Reaction mass of Amines, coco alkyl and β-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs. and β-Alanine, N-coco alkyl derivs.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-11-20 to 2002-07-15

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study (OECD 406)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2002

Report date:

2002

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

the normal range of temperature in the animal room was 22 ± 2°C (instead of 21 ± 2°C as indicated in the Study plan).

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

yes

Remarks:

See above

Principles of method if other than guideline:

These minor deviations were not considered to have compromised the validity or integrity of the study.

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

the study was performed before Reach guidance.

Test material

Test material information

Constituent 1

Reference substance name:

Reaction mass of Amines, coco alkyl and ß-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs. and ß-Alanine, N-coco alkyl derivs.

EC Number:

915-790-0

Molecular formula:

n/a

IUPAC Name:

Reaction mass of Amines, coco alkyl and ß-Alanine, N-(2-carboxyethyl)-, N-coco alkyl derivs. and ß-Alanine, N-coco alkyl derivs.

Test material form:

liquid

Details on test material:

- Purity: 69% (28% isopropanol – 3% water)
- Batch Number: 01652201
- Description: yellow liquid
- Container: one glass flask
- Date of receipt: 28 June 2001
- Storage conditions: at room temperature and protected from light
- Expiry date/stability: June 2002
- Carbon content: 65.8% (weight/weight).

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Saint-Aubin-lès-Elbeuf, France.
- Age at study initiation: 1-3 months old
- Weight at study initiation: 376 ± 22 g for the males and 376 ± 23 g for the females
- Housing: individually in polycarbonate cages
- Diet (e.g. ad libitum): free access to 106 pelleted diet (UAR, Villemoisson, Epinay-sur-Orge, France).
- Water (e.g. ad libitum): Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum.
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
· temperature: 22 ± 2°C
· relative humidity: 30 to 70%
· light/dark cycle: 12 h/12 h
· ventilation: approximately 12 cycles/hour of filtered, non-recycled air.

IN-LIFE DATES: From: December 7, 2001 To: December 31, 2001

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: 0.9% NaCl

Induction 2

Concentration / amount:

Induction (treated group)
• intradermal injections (day 1): test item at the concentration of 1% (w/w) in 0.9% NaCl,
• topical application (day 8): test item at the concentration of 25% (w/w) in 0.9% NaCl.

Challenge (all groups)
• topical application (day 22): test item at the concentration of 1% (w/w) in 0.9% NaCl.

Challenge

Challenge 1

Route:

epicutaneous, open

Vehicle:

other: 0.9% NaCl

Challenge 2

Concentration / amount:

Induction (treated group)
• intradermal injections (day 1): test item at the concentration of 1% (w/w) in 0.9% NaCl,
• topical application (day 8): test item at the concentration of 25% (w/w) in 0.9% NaCl.

Challenge (all groups)
• topical application (day 22): test item at the concentration of 1% (w/w) in 0.9% NaCl.

No. of animals per dose:

-three males and three females for the preliminary test,
- in the main test : control group = 5 animals/sex , treated group = 10 animals/sex/dose

Details on study design:

RANGE FINDING TESTS:
A preliminary test was conducted in order to determine the concentrations to be tested in the main study.
By intradermal route (tested concentrations: 5%, 1% and 0.1% (w/w)):
• 24 hours before treatment, the dorsal region of the animals was clipped,
• intradermal injections of the dosage form preparations (0.1 mL) were performed in the interscapular region,
• cutaneous reactions were evaluated approximately 24, 48 hours and 6 days after the injections.

By cutaneous route
Under the conditions of the induction phase (tested concentration: 25% (w/w)):
• 24 hours before treatment, the interscapular region of the animals was clipped,
• a filter paper (approximately 8 cm2) was fully-loaded with a dosage form preparation and was then applied to the clipped area of the skin. The filter paper was held in place by means of an occlusive dressing for 48 hours,
• cutaneous reactions were evaluated 24 and 48 hours after removal of the dressing.
Under the conditions of the challenge phase (tested concentrations: 25%, 10%, 5% and 1% (w/w)):
• 24 hours before treatment, both flanks of the animals were clipped and shaved,
• the filter paper of a chamber was fully-loaded with a dosage form preparation. The chamber was then applied to the clipped area of the skin (one concentration per flank). The chamber was held in place by means of an occlusive dressing for 24 hours,
• cutaneous reactions were evaluated 24 and 48 hours after removal of the dressings.

In order to respect the criteria for the selection of concentrations (the concentration should be well-tolerated systemically and locally, intradermal injections should cause moderate irritant effect but no necrosis or ulceration of the skin), concentration chosen for the main study was 1% (w/w).
In order to respect the criteria for the selection of concentrations (the concentrations should be well-tolerated systemically and locally, cutaneous application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration, cutaneous application for the challenge phase should be the highest concentration which does not cause irritant effect), concentration chosen for the topical application of the induction phase (day 8) was 25% (w/w). For the challenge application (day 22), it was 1% (w/w).

