Reaction mass of Amines, N-tallow alkyltrimethylenedi-, mono(2-ethylhexanoates), Amines, N-tallow alkyltrimethylenedi-, acetates and n-tallow-1,3-diaminopropane ditallate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

Assay performed on a mixture containing 30-40% of Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates, 25-35% of butylglycol and water until complement to 100%. As the existing data on butylglycol indicate no sensitization potential and a very slight irritating potential, the presence of this solvent does not compromise the interpretation of the results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

On day 1, the third pair of intradermal injections was performed with the test item at the concentration of 50% (v/W) in a mixture FCA/0.9% NaCl (50/50) (control group).

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

yes

Remarks:

See above

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

Data performed before Reach guidance.

Test material

Test material information

Constituent 1

Reference substance name:

Amines,N-(C16-18 and C18 unsaturated alkyl) trimethylenedi-,diacetates.

EC Number:

800-153-0

Cas Number:

1313206-64-2

Molecular formula:

R-NH2+ -(CH2)3-NH3+, (CH3 COO-)2 where R = C18-alkyl

IUPAC Name:

Amines,N-(C16-18 and C18 unsaturated alkyl) trimethylenedi-,diacetates.

Constituent 2

Reference substance name:

Amines, N-tallow alkyltrimethylenedi-, diacetates

EC Number:

272-786-5

EC Name:

Amines, N-tallow alkyltrimethylenedi-, diacetates

Cas Number:

68911-78-4

IUPAC Name:

Amines, N-tallow alkyltrimethylenedi-, diacetates

Test material form:

liquid: viscous

Details on test material:

- Name of test material (as cited in study report): DINORAMAC S36 (D.MAC S36 in the analytical certificate)
- Physical state and appearance: yellow liquid
- Composition of test material, percentage of components: mixture containing 30-40% of Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates, 25-35% of butylglycol and water to complement to 100%.
- Purity test date: 21 May 2003
- Lot/batch No.: 94051339
- Sponsor's filing number: GRL 0061/03
- Expiration date of the lot/batch: June 2004
- Storage condition of test material: at room temperature and from 19 august 2003 protected from light
-Container : one glass flask

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: 1-2 months old
- Weight at study initiation: 335 ± 9 g for the males and 344 ± 11 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: July 30, 2003 To: September 5, 2003

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 0.1% (w/w) in 0.9% NaCl,
• topical application (day 8): test item at the concentration of 25% (w/w) in 0.9% NaCl.

First challenge (all groups): topical application (day 22): test item at the concentration of 10% (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 0.1% (w/w) in 0.9% NaCl,
• topical application (day 8): test item at the concentration of 25% (w/w) in 0.9% NaCl.

First challenge (all groups): topical application (day 22): test item at the concentration of 10% (w/w) in 0.9% NaCl.

No. of animals per dose:

- four males and four 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: 25%, 10%, 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 concentrations: 100%, 50% (w/w), 25% (w/w) and 10% (w/w)):
• 24 hours before treatment, both flanks of the animals were clipped and shaved,
• 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. The chamber 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 dressings.


Under the conditions of the challenge phase (tested concentrations: 100%, 50% (w/w), 25% (w/w) and 10% (w/w)):
• 24 hours before treatment, both flanks of the animals were clipped and shaved,
• 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.

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 0.1% (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) and for the challenge application (day 22) 10% (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
First challenge application
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 10% (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 recent study performed 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:

negative control

Dose level:

0%

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: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

10%

No. with + reactions:

2

Total no. in group:

10

Clinical observations:

discrete erythema

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 10%. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: discrete erythema.

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0%

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: 0%. 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:

10%

No. with + reactions:

4

Total no. in group:

10

Clinical observations:

discrete to moderate erythema and/or dryness of skin

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 10%. No with. + reactions: 4.0. Total no. in groups: 10.0. Clinical observations: discrete to moderate erythema and/or dryness of skin.

