Fatty acids, C16-18 (even numbered), reaction products with tetraethylenepentamine, acetates (salts)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03-Jan-2013 - 22-Feb-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

30 May 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

17th July 1992

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A valid GPMT conducted according to guideline is available, which is reliable without restrictions and adequate for classification and labelling purposes. Potency estimation is not mandatory when existing guideline and GLP conforming data are available, which were conducted before the new annex of the REACH Regulation entered into force.

Test material

Test material information

Constituent 1

Reference substance name:

Fatty acids, C16-18 (even numbered), reaction products with tetraethylenepentamine, acetates (salts)

EC Number:

939-688-0

Molecular formula:

Not applicable

IUPAC Name:

Fatty acids, C16-18 (even numbered), reaction products with tetraethylenepentamine, acetates (salts)

Test material form:

other: solid

Details on test material:

- Name of test material: Fatty acids, C16-18, reaction products with tetraethylenepentamine, acetates (salts)

Specific details on test material used for the study:

- Name of test material: Fatty acids, C16-18, reaction products with tetraethylenepentamine, acetates (salts)

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., Kreuzelweg 53, 5961 NM Horst / The Netherlands
- Females nulliparous and non-pregnant: yes
- Body Weight (at Delivery / Acclimatization Start): Intradermal and epidermal pretests: 342.0 - 404.0 g, Control group and test group: 361.4 - 400.7 g
- Housing: In groups of up to ten in stainless steel cages with standard softwood bedding
- Diet (e.g. ad libitum): Teklad Global Guinea pig diet 2040C (batch nos. 26/12 and 68/12, provided by Provimi Kliba AG, 4303 Kaiseraugst / Switzerland), ad libitum
- Water (e.g. ad libitum): Community tap-water from Itingen ad libitum
- Acclimation period: Twenty-five days under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: purified water

Induction 2

Concentration / amount:

First Intradermal pretest: 10%, 5% and 2%
Second Intradermal pretest: 1%, 0.5% and 0.1%
Epidermal pretest: 25%, 10%, 5% and 2%

Intradermal induction: 0.1%
Dermal induction: 25% (mildly to moderately irritating)
Challenge: 10% (not irritating)

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: purified water

Challenge 2

Concentration / amount:

First Intradermal pretest: 10%, 5% and 2%
Second Intradermal pretest: 1%, 0.5% and 0.1%
Epidermal pretest: 25%, 10%, 5% and 2%

Intradermal induction: 0.1%
Dermal induction: 25% (mildly to moderately irritating)
Challenge: 10% (not irritating)

No. of animals per dose:

- Intradermal pretests: 2 animals
- Epidermal pretest: 2 animals
- Main test: 10 (test group), 5 (control group)

Details on study design:

RANGE FINDING TESTS:
The intradermal pretests were performed during the acclimatization of the main test animals. The test item concentrations used were selected during the preliminary solubility testing and were A = 10%, B = 5% and C = 2% in purified water for the first pretest, and H = 1%, I = 0.5% and J = 0.1% in purified water for the second pretest.
Five days later, four patches of filter paper (3 x 3 cm) were saturated with the test item formulations of D = 25%, E = 10%, F = 5% and G = 2% in purified water. The test item was formulated in the vehicle. The filter paper patches were applied to the clipped and shaved flanks of the same guinea pigs.

intradermal: A concentration of 0.1% in purified water well-tolerated systemically and caused mild to moderate skin reactions.
epidermal: A concentration of 25% in purified water was well-tolerated systemically and caused mild-to-moderate skin irritation.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal + epidermal)
- Exposure period:
- Test groups:
intradermal:
1. 1:1 (v/v) mixture of FCA/0.9% NaCl.
2. The test item at 0.1% in the vehicle.
3. The test item at 0.1% formulated in a 1:1 (v/v) mixture of FCA/0.9% NaCl.
epidermal: 2 x 4 cm patch of filter paper was saturated with the test item, 25% in purified water
- Control group:
intradermal:
1. 1:1 (v/v) mixture of FCA/0.9% NaCl.
2. Vehicle.
3. 1:1 (w/w) mixture of the vehicle in a 1:1 (v/v) mixture of FCA/0.9% NaCl.
epidermal: vehicle only
- Site: scapular area
- Duration: day 1: intradermal indiction, day 8: epidermal induction (48 h under occlusive dressing)
- Concentrations: intradermal: 0.1% in purified water ; epidermal: 25% in purified water

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 22
- Exposure period: 24
- Test groups: 10% test item in purified water
- Control group: 10% test item in purified water
- Site: right flank
- Evaluation (hr after challenge): 24 + 48 h

Challenge controls:

yes

Positive control substance(s):

yes

Remarks:

alpha-Hexylcinnamal, the positive control subsatcne is regularly tested in the laboratory

Results and discussion

Positive control results:

Discrete or patchy erythema was observed in 6 of 10 test group animals 24 hours after treatment with 10% test item in PEG 300. Discrete or patchy erythema was still observed in 4 test group animals 48 hours after treatment with 10% test item in PEG 300. No skin reactions were observed in the control group after treatment with the vehicle.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

3%

No. with + reactions:

6

Total no. in group:

10

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

purified water (left flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no findings

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: purified water (left flank). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no findings.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

10% in purified water (right flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no findings

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 10% in purified water (right flank). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no findings.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

purified water (left flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no findings

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: purified water (left flank). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no findings.

