C8-18 alkylampho(di)propionates

Endpoint summary

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

Study period:

November 20, 2012 - April 5, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study has been performed in accordance with OECD 406 (1992), EU Method B.6 (2008), EPA OPPTS 870.2600 (2003) and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

(1992)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

(2008)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

(2003)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nohsan, Notification No. 8147, April 2011; including the most recent partial revisions

Deviations:

no

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The Maximization test was selected as preferred alternative since the Local Lymph Node Assay has shown to provide false positive results for surfactants.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Kisslegg, Germany
- Age at study initiation: Young adult animals (approx. 5 weeks old) were selected
- Weight at study initiation: 287-353 g (for the 15 animals used in the main study)
- Housing: Group housing of maximally 5 animals per labeled Noryl cage
- Diet: Complete maintenance diet for guinea pigs (SSNIFF® Spezialdiäten GmbH, Soest, Germany); ad libitum
- Water: Free access to tap water
- Acclimation period: at least 5 days

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

IN-LIFE DATES: From: 20 Nov 2012 To: 21 Dec 2012

Route:

intradermal and epicutaneous

Vehicle:

water

Concentration / amount:

See section "Details on study design"

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

See section "Details on study design"

No. of animals per dose:

Test animals: 10
Control animals: 5

Details on study design:

RANGE FINDING TESTS
- A total of 5 animals were used
- The animals were between 4 and 9 weeks old

- For the intradermal induction exposure:
Initially, a series of four test substance concentrations was used (0.1, 0.2, 0.5 and 1%). Each of two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The resulting dermal reactions were assessed 24 and 48 hours after treatment. Based on the results in the initially treated animals, one additional animal was treated in a similar manner with two higher concentrations (2 and 5%) at a later stage. Both of these concentrations induced necrosis at the sites of injection.
- Result (intradermal induction): the 1% concentration showed to be minimal irritating and was used for the main test. Well-defined erythema after 24 hours and slight erythema after 48 hours was observed at this concentration.

- For the epicutaneous induction and challenge exposure (occlusive):
A series of four test substance concentrations was used (10, 20, 50 and undiluted, i.e., 100%). Two different concentrations were applied (0.5 mL each) per animal to the clipped flank, using Metalline patches# (2x3 cm) mounted on Medical tape# which were held in place with Micropore tape# and subsequently Coban elastic bandage#. The animals receiving intradermal injections (0.1, 0.2, 0.5 and 1%) were treated with the lowest concentrations and two further animals with the highest concentrations. After 24 hours, the dressing was removed and the skin cleaned of residual test substance using water. The treated skin areas were assessed for irritation 24 and 48 hours after exposure.
- Result (epidermal application): As no signs of erythema nor oedema were observed to the highest test substance concentration epidermally tested, 100% was chosen as the epidermal induction exposure and as the non-irritating challenge concentration, to use in the main test. One of the 2 animals applied with 100% did show scaliness (desquamation) at 24 hours after exposure. In accordance with the guidelines, the test site of all animals was treated with 10% SDS approximately 24 hours before the epidermal induction in the main study, to provoke a mild inflammatory reaction.

MAIN STUDY
Test animals: 10
Control animals: 5

A. INDUCTION EXPOSURE (test animals)
- No. of exposures: 2

1) Intradermal injections on day 1:
- Concentration: 1% in water
- Site: scapular region (clipped)
Three pairs of intradermal injections:
1) 0.1 mL: FCA (50% in water)
2) 0.1 mL: 1% of the substance in water
3) 0.1 mL: a 1:1 w/w mixture of the substance, at twice the concentration used in 2) and Freunds' Complete Adjuvant
- Readings: On day 3 the dermal reactions caused by the intradermal injections were assessed for irritation

On day 7, the scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium-dodecyl-sulfate (SDS, Boom, Meppel, The Netherlands) in vaseline using a spatula. This concentration of SDS provokes a mild inflammatory reaction.

2)Topical application on day 8:
The 10% SDS treated area between the injection sites was treated with 0.5 mL of a 100% test substance concentration using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage.
The dressing was removed after 48 hours exposure, the skin cleaned of residual test substance using water and the dermal reactions caused by the epidermal exposure were assessed for irritation (on day 10).

INDUCTION (control animals)
The control animals were treated as described for the experimental animals except that, instead of the test substance, vehicle alone was administered.

