Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, N-(hydroxyethyl) derivs.

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

29 July 2003 - 5 September 2003

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

In recently published articles in peer reviewed journals, 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 Maximisation 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 con founders (Basketter et al., 2009) may necessitate the use of guinea pig tests".
References:
Kreiling. R.; Hollnagel. HM .• Hareng.. L.. Eigler. D .. Lee. M.S .• Griem. P Dressen. B.;
Kleber. M .• Albrecht. A,; Garcia C.;Wendel. A .. (2008) Comparison of the skin
sensitizing potential of unsaturated compounds and assessed by the murine
loca llyrnph node assay (LLNA) and the guinea 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 sensitization datasets: Implications for REACH. Reg Tox Pharm. 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 sensitisinng potentials of nonionic sugar lipid surfactants. Reg Tox Pharm 58: 301-307.

N. Hall. S. Cagen. JC. Carillo H. Certa. D. Eigler. F Faulmmmer. C. Garcia. C. Graham. C. Haux. S. Kolle, R. Kreiling. A. Natsch. A. Mehling. (2011) Evaluating the sensitisation 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. Reg Tox Pharm 60: 389·400

Test material

Test material information

Constituent 1

Reference substance name:

Amines, N-(C16-18 (even numbered) and C18-unsatd. alkyl) trimethylenedi-, ethoxylated(NLP)

EC Number:

800-029-6

Cas Number:

1290049-56-7

Molecular formula:

Not applicable

IUPAC Name:

Amines, N-(C16-18 (even numbered) and C18-unsatd. alkyl) trimethylenedi-, ethoxylated(NLP)

Test material form:

liquid: viscous

Details on test material:

name: DINORAMOX S3
batch number: 6751
Chemical name: Ethanol, 2,2'-[[3-[(2-hydroxyethyl)amino]propyl]imino]bis-, N-tallow alkyl derivs.
CAS number: 90367-27-4
Sponsor's filing number: GRL 0060/03
description: thick brown liquid
container: one glass flask
date of receipt: 20 June 2003
storage conditions: at room temperature
purity: 99.76%
expiry date: June 2004.
Certificate of analysis included in report

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 France, L’Arbresle, France.
- Age at study initiation: 1-2 months
- Weight at study initiation: 341 ± 13 g for the males and 354 ± 16 g for the females.
- Housing: individually in polycarbonate cages with stainless steel lid (48 cm x 27 cm x 20 cm) equipped with a polypropylene bottle.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): 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

IN-LIFE DATES: From: 29 July 2003 (start priliminary test) To: 5 September 2003 experimental completion (sacrifice)

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: Intra-dermal: corn oil; topical induction: 80/20 (w/w) mixture of ethanol, water; challenge: acetone

Induction 2

Concentration / amount:

Intradermal: 0.1% (w/w)
topical induction: 25% (w/w)
challenge: 5 (w/w)

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: Intra-dermal: corn oil; topical induction: 80/20 (w/w) mixture of ethanol, water; challenge: acetone

Challenge 2

Concentration / amount:

Intradermal: 0.1% (w/w)
topical induction: 25% (w/w)
challenge: 5 (w/w)

No. of animals per dose:

control group: five males and five females
treated group: 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.

By cutaneous route: 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.

Under the conditions of the challenge phase (tested concentrations: 1%, 5%, 10%, 25%, 50% and 100% (w/w)):
- the filter paper of a chamber (Finn 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.

MAIN STUDY
A. INDUCTION EXPOSURE
Three injections of 0.1 mL were made into each side of this interscapular region (i.e. three pairs of sites), as follows:
1 Anterior site: FCA at 50% (v/v) in 0.9% NaCl
2 Middle location: test item at 0.1% (w/w) in corn oil
3 Posterior site: test item at 0.1% (w/w) in the mixture FCA/0.9% NaCl (50/50)

Cuteneous route:
(No SLS needed as substance was irritating)
On day 8, fully loaded filter paper (approximately 8 cm2) with 25% (w/w), applied on interscapular region for 48 hoursd

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: day 22
- Exposure period: 24 hours
- all animals:
- Posterior right flank: filter paper of a chamber (Finn Chamber) was fully-loaded with the test item
- Posterior left flank: vehicle
- Concentrations: 5% (w/w)
- Evaluation (hr after challenge): 24 and 48 after removal of the dressing

OTHER:

Positive control substance(s):

yes

Remarks:

Mercaptobenzothiazole

Results and discussion

Positive control results:

Concentration :
induction phase: 1% (w/w) on day 1 (intradermal route); 20% (w/w) on day 8 (cutaneous route)
challenge phase: 20% (w/w) on day 22 (cutaneous route)
Vehicle : corn oil
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:

test chemical

Dose level:

5%

No. with + reactions:

5

Total no. in group:

19

Clinical observations:

only discrete erythema (scale 1)

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 5%. No with. + reactions: 5.0. Total no. in groups: 19.0. Clinical observations: only discrete erythema (scale 1) .

Results 2

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 was recorded in 3/19 and 2/19 animals, respectively. Dryness of skin was observed in 6/19 animals.

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5%. No with. + reactions: 5.0. Total no. in groups: 19.0. Clinical observations: discrete or moderate erythema was recorded in 3/19 and 2/19 animals, respectively. Dryness of skin was observed in 6/19 animals..

Any other information on results incl. tables

Preliminary study:

Administration by intradermal route: 25, 10 and 5% resulted to necrosis within 24 hours. At 1% ncrosis became evident after 48 hours.

At 0.1% irritation was observed.

Application by cutaneous route for induction: 100% and 50% resulted to necrosis. At 25% irritation was observed that was resolved by 28 hours.

Application by cutaneous route under conditions of chalenge phase (2 animals / concentration):

100%, 50% and 25% resulted to necrosis.

10% resulted to irritation in 1 of the two animals.

5% did not result to irritation in either animals

1% resulted to discrete or patchy erythema in one of teh two animals.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Testing of Tallow-diamine3EO in a GPMT study resulted to positive reactions in 5/19 (26%) guinea pigs. According classification criteria, no classification is required.

Executive summary:

At the request of CECA SA, Paris-la-Défense, France, the potential of the test item DINORAMOX S3 (batch No. 6751) 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.

 

Methods

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).

 

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.

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.

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.

 

Results

No systemic clinical signs 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. 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.

 

Conclusion

Under our experimental conditions and according to the maximization method of Magnusson and Kligman, the test item DINORAMOX S3 (batch No. 6751) induces delayed contact hypersensitivity in 5/19 (26%) guinea pigs and therefore should be considered as a mild sensitizer.

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.