Results 5

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

10% in purified water (right flank)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no findings

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10% in purified water (right flank). No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no findings.

Results 6

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

purified water (left flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no findings

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: purified water (left flank). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no findings.

Results 7

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10% in purified water (right flank)

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

moderate to marked skin reactions such as discrete or patchy to intense erythema, oedema, scaling and crusts

Results 8

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Purified water (left flank)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no findings

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Purified water (left flank). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no findings.

Results 9

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10% in purified water (right flank)

No. with + reactions:

10

Total no. in group:

10

Clinical observations:

moderate to marked skin reactions such as discrete or patchy to intense erythema, oedema, scaling and crusts

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10% in purified water (right flank). No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: moderate to marked skin reactions such as discrete or patchy to intense erythema, oedema, scaling and crusts.

Any other information on results incl. tables

Pretests

According to the findings observed, the concentration selected for the main study was 0.1%.

According to Magnusson - Kligman and the findings observed, a test item concentration of

25% (weight /weight) in purified water was selected for the epidermal induction and test item

concentrations of 10% (weight /weight) in purified water was selected for the challenge.

VIABILITY / MORTALITY

All animals survived the scheduled observation periods.

 

CLINICAL SIGNS

No clinical signs were recorded in any animal.

 

SKIN REACTIONS

- Skin Reactions in the Intradermal Induction: The expected and commonly observed findings after FCA injection such as erythema, oedema, necrotizing dermatitis, encrustation and exfoliation of encrustation were noted. No detailed description of the skin reactions is given in the report as these effects of FCA are well-known.

- Skin Reactions in the Epidermal Induction: patchy erythema was observed in 5 of 10 test group animals 24 hours after treatment with 25% test item in purified water. Discrete or patchy erythema was still observed in 2 test group animals 48 hours after treatment with 25% test item in purified water. No skin reactions were observed in the control group after treatment with the vehicle.

- Skin Reactions in the Challenge: No skin reactions were observed in the control group after challenge with 10% test item in purified water. All test group animals showed moderate to marked skin reactions such as discrete or patchy to intense erythema, oedema, scaling and crusts 24 hours and 48 hours after treatment with 10% test item in purified water.

 

BODY WEIGHTS

The body weight of the animals was within the range commonly recorded for animals of this strain and age.

 

NECROPSY

No unscheduled deaths occurred, hence no necropsy was performed.

 

		Skin reaction after epidermal induction		Skin reaction after Challenge (10% test item)		Skin reaction after Challenge (vehicle)
Animal number	Group	24 h	48 h	24 h	48 h	24 h	48 h
1	Control	0	0	0	0	0	0
2	Control	0	0	0	0	0	0
3	Control	0	0	0	0	0	0
4	Control	0	0	0	0	0	0
5	Control	0	0	0	0	0	0
6	Test group	0	0	1	2	0	0
7	Test group	1	0	1	1	0	0
8	Test group	1	0	2	2, edema	0	0
9	Test group	0	0	2	2, edema	0	0
10	Test group	1	1	1	2, edema	0	0
11	Test group	0	0	2	1, scale	0	0
12	Test group	0	0	1	3	0	0
13	Test group	1	0	1	1	0	0
14	Test group	0	0	2	3	0	0
15	Test group	1	1	2, crust	2, crust	0	0

 

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

In this study, C16-18FA-TEPA-compound (100% a.i.) was a dermal sensitiser with 100% of the test animals responding to the challenge.

Executive summary:

In a dermal sensitisation study according to OECD guideline 406 (17 July 1992) and EU Method B.6 (30 May 2008) with C16-18FA-TEPA-compound (100% a.i.) in purified water, 15 (10 test and 5 control) young adult female Dunkin-Hartley guinea pigs were tested using the Maximisation test method. The positive control substance was alpha-Hexylcinnamaldehyde with a sensitisation rate of 70%.

The intradermal induction of sensitization in the test group was performed in the nuchal region by injection of 0.1% of the test item in purified water and an emulsion of Freund's Complete Adjuvant and physiological saline (FCA/0.9% NaCl). One week after the intradermal induction, the epidermal induction of sensitization was performed by topical application of 25% of the test item in purified water for 48 hours under occlusion. The animals of the control group were intradermally induced with purified water and FCA/0.9% NaCl and epidermally induced with purified water under occlusion.
Two weeks after epidermal induction the test and control animals were challenged by epidermal application of 10% test item on the right flank and purified water was applied on the left flank. Skin reactions were evaluated at 24 and 48 hours after removal of the dressing.

Epidermal Induction:
Discrete or patchy erythema was observed in 5 of 10 test group animals 24 hours after treatment with 25% test item in purified water. Discrete or patchy erythema was still observed in 2 test group animals 48 hours after treatment with 25% test item in purified water.
No skin reactions were observed in the control group after treatment with the vehicle.

Challenge:
No skin reactions were observed in the control group after challenge with 10% test item in purified water.
All test group animals showed moderate to marked skin reactions such as discrete or patchy to intense erythema, oedema, scaling and crusts 24 hours and 48 hours after treatment with 10% test item in purified water.

In this study, C16-18FA-TEPA-compound (100% a.i.) is a dermal sensitiser.