Registration Dossier

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:
other: 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.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Fatty acids, tall-oil, reaction products with polyethylene-polyamines (Amidoamine)
- Substance type: Bottom phase: yellow to light brown paste / Upper phase: clear amber liquid with flakes of the bottom (determined at NOTOX)
- Physical state: liquid
- Analytical purity: 98%
- Impurities (identity and concentrations): Pentaethylene hexamine (1.5%) / Tetraethylene pentamine (0.6%) / Triethylene tetraamine (3.5%) Free fatty accid (2.0%)
- Isomers composition: C-chain distribution = C16 (<=10%) / C18 (>=85%) / C20-24 (<+5%)
- Purity test date: 11-05-2009
- Lot/batch No.: S001681
- Expiration date of the lot/batch: 23 October 2018
- Stability under test conditions: Stability under storage conditions = Stable / Stability in vehicle (Corn oil) = Not indicated
- Storage condition of test material: At room temperature in the dark under nitrogen

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. 7 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).
The acclimatization period was at least 5 days before the start of treatment under laboratory conditions.
- Diet (e.g. ad libitum): Complete breeding diet for guinea pigs (SSNIFF® MS-Z, V2273; SSNIFF® Spezialdiäten GmbH, Soest, Germany). Hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least twice a week.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: 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: 09 March 2010 To: 16 April 2010

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
Intradermal inductie: 0.2%
Epidermal inductie: 2%
Challange: 0 and 2%

Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
Intradermal inductie: 0.2%
Epidermal inductie: 2%
Challange: 0 and 2%

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 0.2% 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 2% 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 23
One flank of all animals was clipped and treated by epidermal application of a 2% 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- HEXYLCINNAMALDEHYDE

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
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
2%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Scaliness, Scabs
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 2%. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: Scaliness, Scabs.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
2%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Scaliness, Scabs
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 2%. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: Scaliness, Scabs.

Applicant's summary and conclusion

Conclusions:
The skin reactions observed in response to a 2% test substance concentration in 10 (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.
Executive summary:

Assessment of Contact Hypersensitivity to Fatty acids, tall-oil, reaction products with polyethylene-polyamines (Amidoamine) 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 irritation study:

As no signs of necrosis were present, a 0.2%centration was selected for the intradermal induction in the main study. A 2%centration was selected for the epidermal induction exposure.

A 2% test substance concentration was selected for the challenge phase.

 

 

In the main study, ten experimental animals were intradermally injected with a 0.2% concentration and epidermally exposed to a 2% 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 2% test substance concentration and the vehicle.

Skin reactions varying between grades 1 and 2, were observed in all experimental animals in response to the 2% test substance concentration. No skin reactions were evident in the control animals. Scaliness was seen in most treated skin sites among the experimental animals.

No mortality occurred and no symptoms 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

 

The skin reactions observed in response to a 2% test substance concentration in 10 (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 100 per cent.

It is notworthy though that the reactions on 2% concentration that was non-irritating in naive animals in the preliminary test, showed more severe irritating reactions followong the induction exposure 1 week after intradermal injections, then during the challenge exposure again two weeks later.

Based on these results and according to the:

- Globally Harmonized System of Classification and Labeling of Chemicals (GHS) of the United Nations (2007), Fatty acids, tall-oil, reaction products with polyethylene-polyamines (Amidoamine) should be classified as skin sensitizer (Category 1).

- Regulation (EC) No 1272/2008 on classification, labeling and packaging of substances and mixtures, Fatty acids, tall-oil, reaction products with polyethylene-polyamines (Amidoamine) should be classified as skin sensitizer (Category 1) and labeled as H317: May cause an allergic skin reaction.

Classificaton according to CLP (ATP 2): Skin sensitiser Cat.1A (GPMT ≥ 60% positive at > 0.1% i.d. induction)