Amines, di-C16-18-alkylmethyl

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 10-03-1990 to 11-23-1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1991

Report date:

1991

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

Reliability scoring based on 1981 guideline for test n°406

Deviations:

yes

Remarks:

: no data reported on the positive control in the report

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The LLNA method has been proven to give false positives for this type of substances. This test was conducted pre-LLNA. There is a viable GPMT conducted according to GLP and OECD guidelines available.

Test material

Test material information

Constituent 1

Reference substance name:

N-C16-18 (even numbered)-alkyl-N-methyl, C16-18 (even numbered)-alkyl-1-amine

EC Number:

627-132-7

Cas Number:

1227096-04-9

Molecular formula:

No molecular formula

IUPAC Name:

N-C16-18 (even numbered)-alkyl-N-methyl, C16-18 (even numbered)-alkyl-1-amine

Test material form:

solid

Remarks:

waxy

Details on test material:

- Chemical name: N-decyl-N-methyldecan-1-amine
- EC number: 627-132-7

“Based on the qualitative and quantitative information on the composition, the sample used are representative of the boundary composition shared and agree by each registrant.”

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Olac Limited, Bicester, Oxfordshire, England
- Age before acclimatation period: 4-6 weeks old
- Weight at study initiation: 330-451 g
- Housing: in stainless steel cages with grid floors, 5 animals of the same sexe in each cage
- Diet : ad libitum (samples of diet were analysed to detect contamination by PCB and chlorinated hydrocarbons)
- Water: ad libitum
- Acclimation period: yes but no more precision

ENVIRONMENTAL CONDITIONS
- Temperature : 18-20 °C
- Humidity: below 40 %
- Air changes : 15 per hour
- Photoperiod (hrs dark / hrs light):12/12 (no source of natural light)

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

paraffin oil

Induction 2

Concentration / amount:

- Induction exposure:
intrademal: 10% w/v Test Substance (TS) in vehicle
occlusive epicutaneous: 10% w/v
- Challenge exposure:
occlusive epicutaneous: 3 % w/v and 0.5 % w/v

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

paraffin oil

Challenge 2

Concentration / amount:

- Induction exposure:
intrademal: 10% w/v Test Substance (TS) in vehicle
occlusive epicutaneous: 10% w/v
- Challenge exposure:
occlusive epicutaneous: 3 % w/v and 0.5 % w/v

No. of animals per dose:

Control group: 10 animals (5 males and 5 females)
Treated group: 20 animals (10 males and 10 females)

Details on study design:

RANGE FINDING TESTS: conducted to define the concentrations to be tested in the main study.

Intradermal exposure:
24 hours before treatment: dorsal region clipped.
Intradermal administration of 0.1 mL of the test substance (TS) at increasing concentrations.
Evaluation of the potential cutaneous reactions 24 and 48 hours and approximately 168 hours after injection.

Epicutaneous exposure:
24 hours before treatment: dorsal region clipped.
0.4 mL of each concentration applied to a gauze patch of approximately 4 cm2 for 48 hours by occlusive dressing (4 concentrations per animal).
Evaluation of the potential cutaneous reactions 24 and 48 hours after patch removal.


MAIN STUDY

A. INDUCTION EXPOSURE
- No. of exposures: 1 intradermal, 1 epicutaneous
- Exposure period: epicutaneous: 48 hours

-> TEST GROUPS:
Intradermal exposure
Three injections of 0.1 mL were injected into each side of the animal as follows:
. Freund's complete adjuvant (FCA) diluted to 50% with distilled water
. TS at 10% w/v in vehicle
. TS at 5% w/v in a 50/50 (v/v) mixture of FCA and distilled water

Epicutaneous exposure
Application of 0.6 mL of TSat 10% w/v in paraffin oil applied to the scapular region and held in place for 48 hours using an occlusive dressing.

