Amides, C8-18 (even-numbered), C18unsatd, N-[3-(dimethylamino)propyl]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-06-18 to 2009-07-10

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

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:

30 May 2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The study was conducted before the obligation was in place to use in vitro studies as standard information.

Test material

Test material information

Constituent 1

Reference substance name:

Amides, C6-C18, N-[3-(dimethylamino)propyl]

IUPAC Name:

Amides, C6-C18, N-[3-(dimethylamino)propyl]

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 Laboratories, Kissleg, Germany
- Age at study initiation: no data
- Weight at study initiation: 290.6±8.2 g (test group), 305.9±16.1 g (control group)
- Housing: 2-3 animals in Macrolon-cages No.IV with standard softwood bedding Lignocel 9S, Rettenmaier & Söhne GmbH & Co., Holzmühle 1, 73494 Rosenberg, Germany, Batch No.: 0211381001
- Diet: standard laboratory guinea pig diet type "3023" from Altromin, Lage, Germany as pelleted diet (batch No. 0913 and 0743; expiry dates 2009-07-06 and 2009-10-09) (ad libitum)
- Water: normal tap water from municipal source (ad libitum)
- Acclimation period: 10d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 30-70
- Air changes (per hr): 8 times
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: Sesame oil

Induction 2

Concentration / amount:

Pilot experiment:
Intradermal: 0.1 mL of 5 %, 3.5 %, 2 %, 0.5 %,0.1 % and 0.05 % (w/w) concentration in vehicle.
Dermal: soaked patch with 100 %, 75 %, 50 %, 25 %, 5 %, 1 %, 0.5 % and 0.1 % (w/w) concentration in vehicle.
Challenge: soaked patch with Duhring chamber with 1 % and 0.5 % (w/w) concentration in vehicle.
Main experiment:
Based on the results of the pilot experiment the following concentrations were chosen for the main experiment:
Intradermal: 0.1 mL of 0.05 % (w/w) concentration of the test substance in vehicle.
Dermal: soaked patch with 5 % (w/w) concentration of the test substance in vehicle.
Challenge: soaked patch with Duhring chamber with 1 % (w/w) concentration in vehicle.

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: Sesame oil

Challenge 2

Concentration / amount:

Pilot experiment:
Intradermal: 0.1 mL of 5 %, 3.5 %, 2 %, 0.5 %,0.1 % and 0.05 % (w/w) concentration in vehicle.
Dermal: soaked patch with 100 %, 75 %, 50 %, 25 %, 5 %, 1 %, 0.5 % and 0.1 % (w/w) concentration in vehicle.
Challenge: soaked patch with Duhring chamber with 1 % and 0.5 % (w/w) concentration in vehicle.
Main experiment:
Based on the results of the pilot experiment the following concentrations were chosen for the main experiment:
Intradermal: 0.1 mL of 0.05 % (w/w) concentration of the test substance in vehicle.
Dermal: soaked patch with 5 % (w/w) concentration of the test substance in vehicle.
Challenge: soaked patch with Duhring chamber with 1 % (w/w) concentration in vehicle.

No. of animals per dose:

test group: 10,
control group: 5,
pilot experiment: 4
(in total): 19

Details on study design:

Before the main experiment was started, the animals had an acclimatization period of 10 days. The animals were clearly marked with dye and weighed. Food and drinking water was left for the animals ad libitum.
Pilot experiment:
For the purpose of the intradermal application one guinea pig was shorn on the right and left flank over an area of approximately 4 cm x 6 cm. 24 hours later this animal received 0.1 mL of various concentrations of the test substance in vehicle intradermally by disposable syringe.
The skin reactions observed were assessed 24 hours later.
For the epicutaneous application soaked patches of 2 cm x 2 cm size with various concentrations of the test substance in vehicle were applied to shorn right and left flank, covered with Blenderm (3 M company, St. Paul, USA) and bandaged on with Acrylastic (Beiersdorf AG, Hamburg, Germany) which was fixed by Leukoplast (Beiersdorf AG, Hamburg, Germany).
After 24 hours the dressing was removed and the skin reactions which occurred were assessed.
For the challenge dermal application Duhring chambers (0.4 cm) were filled with a soaked patch of 2 cm x 2 cm size with various concentrations of the test substance in vehicle and applied to the skin. 24 hours before the flanks of the animals had been shorn. In order to achieve an occlusive dressing the Duhring chambers were applied each to one flank of a guinea pig, covered with Micropore (3 M company, St. Paul, USA) and bandaged on with
Acrylastic (Beiersdorf AG, Hamburg, Germany) which was fixed by Leukoplast (Beiersdorf AG, Hamburg, Germany). After 24 hours the dressing was removed and the skin reactions which occurred were assessed. In total 4 guinea pigs were used for the pilot experiment.