B. CHALLENGE EXPOSURE (all animals)
One flank of all animals was clipped and treated by epidermal application of a 100% test substance concentration and the vehicle (0.1 mL each), using Patch Test Plasters (Curatest®, Lohmann, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Coban elastic bandage. The dressing was removed after 24 hours exposure and the skin cleaned of residual test substance and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.

Positive control substance(s):

yes

Remarks:

(a reliability check is carried out at regular intervals (<6 months) to check the sensitivity of the test system)

Positive control results:

The latest reliability check was carried out in May/June 2012 using Alpha-Hexylcinnamaldehyde. In this study, a sensitisation rate of 80% was found, showing that the test system is reliable.

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100%

No. with + reactions:

4

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 100%. No with. + reactions: 4.0. Total no. in groups: 10.0.

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

100%

No. with + reactions:

4

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 100%. No with. + reactions: 4.0. Total no. in groups: 10.0.

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Reading:

2nd reading

Hours after challenge:

72

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: negative control. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 5.0.

READINGS (Main study)

Animal number	Intradermal injection (Day 3)						Epidermal exposure (Day 10)		Challenge
	A		B		C		D	D	Day 23		Day 24
Control	E	N	E	N	E	N	Erythema	Oedema	100%	Water	100%	Water
51	3		0		2		0	0	0	0	0	0
52	3		1		2		0	0	0	0	0	0
53	3		0		3		0	0	0	0	0	0
54	3		0		3		0	0	0	0	0	0
55	3		0		2		1	0	0	0	0	0
Experimental
56	3		0		-	2	1	0	1	0	1p	0
57	3		0		3	-	1	0	0	0	0p	0
58	3		1		3	-	2	0	1	0	1p	0
59	3		1		-	3	1	0	0	0	0	0
60	3		1		-	2	1	0	0	0	0p	0
61	3		1		-	2	1	0	0	0	0	0
62	3		1		3	-	3	0	0	0	0	0
63	3		1		3	-	2	0	0	0	0	0
64	3		1		3	-	1	0	1	0	1p	0
65	3		1		3	-	2	0	1	0	1p	0

 

A: 1:1 Mixture of Freunds' Complete Adjuvant and water for injection.

B: A 1% test substance concentration (Experimental); vehicle (Control).

C: 1:1 Mixture of Freunds' Complete Adjuvant and a 2% concentration (Experimental) or vehicle (Control).

D: A 100% test substance concentration (Experimental); vehicle (Control).

E: Erythema (grade)

N: Signs of necrosis (mm in diameter)

p: Scaliness (desquamation)

 

Other information

- No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study

- Body weights and body weight gain of experimental animals remained in the same range as controls over the study period

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The skin sensitization potential of the substance (a surfactant) was investigated using a GPMT study in accordance with OECD 406 (1992) and according to GLP principles. Based on the results of the study, the substance needs to be classified as a skin sensitiser in accordance with the CLP Regulation (sub-category 1B). The observed sensitization rate was 40%.

Executive summary:

The skin sensitization potential of the substance (a surfactant) was investigated using a GPMT study in accordance with OECD 406 (1992), EU Method B.6 (2008), EPA OPPTS 870.2600 (2003) and according to GLP principles. The Maximization test was selected as preferred alternative since the Local Lymph Node Assay has shown to provide false positive results for surfactants.

Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, ten experimental animals were intradermally injected with a 1% concentration with concurrent intradermal injections containing Freund’s Complete Adjuvant, then epidermally exposed to a 100% concentration. Five control animals were similarly treated, but with vehicle alone (water). Approximately 24 hours before the epidermal induction exposure all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were challenged with a 100% test substance concentration and the vehicle.

Skin reactions of grade 1 accompanied by scaliness (desquamation) were observed in four experimental animals in response to the 100% test substance concentration. Scaliness was also noted for two other experimental animals. No skin reactions were evident in the control animals.

Skin reactions were observed in six (of the ten) experimental animals in response to a 1% intradermal induction dose and subsequent 100% epidermal induction and challenge dose, and consisted of slight erythema and scaliness in four (of the ten) experimental animals and scaliness only in two (of the ten) experimental animals. Scaliness observed as the sole skin reaction in two (of the ten) experimental animals was not considered to be clearly evident of sensitization, since this skin reaction was also noted in the prescreen test in response to the same test substance concentration applied epidermally to untreated animals. Therefore, a sensitization rate of 40 per cent was calculated.

