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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 March 2010 - 16 April 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
other: OECD Guideline 406 (Skin Sensitisation) EU Method B.6 (Skin Sensitisation) EPA OPPTS 870.2600 (Skin Sensitisation) Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147, November 2000, including the most recent
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This is the scientific justification for performing the OECD Test No. 406 for Skin Sensitisation, i.e. the Guinea Pig Maximization Test (GPMT), instead of1he required OECD Test No. 429, i.e. Skin Senitisation according to the Local Lymph Node Assay.

In recently published articles in peer reviewed journals. see reference list, it is clearly demonstrated that surfactants are more likely to give rise to false positives in the LLNA. Consequently, in the evaluation of such substances for sensitizing properties the LLNA test is not an appropriate assay and would not represent an optimum use of test animals. It is therefore recommended that the Guinea Pig Maximization Test (GPMT) is used instead. This is also supported by the TG OECD 406 ”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".
References:
Kreiling. R .. Hollnagel, H..M ; Hareng. L.. Eigler D. .. Lee. M.S . Griem. P .. Dreesen B,
Klebcr. M ., Albrecht. A.. Garcia. C .• Wendel, A (2008) ,Comparison of the skin
sensitizing potential of unsaturated compounds and assessed by the murine
local lymph node assay (LLNA) and the guinca pig maximization test (GPMT).
Food Chem. Toxicol. 46. 1896 – 1904

D. Basketter. N. Ball. S. Cagen,. JC Carrillo. H. Certa. D. Eigler. H. Esch. C. Garcia, C. Graham. C.Haux. R.
Kreiling. A. Mehling. (2009) Application of a weight of evidence approach to assessing discordant sensitisation
datasets: Jmplications for REACH. Reg Tox Pharrn. 55:90-96.

C. Garcia. . N. Ball. S. Cagen,. JC Carrillo. H. Certa. D. Eigler. H. Esch C. Graham.. C.Haux, R. Kreiling. A.
Mehling. (2010) Comparative testing for the identification of skin sensitizing potentials of nonionic sugar lipid
surfactants. Reg Tox Pharrn 58: 301-307
Note: Ref list not complete due to lack of space.

Test material

Constituent 1
Chemical structure
Reference substance name:
Fatty acids, C18 unsat, reaction products with diethylenetriamine
EC Number:
629-715-1
Cas Number:
1226892-43-8
Molecular formula:
UVCB substance - not applicable
IUPAC Name:
Fatty acids, C18 unsat, reaction products with diethylenetriamine
Test material form:
liquid
Details on test material:
Name: Fatty acids, C18 unsat, reaction products with diethylenetriamine
CAS: 1226892-43-8 (former CAS number 68442-97-7)
- Name of test material (as cited in study report): Tall oil diethylenetriamine imidazoline
- Substance type: Clear slightly viscous amber liquid (determined at NOTOX)
- Physical state: Liquid
- Analytical purity: 98%
- Impurities (identity and concentrations): Free diethylenetriamine (3.6%) + Free fatty accid (2.0%)
- Lot/batch No.: S000922
- Expiration date of the lot/batch:02 July 2017
- Stability Tall oil diethylenetriamine imidazoline under storage conditions = Stable
- Storage condition of test material: At room temperature in the dark under nitrogen

- Specific Gravity / Density: 0.926
- pH: 10-12 at concentration of 75%
- Solubility in Propylene glycol: Up to 200 mg/g
Stability in Propylene glycol:
- at least 6 hours at RT over the concentration range 2 to 30 mg/mL (WIL report 491556 & 503870)
- at least 8 days in the refrigerator under nitrogen over the concentration range 2 to 20 mg/mL (WIL report 503866)

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Kisslegg, Germany.
- Age at study initiation: approx. 6 weeks old
- Weight at study initiation: no data
- Housing: Group housing of maximally 5 animals per labeled cage (74 cm x 54 cm x 25 cm height) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France).
- Diet (e.g. ad libitum):Complete breeding diet for guinea pigs (SSNIFF® MS-Z, V2273; SSNIFF® Spezialdiäten GmbH, Soest, Germany). Hay (TecniLab-B MI BV, Someren, The Netherlands) was provided at least twice a week.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17.5 – 20.7ºC
- Humidity (%): 23 - 95%)
- Air changes (per hr): approximately 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial fluorescent light and 12 hours darkness per day.