Main Experiment:
Intradermal induction
Each guinea pig was shorn 24 hours before the beginning of the experiment on the back over an area of 4 cm x 6 cm below the shoulder blade, without damaging the skin. The next day the following injections were made at left and right shoulder.
(1) 0.1 mL per side FCA (mixed at a ratio of 1+1 in vehicle)
(2) 0.1 mL per side of the concentration of the test substance found to be slightly irritant in the pilot experiment.
(3) 0.1 mL per side of the concentration of the test substance found to be slightly irritant in the pilot experiment in FCA (mixed at a ratio of 1+1 in vehicle).
The control animals were treated similarly - with the exception that they received only the diluting vehicle agent instead of the concentration of the test substance found to be slightly irritant. 24 hours later skin reactions observed were classified according to the following scheme:

Erythema and scurf formation:
no reddening - 0
slight reddening (difficult to detect) - 1
easily detectable (erythema) - 2
medium (erythema) - 3
strong (erythema) (dark red) to slight formation of scurf (damage deep down) - 4

Edema formation:
no swelling - 0
slight swelling (hardly detectable) - 1
easily recognizable swelling (clearly marked by elevation) - 2
medium edema (swelling of approx. 1 mm) - 3
strong edema (swelling over 1 mm and clearly stretching beyond the treated area) - 4
Epicutaneous induction
One week (day seven of the treatment period) after the intradermal application the same part of the skin was dermally treated. A soaked patch (2 cm x 4 cm) with the chosen concentration of the test substance in vehicle was placed over the injection area and fixed in the way described in the pilot experiment. The control animals received only the vehicle instead of the test substance. The dressing was removed after 48 hours and the skin reactions observed were classified according to the assessment scheme shown above.

Challenge:
The challenge application was carried out on control and test group animals 2 weeks (corresponding to day 21 of the treatment period) after the dermal application. Areas of 5 cm x 5 cm on the flanks of the animals were shorn 24 hours before application. Duhring chambers (0.4 cm) were filled with soaked patches of 2 cm x 2 cm size with the chosen concentration of the test substance in vehicle and were laid on the left flank and fixed in the described manner. On the right flank the vehicle only was applied in the same way.
After 24 hours the dressing was removed. After a further 21 hours after removal of the dressing the skin was shorn. Three hours later the skin reaction was assessed according to the assessment scheme described above. This assessment was repeated after a further 24 hours.
All animals were finally weighed on day 24 of the treatment period.

Positive control substance(s):

yes

Remarks:

2-Mercaptobenzothiazole 97%

Results and discussion

Positive control results:

Sensitisation rate of 100% (10 of 10 animals positive)

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

1% (W/W) concentration of the test substance in vehicle

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

slight and easily detectable reddening and swelling

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1% (W/W) concentration of the test substance in vehicle. No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: slight and easily detectable reddening and swelling.

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

1% (W/W) concentration of the test substance in vehicle

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no visible skin reaction

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 1% (W/W) concentration of the test substance in vehicle. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no visible skin reaction.

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1% (W/W) concentration of the test substance in vehicle

No. with + reactions:

6

Total no. in group:

10

Clinical observations:

slight and easily detectable reddening and swelling

Remarks on result:

other: see Remark

Remarks:

Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1% (W/W) concentration of the test substance in vehicle. No with. + reactions: 6.0. Total no. in groups: 10.0. Clinical observations: slight and easily detectable reddening and swelling.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

1% (W/W) concentration of the test substance in vehicle

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

no visible skin reaction

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 1% (W/W) concentration of the test substance in vehicle. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no visible skin reaction.

