Reaction mass of ethoxylated N-oleyl-1,3-propanediamine, hydrofluorides of and ethoxylated N-palmityl-1,3-propanediamine, hydrofluorides of and ethoxylated N-stearyl-1,3-propanediamine, hydrofluorides of and olaflur

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-07-29 to 2003-09-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

17 July 1992

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

96/54/EEC, 30 July 1996

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Due to animal welfare, the already existing study (OECD 406 Skin Sensitisation) was used. This existing study contains a robust study summary, was performed under GLP-conditions, and deserves the highest reliability of '1'. Therefore, performing an additonal study LLNA according to OECD 429 is, not justified.

Test material

Test material information

Constituent 1

Test material form:

liquid

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: Hartley Crl: (HA) BR

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories France, L` Arbresle, France
- Age at study initiation: the animals of the main test were 1-2 months old
- Weight at study initiation: male: 341 +/- 13 g; female: 354 +/- 16 g (the animals of the main test)
- Diet (e.g. ad libitum): pelleted diet
- Water (e.g. ad libitum): drinking water filtered; ad libitum
- Acclimation period: at least 5 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal and epicutaneous

Vehicle:

corn oil

Concentration / amount:

Induction (treated group)
- intradermal route: Ethanol, 2,2`-[[3-(2-hydroxyethyl)amino]propyl]imino]bis-N-tallow alkyl derivs. at 0.1 % (w/w)
- cutaneous route: Ethanol, 2,2`-[[3-(2-hydroxyethyl)amino]propyl]imino]bis-N-tallow alkyl derivs. at the concentration of 25 % (w/w)

Challenge (all groups):
- Ethanol, 2,2`-[[3-(2-hydroxyethyl)amino]propyl]imino]bis-N-tallow alkyl derivs.: at the concentration of 5 %

Challenge

Route:

epicutaneous, open

Vehicle:

corn oil

Concentration / amount:

Induction (treated group)
- intradermal route: Ethanol, 2,2`-[[3-(2-hydroxyethyl)amino]propyl]imino]bis-N-tallow alkyl derivs. at 0.1 % (w/w)
- cutaneous route: Ethanol, 2,2`-[[3-(2-hydroxyethyl)amino]propyl]imino]bis-N-tallow alkyl derivs. at the concentration of 25 % (w/w)

Challenge (all groups):
- Ethanol, 2,2`-[[3-(2-hydroxyethyl)amino]propyl]imino]bis-N-tallow alkyl derivs.: at the concentration of 5 %

No. of animals per dose:

- a control group of ten animals (five males and five females)
- treated group of 20 animals (ten males and ten females)

Details on study design:

RANGE FINDING TESTS:
By intradermal route (tested concentrations: 0.1%, 1%, 5%, 10% and 25% (w/w)):
• intradermal injections of the dosage form preparations (0.1 mL) were performed in the interscapular region,
• local reactions were evaluated approximately 24, 48 hours and 6 days after the injections.

Under the conditions of the induction phase (tested concentrations: 10%, 25%, 50% (w/w) and 100%):
• 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.


MAIN STUDY
A. INDUCTION EXPOSURE
- Intradermal route:
On day 1, six 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).

- Cutaneous route:
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.
On removal of the dressing (day 10), no residual test item was observed. A local irritation was recorded in all the animals of both groups.

B. CHALLENGE EXPOSURE
On day 22, the animals of the treated and control groups received an application of the test item and vehicle. The filter paper of a chamber (Finn Chamber) was fully-loaded with the test item at the concentration of 5% (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:

NA

Positive control substance(s):

yes

Remarks:

Mercaptobenzothiazole

Results and discussion

Positive control results:

Under the experimental conditions and according to the Magnusson and Kligman method, the test item Mercaptobenzothiazole at the concentration of 20% (w/w) induced positive skin sensitization reactions in 80% (8/10) guinea pigs.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

5 %

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

none

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

5 %

No. with + reactions:

0

Total no. in group:

0

Clinical observations:

none

Results 3

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

5 %

No. with + reactions:

5

Total no. in group:

19

Clinical observations:

discrete erythema

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5 %

No. with + reactions:

5

Total no. in group:

19

Clinical observations:

discrete or moderate erythema

Results 5

Reading:

2nd reading

Hours after challenge:

24

Group:

positive control

Dose level:

20 % of mercaptobenzothiazole

No. with + reactions:

8

Total no. in group:

10

Clinical observations:

oedema, crusts, dryness of the skin

Any other information on results incl. tables

NA

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under this experimental conditions and according to the maximization method of Magnusson and Kligman, the test item Ethanol, 2,2`-[[3-(2-hydroxyethyl)amino]propyl]imino]bis-N-tallow alkyl derivs. induces delayed contact hypersensitivity in 5/19 guinea pigs and therefore should be considered as a mild sensitizer.

Executive summary:

Ethanol, 2,2`-[[3-(2-hydroxyethyl)amino]propyl]imino]bis-N-tallow alkyl derivs. was tested to thirty guinea pigs according to the maximization method of Magnusson and Kligman, EU-method B.6 and OECD guideline 406.

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 to 50% (v/v) with 0.9% NaCl (both groups),

• test item at the concentration of 0.1% in corn oil (treated group) or vehicle alone (control group),

• test item at the concentration of 0.1% in a mixture FCA/0.9% NaCl (50/50, w/w) (treated group) or vehicle at the concentration of 50% (v/w) in a mixture FCA/0.9% NaCl (50/50, v/v) (control group)

based on pretest results revealing slight irritation at this test condition.

On day 8, the animals of the treated group received a topical application of the test item at the concentration of 25% (w/w) in ethanol/water (80/20) to the same test site, which was then covered by an occlusive dressing for 48 hours. The animals of the control group received an application of the vehicle under the same experimental conditions. This is based on slight irritation noted at this concentration in the pretest.

On day 22, all animals of both groups were challenged by a cutaneous application of the test item at the concentration of 5% (w/w) in acetone to the right flank. The test item was maintained under an occlusive dressing for 24 hours. The vehicle was applied to the left flank under the same experimental conditions. Again this concentration was based on pretest results indicating non-sensitising effect at

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

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 of systemic toxicity and no deaths related to treatment 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, at the 24-hour reading, a discrete erythema was noted in 5/19 animals (~ 25%). At the 48-hour reading, a discrete or moderate erythema was recorded in 3/19 and 2/19 animals, respectively. Dryness of the skin, which masked the evaluation of the erythema in one animal, was also observed in 5/19 animals at the 48-hour reading. The cutaneous reactions observed at the 48-hour reading in 5/19 animals of the treated group were attributed to delayed contact hypersensitivity.

Ethanol, 2,2`-[[3-(2-hydroxyethyl)amino]propyl]imino]bis-N-tallow alkyl derivs. induces delayed contact hypersensitivity in 5/19 guinea pigs and therefore should be considered as a mild sensitizier. However, according to the classification criteria laid down in Council Directive 67/548/EEC (and subsequent adaptations), the test item should not be considered as a skin sensitizer.