1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C16-18 (even numbered) and C18 unsatd., Me sulfates (salts)

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:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

May 2008

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

adopted 17th July 1992

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A valid GPMT conducted according to guideline is available, which is reliable without restrictions and adequate for classification and labelling purposes. Potency estimation is not mandatory when existing guideline and GLP conforming data are available, which were conducted before the new annex of the REACH Regulation entered into force. Moreover, no indication for skin sensitisation was observed in this study, thus, no dose response information is needed. For this reason and for reasons of animal welfare no additional LLNA was conducted.

Test material

Test material information

Constituent 1

Reference substance name:

1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C16-18 (even numbered) and C18 unsatd., Me sulfates (salts)

EC Number:

941-174-6

IUPAC Name:

1-Propanaminium, 2-hydroxy-N-(2-hydroxypropyl)-N,N-dimethyl-, esters with fatty acids, C16-18 (even numbered) and C18 unsatd., Me sulfates (salts)

Test material form:

other: white solid wax

Details on test material:

- Name of test material: MDIPA-Esterquat C16-18 and C18 unsatd.
- Physical state: waxy solid
- Analytical purity: 100%

Specific details on test material used for the study:

- Name of test material: MDIPA-Esterquat C16-18 and C18 unsatd.
- Physical state: solid
- Analytical purity: 100%

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 France, L’arbresle Cedex, France
- Age at study initiation: approx. 8 weeks
- Housing: Group housing of maximally 5 animals per labeled Noryl cage (Tecniplast; 74 cm x 54 cm x 25 cm height) containing sterilized sawdust as bedding material (Litalabo, S.P.P.S., Argenteuil, France) and shelters (CS3B02A Play tunnels (90 mm x 5 mm x 125 mm), Datesand, Manchester, UK) as cage enrichment
- Diet (e.g. ad libitum): Complete maintenance diet for guinea pigs (SSNIFF® Spezialdiäten GmbH, Soest, Germany) ad libitum; hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least twice a week
- Water (e.g. ad libitum): tap weater, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-24
- Humidity (%): 40-70
- Air changes (per hr ):approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

water

Induction 2

Concentration / amount:

Preliminary irritation study
Intradermal: 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05%
Epidermal: 50, 40, 30, 20, 10, 5, 2, 1%

Main study
Intradermal induction: 0.2%
Epidermal induction: 10%
Challenge: 1%

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

water

Challenge 2

Concentration / amount:

Preliminary irritation study
Intradermal: 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05%
Epidermal: 50, 40, 30, 20, 10, 5, 2, 1%

Main study
Intradermal induction: 0.2%
Epidermal induction: 10%
Challenge: 1%

No. of animals per dose:

Experimental group: 10 females
Control group: 5 females

Details on study design:

RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select test substance concentrations to be used in the main Study. The selection of concentrations was based on the following criteria:
-The concentrations are well-tolerated systemically by the animals.
-For the induction exposures: the highest possible concentration that produced mild to moderate irritation (grades 2 - 3).
-For challenge exposure: the maximum non-irritant concentration.
Intradermal: 10, 5, 2, 1, 0.5, 0.2, 0.1, 0.05%
Epidermal: 50, 40, 30, 20, 10, 5, 2, 1%

Based on the results, the test substance concentrations selected for the main study were a 0.2% concentration for the intradermal induction and a 10% concentration for the epidermal induction exposure. A 1% test substance concentration was selected for the challenge phase.

MAIN STUDY
A. INDUCTION EXPOSURE
intradermal
- Test groups:
A) A 1:1 w/w mixture of Freunds Complete Adjuvant with water for injection
B) The test substance at a 0.2% concentration
C) A 1:1 w/w mixture of the test substance, at twice the concentration used in (B) and Freunds Complete Adjuvant
- Control group: animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered
- Site: scapular region
- Frequency of applications: 1x

epidermal
- No. of exposures: 1
- Exposure period: 48 h
- Test groups: 0.5 mL of a 10% test substance concentration
- Control group: 0.5 mL vehicle
- Site: scapular area
- Frequency of applications: 1x


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: 24, 25
- Exposure period: 24 h
- Test groups: 0.1 mL 1% test substance, 0.1 mL vehicle
- Control group: 0.1 mL 1% test substance, 0.1 mL vehicle
- Site: flank
- Evaluation (hr after challenge): 24, 48 h

Positive control substance(s):

yes

Remarks:

Alpha- Hexylcinnamaldehyde

Results and discussion

Positive control results:

A reliability check with the positive control substance Alpha- Hexylcinnamaldehyde is carried out at regular intervals (last check Jan/Feb 2013) to check the sensitivity of the test system.
Concentrations selected for this study were:
Intradermal induction: A 20% solution in water (w/w).
Epidermal induction: undiluted.
Challenge: a 20% solution in water (w/w).
The positive control did induce skin sensitisation.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

20% Alpha- Hexylcinnamaldehyde

No. with + reactions:

7

Total no. in group:

10

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

1%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1%

No. with + reactions:

1

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 1%. No with. + reactions: 1.0. Total no. in groups: 10.0.

