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

Endpoint summary

Description of key information

Guinea pig maximisation test: Not sensitising (OECD guideline 406 / EU method B.6; GLP), Induction: intradermal with 0.2% test substance concentration, epidermal with 10% test substance concentration; Challenge: topical with 1% test substance concentration; discrete or patchy erythema (grade 1) in 1/10 animals

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

May 2008

Deviations:

no

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.

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%

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

Route:

intradermal and epicutaneous

Vehicle:

water

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%

Route:

epicutaneous, occlusive

Vehicle:

water

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

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.

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

20% Alpha- Hexylcinnamaldehyde

No. with + reactions:

7

Total no. in group:

10

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.

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.

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.

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.

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

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

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. The positive control α-hexyl cinnamic aldehyde produced a sensitisation rate of 60%.

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. According to the Magnusson and Kligman grading scale, discrete or patchy erythema (grade 1) as shown by one animal is considered as a positive reaction.

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.

 

There is supporting information available from the two structurally related substances MDEA-Esterquat C16-18 and C18 unsatd. and MDIPA Esterquat C18 unsatd. which also were not sensitising in dermal sensitisation studies according to OECD guideline 406 using the methods of Magnusson & Kligman and Bühler.

 

In a dermal sensitisation study according to OECD guideline 406 (17 July 1992) and EU method B.6 (30 May 2008) with MDIPA Esterquat C18 unsatd. (100% a.i.) 10 young adult Dunkin Hartley, HsdPoc:DH guinea pigs were tested using the method of method of Magnusson & Kligman (Guinea Pig Maximisation Test). The positive controlα-hexyl cinnamic aldehyde produced a sensitisation rate of 40%. Test concentrations were selected based on the results of a pilot study: intradermal induction 0.5% in sesame oil; epicutaneous induction 25% on sesame oil; challenge 1% in sesame oil. No pretreatment to create local skin irritation was necessary before epicutaneous induction as the selected concentration was slightly irritating. After challenge no visible changes of the treated skin sites were observed in any of the test and control group animals 24 and 48 h after patch removal (= grade 0).

In two dermal sensitisation studies according to OECD Guideline 406 (12 May 1981) with MDEA-Esterquat C16-18 and C18 unsatd. (80 % a. i.) in isopropanol, Pirbright-Hartley guinea pigs (10 male and 10 female in each study) were tested using the method of Bühler.

In the first study no skin alterations were observed during the induction period with 15 % test substance in 96 % ethanol. At challenge with 15 % test substance in acetone, slight erythema was observed in 1/20 test animals. No skin reactions were observed in the control animals. Both test and control animals were re-challenged with 15 % and 7.5 % MDEA-Esterquat C16-18 and C18 unsatd. in acetone. No skin responses were observed upon re-challenge.

In the second study no skin alterations were observed during the induction period with 15 % test substance in 96 % ethanol. 48 hours after challenge with 7.5 % test article, partly slight, patchy erythema (score 0.5) was observed in the test group in 3/20 animals. No skin reactions were observed in the control animals. There was a very slight intergroup difference in the skin alterations between test- and control group.

MDEA-Esterquat C16-18 and C18 unsatd. did not produce skin sensitisation in human repeat insult patch tests as well.

 

There is no data gap for skin sensitisation. Although no human data are available for MDIPA-Esterquat C16-18 and C18 unsatd. there is no reason to believe that results obtained in guinea pigs would not be applicable to humans.

 

 

Justification for the Guinea Pig Maximisation Test (GPMT)

The test substance MDIPA-Esterquat C16-18 and C18 unsatd. is a surface active chemical with the alkyl moieties being predominantly composed of C16-18 and C18 usaturated residues.

In recently published articles in peer reviewed journals (see reference list below, and publications cited in the revised OECD guideline 429 (July 2010)) it is clearly demonstrated that for the realistic assessment of the skin sensitization potential of surfactants the LLNA (OECD 429) is much less suitable than the Magnusson & Kligman method (OECD 406) and could lead to confounding results.

In particular, the OECD 429 guideline specifies that “In addition, test substance classes or substances containing functional groups shown to act as potential confounders (Basketter et al., 2009) may necessitate the use of guinea pig tests”.

Consequently, in the evaluation of such surface active substances for sensitising properties, the LLNA is not an appropriate assay and would not represent an optimum use of test animals. The mechanism underlying confounding or false positive result is not fully understood, but it may be that an unspecific, non-immunologically triggered mechanism may be the cause of an increased lymphocyte proliferation.

In contrast to the GPMT, the LLNA focuses on the induction process of skin sensitisation (i.e. lymphocyte proliferation), and does not capture the process of elicitation which checks if the organism had actually been sensitized or not. The design of the LLNA does not allow to assess whether lymphocyte proliferation is a specific response or unspecific (false positive) response.

 

References:

Kreiling, R et al., Food and Chemical Toxicology 46 (2008): 1896-1904: Comparison of the skin sensitizing potential of unsaturated compounds as assessed by the local lymph node assay (LLNA) and the guinea pig maximization test (GPMT)

 

Basketter, D et al., Regulatory Toxicology and Pharmacology 55 (2009): 90-96: Application of a weight of evidence approach to assessing discordant sensitisation data sets: Implications for REACH

 

Garcia, C et al., Regulatory Toxicology and Pharmacology 58 (2010): 301-307: Comparative testing for the identification of skin sensitizing potentials of nonionic sugar lipid surfactants

 

Ball, N et al., Regulatory Toxicology and Pharmacology 60 (2011): 389-400: Evaluating the sensitizing 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

 

 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

MDIPA-Esterquat C16-18 and C18 unsatd. did not show any skin sensitising properties in guinea pigs. There is no information available for respiratory sensitisation. Therefore, there is a data gap in this respect. However, the data gap cannot be fulfilled with experimental data, since there is no internationally accepted animal model for respiratory sensitisation. In case human data for respiratory sensitisation emerges, this will be taken into account.

Justification for classification or non-classification

Based on relevant, reliable and adequate data, MDIPA-Esterquat C16-18 and C18 unsatd. does not need to be classified and labelled according to the CLP Regulation (EC) No 1272/2008 with respect to skin sensitisation.