-> CONTROL GROUP:
Intradermal exposure
Three injections of 0.1 mL were injected into each side of the animal as follows:
. Freund's complete adjuvant (FCA) diluted to 50% with distilled water
. vehicle
. A mixture 50/50 (v/v) FCA diluted to 50% with distilled water, and vehicle

Epicutaneous exposure
Application of 0.6 mL of vehicle applied to the scapular region and held in place for 48 hours using an ocular dressing.

- Site:
Intradermal exposure
6 injections in the clipped area (4 x 2 cm) in the scapular region

Epicutaneous exposure
4 x 2 cmarea over the scapulae

- Frequency of applications:
One intradermal injection and one epicutaneous application 8 days after on the same site.

- Duration:
Epicutaneous exposure: 48 hours


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 22
- Exposure period: 24 hours

-> TEST GROUPS:
0.03 mL of the TS at 3% w/v in vehicule to one site and 0.03 mL of the TS at 0.5% w/v in vehicule to a second site on the posterior right flank (occlusiv e epicutaneous application)
0.03 mL of the vehicle on the posterior left flank (occlusive epicutaneous application)

-> CONTROL GROUPS:
Same treatment as test group
- Site: posterior right and left flanks
- Evaluation (hr after challenge): 24 and 48 hours after removal of the dressing

Challenge controls:

Vehicle was applied at the challenge application on the posterior left flank of both test and control groups of animals.

Positive control substance(s):

not specified

Remarks:

Positive controls were conducted in two other studies within the 6 month validation period on Armoblen 600 and NAPAA. See section 7.12 "Addtional toxicological information" for details.

Results and discussion

Positive control results:

No data generated during this test. The study is however within 6 months of two other valid positive controls being conducted during tests on diffrent substances following the same guideline at the same testing facility. Therefore in light of this evidence the animals in this study are considered to have valid positive reactions to appropriate postive control test material. Inclusion of positive controls within the study itself is therefore considered not necessary. See 7.12 "Addtional toxicological infromation" for details on positive controls.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

challenged with Armeen M2HT 3%

No. with + reactions:

0

Total no. in group:

10

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

Challenged with Armeen M2HT 3%

No. with + reactions:

0

Total no. in group:

10

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Induced with Armeen M2HT and challenged with Armeen M2HT 3%

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

slight erythema

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Induced with Armeen M2HT and challenged with Armeen M2HT 3%

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

slight erythema

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

challenged with Armeen M2HT 0.5 %

No. with + reactions:

0

Total no. in group:

10

Results 6

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

challenged with Armeen M2HT 0.5 %

No. with + reactions:

0

Total no. in group:

10

Results 7

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

Induced with Armeen M2HT and challenged with Armeen M2HT 0.5 %

No. with + reactions:

3

Total no. in group:

20

Clinical observations:

slight erythema

Results 8

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Induced with Armeen M2HT and challenged with Armeen M2HT 0.5 %

No. with + reactions:

1

Total no. in group:

20

Clinical observations:

slight erythema

Results 9

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

challenged with paraffin oil

No. with + reactions:

0

Total no. in group:

10

Results 10

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

challenged with paraffin oil

No. with + reactions:

0

Total no. in group:

10

Results 11

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

induced with Armeen M2HT and challenged with paraffin oil

No. with + reactions:

0

Total no. in group:

20

Results 12

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

Induced with Armeen M2HT and challenged with paraffin oil

No. with + reactions:

0

Total no. in group:

20

Any other information on results incl. tables

CHALLENGE PHASE: individual dermal responses after removal of occlusive dressings