IN-LIFE DATES: From: To: 09 March 2010 - 16 April 2010

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:

intradermal inductie: 0.1%
epidermal inductie: 0.5%
Challange: 0 and 0.5%
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:

intradermal inductie: 0.1%
epidermal inductie: 0.5%
Challange: 0 and 0.5%
No. of animals per dose:
Experimental group: 10 females.
Control group: 5 females.
Details on study design:
RANGE FINDING TESTS:
Series of test substance concentrations were tested (0.5, 1, 2 and 5%. )
The test system and procedures were identical to those used during the main study, unless otherwise specified. The six animals selected were between 4 and 9 weeks old. No body weights were determined.


MAIN STUDY
A. INDUCTION EXPOSURE
-Day 1
The scapular region was clipped and three pairs of intradermal injections (0.1 mL/site) were made in this area as follows:
A) A 1:1 w/w mixture of Freunds' Complete Adjuvant (Difco, Detroit, U.S.A.) with water for injection (Fresenius AG, Bad Homburg, Germany).
B) The test substance at a 0.1% concentration.
C) A 1:1 w/w mixture of the test substance, at twice the concentration used in (B) and Freunds' Complete Adjuvant.
Note: One of each pair was on each side of the midline and from cranial A) to caudal C).

Day 3
The dermal reactions caused by the intradermal injections were assessed for irritation.


Day 8
The scapular area between the injection sites was clipped and subsequently treated with 0.5 mL of a 0.5% test substance concentration using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage.

The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance using water and the dermal reactions caused by the epidermal exposure were assessed for irritation.


B. CHALLENGE EXPOSURE
-Day 21
One flank of all animals was clipped and treated by epidermal application of a 0.5% test substance concentration and the vehicle (0.1 mL each), using Patch Test Plasters (Curatest®, Lohmann, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Coban elastic bandage.

The dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.

After termination, animals were sacrificed by intra peritoneal injection of pentobarbital (Euthesate®; Sanofi Sante B.V., Maassluis, The Netherlands).

In life examinations:

Mortality/Viability: Twice daily

Toxicity: At least once daily.

Body weights: Prior to start and at termination of the study.

Irritation:
Skin reactions were graded according to the following numerical scoring systems. Furthermore, a description of all other (local) effects was recorded.
To facilitate scoring, the epidermally treated skin-areas were clipped at least 3 hours before the 48-hour reading of these areas in the preliminary irritation study and challenge phase.

Positive control substance(s):
yes
Remarks:
Alpha-Hexylcinnamicaldehyde

Results and discussion

Positive control results:
Reliability test:

Was performed not more than 6 months previously. Similar procedures were used in the reliability test and in this study.

The skin reactions observed in seven experimental animals in response to the 20% test substance concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 70 per cent to the 20% concentration.

From these results, it was concluded that the female guinea pig of the Dunkin Hartley strain is an appropriate animal model for the performance of studies designed to evaluate the sensitizing potential of a substance in a Maximization type of test.


In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
0.5%
No. with + reactions:
4
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
0.5%
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
24
Group:
negative control
Dose level:
0.5%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
Alpha- Hexylcinnamaldehyde 20%
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
Alpha- Hexylcinnamaldehyde 20%
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
6/10 animals showed scaliness, whereas 4/10 showed no reactions
Remarks on result:
positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The results indicate a sensitization rate of 40 per cent.
Executive summary:

Assessment of Contact Hypersensitivity to Tall oil diethylenetriamine imidazoline in the Albino Guinea Pig( Maximization Test).

The study was carried out based on the guidelines described in:

OECD No. 406 (1992) "Skin Sensitization"

EC No 440/2008; B6: "Skin Sensitization: Guinea-Pig Maximization Test (GPMT)"

EPA OPPTS 870.2600 (2003) “Skin Sensitization”

JMAFF Guidelines (2000) including the most recent partial revisions.

The study was based on the method described by Magnusson and Kligman, "Allergic Contact Dermatitis in the Guinea Pig - Identification of Contact Allergens" (1970).

 

Test substance concentrations selected for the main study were based on the results of a preliminary study.

 

In the main study, ten experimental animals were intradermally injected with a 0.1% concentration and epidermally exposed to a 0.5% concentration. Five control animals were similarly treated, but with vehicle alone (corn oil).

 

Two weeks after the epidermal application all animals were challenged with a 0.5% test substance concentration and the vehicle.

 

Skin reactions of grade 1 were observed in four experimental animals in response to the 0.5% test substance concentration. No skin reactions were evident in the control animals.

 

The skin reactions observed in response to a 0.5% test substance concentration in 4 (of the ten) experimental animals in the challenge phase were considered indicative of sensitization, based on the absence of any response in the control animals. These results indicate a sensitization rate of 40 per cent.

These results lead to classification according to CLP (ATP 2): Skin sensitiser Cat.1A (GPMT ≥ 30% positive at ≤ 0.1% i.d. induction)