Results 4

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

1%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 5

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

1%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 1%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

Preliminary irritation study

SKIN REACTIONS AFTER INTRADERMAL INJECTION

Animal	Conc %	24 hours after injection		48 hours after injection
number		Erythema	Necrosis	Erythema	Necrosis
		(grade)	(mm)	(grade)	(mm)
48	10		5		6
	5		4		5
49	2		3		4
	1		2		3
45	0.5	3			1
	0.2	2		1
50	0.1	2		1
	0.05	1		1

 

SKIN REACTIONS AFTER EPIDERMAL EXPOSURE

Animal	Conc. %	24 hours after exposure		48 hours after exposure
number		Erythema	Oedema	Erythema	Oedema
		(grade)	(grade)	(grade)	(grade)
46	10	0	0	0	0
	5	0	0	0	0
47	10	0	0	0	0
	5	0	0	0	0
48	2	0	0	0	0
	1	0	0	0	0
49	2	0	0	0	0
	1	0	0	0	0
45	50	N	1	N	2
	40	N	1	N	2
50	30	N	1	N	2
	20	N	0	N	1

Main Study

Induction readings

Animal number	Intradermal injection (Day 3)						Epidermal exposure (Day 10)
	A		B		D		D
Control group	E	N	E	N	E	N	Erythema	Oedema
31	3		0		3		0	0
32	3		1		2		0	0
33	3		1		3		0	0
34	3		0		3		0	0
35	3		0		3		0	0
Experimental group	E	N	E	N	E	N
36	3		2		3		3	0
37	3		3		3		3	0
38	3		2		2		3	0
39	3		2		3		3	0
40	3		3		3		3	0
41	3		3		3		3	0
42	3		3		3		3	0
43	3		3		3		3	0
44	3		3		3		3	0
45	3		3		3		3	0

 

A. 1:1 Mixture of Freund’s Complete Adjuvant and water for injection.

B. A 0.2% test substance concentration (Experimental); vehicle (Control).

C. 1:1 Mixture of Freund’s Complete Adjuvant and a 0.4% concentration (Experimental) or vehicle (Control).

D. A 10% test substance concentration (Experimental); vehicle (Control).

 

Skin effects intradermal injections:

E. Erythema (grade)

N. Signs of necrosis (mm in diameter)

Challenge readings

Animal number	DAY 24		DAY 25
	1%^#	Vehicle*	1%^#	Vehicle*
Control group
1	0	0	0	0
2	0	0	0	0
3	0	0	0	0
4	0	0	0	0
5	0	0	0	0
Experimental group
36	0	0	0	0
37	0	0	0	0
38	0	0	0	0
39	0	0	0	0
40	0	0	0	0
41	0	0	0	0
42	0	0	1p	0
43	0	0	0	0
44	0	0	0p	0
51	0	0	0	0

 

#. Test substance concentration.

 *. Water (Elix)

p. Scaliness

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In this Guinea Pig Maximisation Test, MDIPA-Esterquat C16-18 and C18 unsatd. was not sensitising.

Executive summary:

In a dermal sensitisation study according to OECD Guideline 406 (adopted 17th July 1992) and EU method B.6 (May 2008) with MDIPA-Esterquat C16-18 and C18 unsatd. (100% a.i.) in water, young adult female Dunkin-Hartley guinea pigs (10 in test group, 5 in control group) were tested using the method of Magnusson and Kligman. Alpha-Hexylcinnamaldehyde was used as positive control.

Based on the results of a preliminary study, the test substance concentrations selected for the main study were a 0.2% concentration for the intradermal induction and a 10% concentration for the epidermal induction exposure. A 1% test substance concentration was selected for the challenge phase.

After epidermal induction all animals of the experimental group showed signs of irritation. Following a challenge exposure to a 1% test substance concentration, one experimental animal showed discrete or patchy erythema, with scaliness, and one experimental animal showed scaliness only, both at 48 hours after exposure. No skin reactions were evident in the control animals and all other experimental animals. The sensitisation rate was 10%.

According to CLP, EU GHS (Regulation (EC) No 1272/2008), a response of at least 30% of the test animals of an adjuvant type guinea pig test method for skin sensitisation is considered as positive.

MDIPA-Esterquat C16-18 and C18 unsatd. is not a dermal sensitiser in this study.