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Diss Factsheets

Administrative data

Description of key information

Guinea pig maximisation test: sensitising; OECD guideline 406; GLP; induction: 0.1% in purified water (intradermal) / 25% in purified water (epicutaneous; mildly to moderately irritating); challenge: 10% in purified water

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
30 May 2008
Deviations:
no
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.
Specific details on test material used for the study:
- Name of test material: Fatty acids, C16-18, reaction products with tetraethylenepentamine, acetates (salts)
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
Route:
intradermal and epicutaneous
Vehicle:
other: purified water
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)
Route:
epicutaneous, occlusive
Vehicle:
other: purified water
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
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.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
3%
No. with + reactions:
6
Total no. in group:
10
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.
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.
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.
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.
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.
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
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.
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.

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

 

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.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Skin sensitisation


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.


 


Justification for the Magnusson & Kligman method test according to OECD guideline 406


The test substance C1618FA-TEPA-compound is a surface active chemical. In recently published articles in peer reviewed journals (see reference list below, and publications cited in the revised OECD guideline 429 (July 2010)) it is clearly demonstrated that for the realistic assessment of the skin sensitisation potential of surfactants the LLNA (OECD 429) is much less suitable than the Magnusson & Kligman method (OECD 406) and could lead to confounding results.


In particular, the OECD 429 guideline specifies that “In addition, test substance classes or substances containing functional groups shown to act as potential confounders (Basketter et al., 2009) may necessitate the use of guinea pig tests”.


Consequently, in the evaluation of such surface active substances for sensitising properties, the LLNA test is not an appropriate assay and would not represent an optimum use of test animals. The mechanism underlying confounding or false positive result is not fully understood, but it may be that an unspecific, non-immunologically triggered mechanism may be the cause of an increased lymphocyte proliferation.


In contrast to the M&K, the LLNA focuses on the induction process of skin sensitisation (i.e. lymphocyte proliferation), and does not capture the process of elicitation which checks if the organism had actually been sensitised or not. The design of the LLNA study does not allow to assess whether lymphocyte proliferation is a specific response or unspecific (false positive) response.


 


Respiratory sensitisation


There is no information available for respiratory sensitisation. Therefore, there is a data gap in this respect. However, the data gap cannot be fulfilled with experimental data, since there is no internationally accepted animal model for respiratory sensitisation. In case human data for respiratory sensitisation emerges, this will be taken into account.


 


No human information is available for skin sensitisation. However, there is no reason to believe that these results would not be applicable to humans.


 


References


Ball, N et al., Regulatory Toxicology and Pharmacology 60 (2011): 389-400 Evaluating the sensitizing 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


 


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


 


Garcia, C et al., Regulatory Toxicology and Pharmacology 58 (2010): 301-307 Comparative testing for the identification of skin sensitizing potentials of nonionic sugar lipid surfactants


 


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




Justification for selection of skin sensitisation endpoint:
OECD & EC guideline study; no deviations; GLP

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on relevant, reliable and adequate data, C16-18FA-TEPA-compound is classified as skin sensitiser (Category 1) and labelled with H317 according to regulation (EC) 1272/2008.