Based on the results of the study, the substance needs to be classified as a skin sensitiser in accordance with the CLP Regulation (sub-category 1B).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Guinea pig maximisation test (OECD 406)

The skin sensitization potential of the substance (a surfactant) was investigated using a GPMT study in accordance with OECD 406 (1992), EU Method B.6 (2008), EPA OPPTS 870.2600 (2003) and according to GLP principles. The Maximization test was selected as preferred alternative since the Local Lymph Node Assay has shown to provide false positive results for surfactants.

Test substance concentrations selected for the main study were based on the results of a preliminary study. In the main study, ten experimental animals were intradermally injected with a 1% concentration with concurrent intradermal injections containing Freund’s Complete Adjuvant, then epidermally exposed to a 100% concentration. Five control animals were similarly treated, but with vehicle alone (water). Approximately 24 hours before the epidermal induction exposure all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were challenged with a 100% test substance concentration and the vehicle.

Skin reactions of grade 1 accompanied by scaliness (desquamation) were observed in four experimental animals in response to the 100% test substance concentration. Scaliness was also noted for two other experimental animals. No skin reactions were evident in the control animals.

Skin reactions were observed in six (of the ten) experimental animals in response to a 1% intradermal induction dose and subsequent 100% epidermal induction and challenge dose, and consisted of slight erythema and scaliness in four (of the ten) experimental animals and scaliness only in two (of the ten) experimental animals. Scaliness observed as the sole skin reaction in two (of the ten) experimental animals was not considered to be clearly evident of sensitization, since this skin reaction was also noted in the prescreen test in response to the same test substance concentration applied epidermally to untreated animals. Therefore, a sensitization rate of 40 per cent was calculated.

Based on the results of the study, the substance needs to be classified as a skin sensitiser in accordance with the CLP Regulation (sub-category 1B).

Other information (incl. HRIPT)

Despite a long history of use, observation of human skin sensitization is limited to one case report in the open literature. Absence of skin sensitization potential for humans is supported by data from a human RIPT. In this RIPT test, the material was diluted to a concentration of 10% w/v in distilled water prior to testing. During induction, the material was applied to the back three times per week for three successive weeks. Sites were covered for 24 h with non-occlusive patches secured with surgical tape. Repeated applications were made to the same test sites. Reactions were scored 48 or 72 h after each induction application according to the Draize scale. The challenge phase was initiated 10 to 15 days after application of the final induction patch. Challenge patches (non-occlusive) were applied for 24 h to new sites on the back; reactions were scored 48 and 96 h later. It was reported that it did not induce skin irritation or sensitization in any of the subjects tested.

LLNA study with a marketed formulation

With a marketed formulation containing the registered substance at 8.2% a LLNA study has been performed in accordance with OECD 429 (2002), EU Method B.42 (2004) and EPA OPPTS 870.2600 (2003). Proper conduct of the LLNA was confirmed via a positive response using 30% α-hexylcinnamaldehyde (HCA), a moderate contact sensitizer, which elicited proliferation that was 9.6 in comparison to vehicle-treated mice. As the formulation elicited proliferative responses with stimulation indices (SI) below 3 at all test concentrations (including undiluted formulation) in comparison to vehicle-treated mice, the formulation did not demonstrate potential to induce dermal sensitization in the mouse LLNA. The results therefore support the conclusion that the registered substance at a concentration of 8.2% or below does not induce skin sensitization.

Migrated from Short description of key information:
The skin sensitization potential of the substance (a surfactant) was investigated using a GPMT study in accordance with OECD 406 (1992) and according to GLP principles. Based on the results of the study, the substance needs to be classified as a skin sensitizer in accordance with the CLP Regulation (sub-category 1B). Despite a long history of use, observation of human skin sensitization is limited to one case report in the open literature. Absence of skin sensitization potential for humans is supported by data from a human RIPT with 10%. Furthermore, in a LLNA assay, a formulation containing the registered substance at 8.2% did not induce sensitization.

Justification for selection of skin sensitisation endpoint:
Key study conducted in compliance with OECD guideline and GLP.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results of the GPMT study, the substance needs to be classified as a skin sensitiser in accordance with the CLP Regulation (sub-category 1B).

At a concentration of 8.2% or below, the substance does not induce skin sensitization, based on the results of the LLNA study with a formulation. This is also supported by the absence of sensitization reactions in a human RIPT study at 10%.