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0%

No. with + reactions:

0

Total no. in group:

19

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 19.0.

Results 6

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10%

No. with + reactions:

9

Total no. in group:

19

Clinical observations:

discrete to moderate erythema

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10%. No with. + reactions: 9.0. Total no. in groups: 19.0. Clinical observations: discrete to moderate erythema.

Results 7

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0%

No. with + reactions:

0

Total no. in group:

19

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 19.0.

Results 8

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

12

Total no. in group:

19

Clinical observations:

discrete to moderate erythema and/or dryness of skin

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10%. No with. + reactions: 12.0. Total no. in groups: 19.0. Clinical observations: discrete to moderate erythema and/or dryness of skin.

Any other information on results incl. tables

No systemic clinical signs and no deaths related to treatment were noted during the study. The body weight gain of the treated animals was similar to that of controls. Marked local reactions at the intradermal injection sites were noted in a few animals of the treated group, between days 10 and 14. Therefore, one of these animals (Male No. 156) was sacrificed on day 14 for ethical reasons.

A discrete erythema (grade 1) was noted in 2/10 and 1/10 animals of the control group at the 24 and 48 -hour readings, respectively. Dryness of the skin, which masked the evaluation of the erythema in one animal, was also observed in 4/10 animals at the 48 -hour reading. In the treated group, at the 24 -hour reading, a discrete or moderate erythema (grade 1 or 2) was recorded in 7/19 and 2/19 animals, respectively. At the 48 -hour reading, a discrete or moderate erythema (grade 1 or 2) was seen in 8/19 and 2/19 animals, respectively. Dryness of the skin was also noted in 8/19 animals at the 48 -hour reading.

The persistent cutaneous reactions observed in 3/19 animals of the treated group, which were of higher severity than those recorded in the animals of the control group, were attributed to possible delayed contact hypersensitivity. According to the classification criteria laid down in Regulation EC n°1272/2008 (CLP), the test item should not be considered as a skin sensitizer.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

According to the classification criteria laid down in Regulation EC n°1272/2008 (CLP), the test item, Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates should not be considered as a skin sensitizer.

Executive summary:

The potential of the test item, Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates to induce delayed contact hypersensitivity was evaluated in guinea pigs according to the maximization method of Magnusson and Kligman and to OECD 406 guideline. The assay was performed on a mixture containing 30-40% of Amines, N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-, diacetates, 25-35% of butylglycol and water until complement to 100%. As the existing data on butylglycol indicate no sensitization potential and a very slight irritating potential, the presence of this solvent does not compromise the interpretation of the results.

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), and 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 0.1% (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 10% (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 of all the animals.

No systemic clinical signs and no deaths related to treatment were noted during the study. The body weight gain of the treated animals was similar to that of controls. Marked local reactions at the intradermal injection sites were noted in a few animals of the treated group, between days 10 and 14. Therefore, one of these animals was sacrificed on day 14 for ethical reasons.

A discrete erythema (grade 1) was noted in 2/10 and 1/10 animals of the control group at the 24 and 48 -hour readings, respectively. Dryness of the skin, which masked the evaluation of the erythema in one animal, was also observed in 4/10 animals at the 48 -hour reading. In the treated group, at the 24 -hour reading, a discrete or moderate erythema (grade 1 or 2) was recorded in 7/19 and 2/19 animals, respectively. At the 48 -hour reading, a discrete or moderate erythema (grade 1 or 2) was seen in 8/19 and 2/19 animals, respectively. Dryness of the skin was also noted in 8/19 animals at the 48 -hour reading.

The persistent cutaneous reactions observed in 3/19 (16%) animals of the treated group, which were of higher severity than those recorded in the animals of the control group, were attributed to possible delayed contact hypersensitivity.

According to the classification criteria laid down in Regulation EC n°1272/2008 (CLP), the test item,Amines, N-(C16-18 and C18 -unsatd. alkyl)trimethylenedi-, diacetates should not be considered as a skin sensitizer.