Group	Sexe	Animal	Topical application of 0.03 ml 3 %w/v Armeen in paraffin oil		Topical application of 0.03 ml 0.5 %w/v Armeen in paraffin oil		Topical application of 0.03 ml paraffin oil
			24 hours after removal of dressing	48 hours after removal of dressing	24 hours after removal of dressing	48 hours after removal of dressing	24 hours after removal of dressing	48 hours after removal of dressing
Control	M	601	0	0	0	0	0	0
		602	0	0	+-	+-	0	0
		603	0	0	0	0	0	0
		604	0	0	0	0	+-	+-
		605	0	0	0	0	0	0
Control	F	606	0	0	0	0	0	0
		607	0	0	0	0	0	0
		608	0	0	0	0	+-	+-
		609	0	0	0	0	0	0
		610	0	0	0	0	0	0
Treated	M	611	+-	0	0	0	0	0
		612	+-	0	0	0	0	0
		613	0	0	0	+-	0	0
		614	+-	+-	+-	+-	+-	+-
		615	+-	+-	+-	0	+-	0
		616	0	0	0	+-	0	0
		617	+-	0	0	0	+-	0
		618	0	0	0	0	+-	+-
		619	+-	0	+-	0	0	0
		620	0	+-	0	+-	+-	0
Treated	F	621	0	0	0	0	0	0
		622	0	0	0	0	0	0
		623	0	0	0	0	+-	+-
		624	+-	0	0	0	0	0
		625	+-	1	0	1	+-	+-
		626	+-	+-	+-	1	0	0
		627	+-	0	+-	0	+-	+-
		628	1	0	0	+-	0	0
		629	+-	0	0	0	+-	0
		630	0	0	0	1	0	0

0: No response

+-: Barely perceptible erythema

1: Slight erythema

2: Moderate erythema

3: Severe erythema

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

In the treated group, following cutaneous reactions were noted:
-Challenge application of 3 % w/v Armeen M2HT in paraffin oil caused slight erythema in 1 out of 20 animals in the treated group after the 24 and the 48-hour readings and no cutaneous reactions in the control group.
-Challenge application of 0.5 % w/v Armeen M2HT in paraffin oil caused slight erythema in 3 and 1 out of 20 treated animals after the 24 and 48-hour readings respectively. No cutaneous reactions were observed in the control group.
-Challenge application of paraffin oil alone caused caused no significant cutaneous reaction.

Executive summary:

The potential of Armeen M2HT to induce delayed contact hypersensitivity was assessed in guinea pigs according to the OECD guideline 406 and conducted in compliance with the principles of Good Laboratory Practice regulations.

Positive controls were not included in the study though guideline equivalent GPMT studies on Armoblen 600 & NAPAA were conducted with positive controls that were successful 6 months prior to and 3 months after respectively of this test in the same contract laboratory. This is consistent with the OECD guideline need for reliability checks in the place of positive controls. Further information on these studies can be found in section 7.12 "Additional toxicological information".

The induction phase has been realized both by intradermal route on day 1 (Armeen M2HT 10 % w/v in paraffin oil) and by cutaneous route on day 8 (Armeen M2HT 10% w/v in paraffin oil ) in 2 groups of guinea pigs: 5 males and 5 females for control group and 10 males and 10 females for treated group. The challenge phase was realized on day 22 by cutaneous application of Armeen M2HT 3% and Armeen M2HT 0.5% w/v in paraffin oil on two sites on the right flank (vehicle on the left flank). the cutaneous reactions were scored 24 and 48 hours after the challenge phase.

 

After the challenge application with 3 % w/v Armeen 2HT in paraffin oil, a slight erythema was observed in 1/ 20 animals of the treated group at the 24 and 48-hour readings. No cutaneous reactions were recorded in the control group.

 

After the challenge application with 0.5 % w/v Armeen 2HT in paraffin oil, a slight erythema was observed in 3 and 1 out the 20 animals of the treated group at the 24 and 48-hour readings respectively.

 

The persistent cutaneous reactions observed in 4/20 animals of the treated group after the challenge application may be attributable to delayed contact hypersensitivity but as the cutaneous reactions were more confined to the 24 hour examination, they were more consistent with irritation than delayed contact hypersensitivity.

 

Therefore, the cutaneous reactions observed in 4/20 animals (20%) of the treated groups were considered as irritative response and the substance was not considered as a skin sensitiser.