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

2-Mercaptobenzothiazole

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 2-Mercaptobenzothiazole. No with. + reactions: 10.0. Total no. in groups: 10.0.

Any other information on results incl. tables

Results:

Symptoms:

Symptoms of systemic toxicity caused by the test substance were not observed.

Body Weight:

Body weight development was positive and within normal range.

Pilot Experiment:

The intradermal application of the 5 %, 3.5 % and 2 % (w/w) solutions of the test substance in vehicle showed a strong skin reaction and moderate skin reaction was observed with the 0.5 % and 0.1 % (w/w) concentration. Slight skin reaction was observed with the 0.05 % (w/w) concentration.

Dermal application of the 100 %, 75 %, 50 % and 25 % (w/w) concentration of the test substance in vehicle without Duhring Chambers led to necrosis of the treated skin sites. The test substance preparation of 5 % (w/w) concentration showed a very slight reaction. Application of the 1 %, 0.5 % and 0.1 % (w/w) concentration in vehicle showed no reaction on the skin.

Dermal application with Duhring Chambers with the 1 % and 0.5 % (w/w) concentrations of the test substance in vehicle showed no reactions on the skin.

Two animals of the pilot experiment have been sacrificed by an overdose of a narcotic substance shortly after observation of the strong skin reactions.

Induction:

The intradermal and the epicutaneous induction resulted in slight skin reactions in a various number of animals indicating that the chosen test substance concentrations led to slight irritation.

In the control group animals no vehicle related skin reaction was observed after the application of the vehicle. The results on each animal are presented in the following table:

	Intardermal induction		Epicutan induction
	Erythema	Edema	Erythema	Edema
Test group animals	1/1/0/1/1/0/0/1/1/1	0/0/0/0/0/0/0/0/0/0	1/1/0/0/1/1/1/0/1/0	0/0/0/0/0/0/0/0/0/0
Control group animals	0/0/0/0/0	0/0/0/0/0	0/0/0/0/0	0/0/0/0/0

	First reading after 24h		Second reading after 48h
	Erythema	Edema	Erythema	Edema
Test group animals	0/1/0/0/2/2/2/0/1/0	0/1/0/0/2/1/1/0/0/0	0/1/0/0/1/2/2/1/1/0	0/1/0/0/1/1/1/0/0/0
Control group animals	0/0/0/0/0	0/0/0/0/0	0/0/0/0/0	0/0/0/0/0

Applicant's summary and conclusion

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: other: CLP, EU GHS (Regulation (EC) No 1272/2008)

Conclusions:

The test substance Amides, C6-C18, N-[3-(dimethylamino)propyl] (a.i. 98.4%) has to be regarded as sensitising under the applied test conditions when exposed to the skin of experimental animals.

Executive summary:

In a sensitization study with Amides, C6-C18, N-[3-(dimethylamino)propyl] (a.i. 98.4%) in sesame oil, young adult Dunkin Hartley guinea pig were tested using the maximization test method according to OECD Guideline 406, 17 July, 1992 and EU Method B.6, 30 May 2008.

The maximum compatible doses which led to slight irritation after intradermal and dermal application as well as the subirritative dose for the challenge were determined in a pilot experiment. Sesame oil was used as vehicle during induction and challenge. Based on the results of the pilot study, for the intradermal and epicutaneous induction procedure test substance concentrations of 0.05 % and 5 % were used, respectively. The test article concentration for the challenge procedure was 1 %. In the challenge visible changes of the skin like slight and easily detectable erythema (= grading 1 and 2) and slight to easily recognizable swelling (= grading 1 and 2) were observed in five out of ten test group animals 24 h after patch removal; 48 h after the challenge these skin reactions were still observed and one additional animal showed slight erythema (= grading 1). The five control animals were free of skin reactions. Thus, the sensitisation rate was 60 %. In the remaining four test group animals and in all control group animals no visible skin reactions were noted at any time (= grading 0).

In this study the test substance has to be regarded as sensitising under the applied test conditions when exposed to the skin of experimental animals.