

0

0

0

0

0

22

0

0

0

0

0

0

0

0

23

0

0

0

0

0

0

0

0

24

0

0

0

0

0

0

0

0

25

0

0

0

0

0

0

0

0

26

0

0

0

0

0

0

0

0

27

0

0

0

0

0

0

0

0

28

0

0

0

0

0

0

0

0

29

0

0

0

0

0

0

0

0

30

0

0

0

0

0

0

0

0

No deaths occured. No significant differences in the gain of body weight was observed between treatment and control group.

Interpretation of results:
other: CLP/EU GHS criteria not met, no classification required according to Regulation (EC) No 1272/2008.
Executive summary:

A Guinea pig maximization test was performed according to OECD Guideline 406. 20 test and 10 control animals (Dunkin-Hartley guinea pigs) were induced intradermally with 25% substance solution and epicutaenously with 100% liquid test substance. Epicutaneous challenge was with a 50% test substance solution in paraffin oil. 24 and 48 hours after termination of challenge exposure skin readings revealed no indications for a skin sensitising potential of Fatty acids, C16-18, 2-ethylhexyl esters.

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
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
epicutaneous induction and challenge performed under semi-occlusive conditions
Qualifier:
according to guideline
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:
There is data available for an in vivo Guinea Pig Maximisation Test that was performed prior to the amendment to Regulation (EC) No 1097/2006 stating the LLNA is 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
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
Day(s)/duration:
Day 1
Adequacy of induction:
highest technically applicable concentration used
Route:
epicutaneous, semiocclusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
Day(s)/duration:
Day 8
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100%
Day(s)/duration:
Day 21
Adequacy of challenge:
highest non-irritant concentration
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 was 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 chemical
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
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 chemical
Dose level:
undiluted
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
other: study performed in 1998
Group:
positive control
Dose level:
10%
No. with + reactions:
8
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
alpha-hexylcinnamic aldehyde

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)
Additional information:

QSAR

CAS 91031-57-1

The skin sensitising potential of Fatty acids, C16-18, isononyl esters (CAS 91031-57-1) was predicted in the QSAR OECD Toolbox v4.1 (WoE, 2017). The test substance falls within the model applicability domain for the database 'Skin sensitisation (Danish EPA DB)'. The result of the prediction was negative, no skin sensitising potential was predicted.

Animal Data

CAS 93803-87-3

A Guinea pig maximisation test (GPMT) was performed with 2-octyldodecyl isooctadecanoate (CAS 93803-87-3) according to OECD guideline 406 and under GLP conditions (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-h 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 h, under semi-occlusive conditions. Skin reactions were evaluated 24 and 48 h after the challenge application. During the study, no test substance-related clinical signs and no effects on body weight gain were observed. 8 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 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 had no sensitising effect in guinea pigs under the experimental conditions.

CAS 3687-46-5

An in vivo skin sensitisation study (Local Lymph Node Assay, LLNA) was performed with Decyl oleate (CAS 3687-46-5) according to OECD guideline 429 and under GLP conditions (WoE, 2010). Five female CBA/J mice per dose were treated with 0, 25, 50 and 100% of the test substance in acetone/olive oil. The test substance formulations or the vehicle were applied epicutaneously onto the dorsal part of each ear (25 µL/ear) for three consecutive days. All mice were sacrificed three days after the last treatment (on Day 6) and the weight of the lymph nodes was determined. The cell proliferation of pooled lymph nodes from individual animals was measured as 3H-methyl thymidine incorporation and determined by beta-scintillation counting. The stimulation indices were 1.0, 1.2, 2.0 and 2.1 for the control, 25, 50 and 100% groups respectively. The SI slightly increased with the dose level, but not statistically significant. Positive and vehicle controls were valid. An EC3 value of the test substance could not be calculated, as all stimulation indices were <3. Based on the study results, the test substance was not skin sensitising.

CAS 91031-48-0

A Guinea pig maximisation test was performed with Fatty acids, C16-18, 2-ethylhexyl esters (CAS 91031-48-0) according to OECD guideline 406 and under GLP conditions (WoE, 1991). On the first day 20 test and 10 control Dunkin-Hartley guinea pigs were induced intradermally with 25% test substance diluted in paraffin oil on both sides of the scapular region with and without Freud's complete adjuvant. On Day 7, the animals were treated with 10% sodium dodecyl sulphate to induce mild skin irritation. On Day 8, a 48-h epicutaneous induction treatment with the undiluted test substance was performed under occlusive conditions. On Day 22, the challenge treatment was performed by topical application of the test substance at 50% in paraffin oil (right flank) and a blank patch (left flank) to all animals for 24 h, under occlusive conditions. Skin reactions were evaluated 24 and 48 h after removal of the dressing. During the study, no test substance-related clinical signs and no effects on body weight gain were observed. No skin reactions were observed after the challenge treatment in any of the animals of the test and control groups. The test substance had no sensitising effect in guinea pigs in this study.

Overall conclusion for skin sensitisation

No sensitising potential was seen in experimental studies in mice (LLNA) and guinea pigs (GPMT) performed with source substances. Based on the available information, Fatty acids, C16-18, isononyl esters (CAS 91031-57-1) is not expected to be skin sensitising.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

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, C16-18, isononyl esters (CAS 91031-57-1), 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.