MAIN STUDY
A. INDUCTION EXPOSURE (intradermal injection and topical administration)
On day 1, six intradermal injections were made deep into the dermis of a 4 cm x 2 cm clipped interscapular area, using a needle (diameter: 0.50 x 16 mm) mounted on a 1 mL plastic syringe (0.01 mL graduations). 2 injections per site.
As the test item was shown to be irritant during the preliminary test, a topical application with sodium lauryl sulfate was not necessary on day 7.
On day 8, a pad of filter paper (approximately 8 cm2) was fully-loaded with the test item at the concentration of 25% (w/w) and was then applied to the interscapular region of the animals of the treated group.
The animals of the control group received an application of the vehicle alone under the same experimental conditions.
The pad was held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive anallergenic waterproof plaster.

B. CHALLENGE EXPOSURE
On day 22, the animals of treated and control groups received an application of the test item and vehicle. The filter paper of a chamber was fully-loaded with the test item at the concentration of 1% (w/w) and was then applied to a clipped area of the skin of the posterior right flank of all animals.
The vehicle was applied under the same experimental conditions to the skin of the posterior left flank.
The chambers were held in contact with the skin for 24 hours by means of an adhesive anallergenic waterproof plaster.

Challenge controls:

no

Positive control substance(s):

yes

Remarks:

MERCAPTOBENZOTHIAZOLE. In a study performed in October 2001 under CIT experimental conditions, the strain of guinea pigs used showed a satisfactory sensitization response in 100% animals.

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

1%

No. with + reactions:

1

Total no. in group:

20

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1%. No with. + reactions: 1.0. Total no. in groups: 20.0.

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1%

No. with + reactions:

2

Total no. in group:

20

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1%. No with. + reactions: 2.0. Total no. in groups: 20.0.

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

1%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 1% . No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

1%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Any other information on results incl. tables

No clinical signs and no deaths were observed during the study.

The body weight gain of the treated animals was similar to that of controls.

After the challenge phase, on removal of the dressing, no residual test item was observed.

No cutaneous reactions were observed in the animals of the control group.

In the treated group, a discrete or moderate erythema (grade 1 or 2), together with dryness of the skin and crusts, was noted in a single animal.

No other relevant skin reactions were recorded.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Thes test item should not be considered as a skin sensitizer.

Executive summary:

The potential of the test item to induce delayed contact hypersensitivity was evaluated in guinea pigs according to the maximization method of Magnusson and Kligman and to OECD (No. 406, 17th July 1992) and EC (96/54/EEC, B.6, 30 July 1996) guidelines. The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

Thirty guinea pigs were allocated to two groups: a control group of five males and five females and a treated group of ten males and ten females.

On day 1, three pairs of intradermal injections were performed in the interscapular region of all animals:

• Freund's complete adjuvant (FCA) diluted at 50% (v/v) with 0.9% NaCl (both groups),

• test item at the chosen concentration in the chosen vehicle (treated group) or vehicle alone (control group),

• test item at the chosen concentration in a mixture FCA/0.9% NaCl (50/50, v/v) (treated group) or vehicle at the concentration of 50% (w/v) in a mixture FCA/0.9% NaCl (50/50, v/v) (control group).

On day 8, the test item (treated group) or the vehicle (control group) was applied topically to the same test site, which was then covered by an occlusive dressing for 48 hours.

On day 22, all animals of both groups were challenged by a cutaneous application of the test item to the right flank. The left flank served as control and received the vehicle only. The test item and vehicle were maintained under an occlusive dressing for 24 hours.

Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing.

Test item concentrations were as follows:

Induction (treated group)

• intradermal injections (day 1): test item at the concentration of 1% (w/w) in 0.9% NaCl,

• topical application (day 8): test item at the concentration of 25% (w/w) in 0.9% NaCl.

Challenge (all groups)

• topical application (day 22): test item at the concentration of 1% (w/w) in 0.9% NaCl.

At the end of the study, animals were killed without examination of internal organs.

No skin samples were taken from the challenge application sites.

No clinical signs and no deaths were noted during the study.

After the challenge application, no cutaneous reactions were observed in the animals of the control group.

In the treated group, a discrete or moderate erythema, together with dryness of the skin and crusts, was noted in a single animal.

No other relevant skin reactions were recorded.

Under these experimental conditions and according to the classification criteria laid down in Directive 93/21/EEC (27th April 1993) adapting to technical progress for the eighteenth time Council Directive 67/548/EEC, the test item should not be considered as a skin sensitizer.