Salts of 2-ethylhexyl phosphate esters with (Z)- octadec-9-enylamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24 October - 27 november 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study conducted in accordance with OECD guidance and GLP. All guideline validity criteria were met.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

July 2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

Commission Regulation (EC) No. 440/2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Salts of 2-ethylhexyl phosphate esters with (Z)- octadec-9-enylamine

EC Number:

949-054-5

Molecular formula:

not applicable, UVCB substance

IUPAC Name:

Salts of 2-ethylhexyl phosphate esters with (Z)- octadec-9-enylamine

Test material form:

liquid: viscous

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands and Envigo RMS (UK) Limited, Oxon, UK
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: The animals were housed in suspended solid floor polypropylene cages furnished with softwood flake bedding.
- Diet (e.g. ad libitum): Free access to food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK) was allowed throughout the study.
- Water (e.g. ad libitum): Free access to mains tap water was allowed throughout the study.
- Acclimation period: ≥ 5 days
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25 ºC
- Humidity (%): RH = 30-70 %
- Air changes (per hr): ≥15/h
- Photoperiod (hrs dark / hrs light): 12:12 h
- IN-LIFE DATES: Not reported

Study design: in vivo (LLNA)

Vehicle:

unchanged (no vehicle)

Remarks:

Acetone: olive oil (4: 1 v/v) was used to prepare 50%, 10%, 5%, 2.5% or 1% dilutions

Concentration:

Prelim = 100 (undiluted), 50, 10, 5, 2.5 and 1 % v/v (in acetone:olive oil 4:1 v/v)
Main test = 2.5, 1 and 0.5 % v/v (in acetone:olive oil v/v)

No. of animals per dose:

Prelim = 1 mouse/ concentration
Main test = 4 mice/ concentration

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Test item formulated in acetone:olive oil 4:1 v/v - homogeneity not confirmed.
- The animals treated with the undiluted test item or the test item at concentrations of 50 % or 10 % v/v in acetone/olive oil 4:1, were humanely killed, on Days 4 or 5, due to the occurrence of clinical signs of toxicity that were considered to approach the moderate severity limit set forth in the UK Home Office Project License. Hunched posture and emaciation were noted in the animals treated with the test item at concentrations of 50 % or 10 % v/v in acetone/olive oil 4:1. Body weight loss was also noted.
A greater than 25 % increase in mean ear thickness was noted in the animal treated with the test item at a concentration of 5 % v/v in acetone/olive oil 4:1.
No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25 % increase in mean ear thickness were noted in the animals treated with the test item at concentrations of 2.5 % or 1% v/v in acetone/olive oil 4:1 .
Based on this information the dose levels selected for the main test were 2.5 %, 1 % and 0.5 % v/v in acetone/olive oil 4:1

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: LSC of pooled lymph node cells 3 days post administration of 3H-methylthymidine. The administration of 3H-methylthymidine was injected into the tail vein of the mice 5 days after the topical application of the test formulation. The stimulation index and EC3 values were determined from the results of the LSC analysis.

TREATMENT PREPARATION AND ADMINISTRATION:

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

N/A

Results and discussion

Positive control results:

A 25 % formulation of α-Hexylcinnamaldehyde, tech., 85% illicited a stimulation index = 5.54, indicative of a positive result for sensitisation, thus, demonstrating the sensitivity and reliability of the test system.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.28

Test group / Remarks:

0.5 % v/v / Negative

Results 2

Key result

Parameter:

SI

Value:

1.85

Test group / Remarks:

1 % v/v / Negative

Results 3

Key result

Parameter:

SI

Value:

4.26

Test group / Remarks:

2.5 % v/v / Positive

Results 4

Key result

Parameter:

EC3

Value:

1.7

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION :

See Table 4

EC3 CALCULATION :

EC3 = 1 + [[(3 - 1.85) / (4.26 - 1.85)] x (2.5 - 1)] = 1.7

CLINICAL OBSERVATIONS:

Prelim test = The animals treated with the undiluted test item or the test item at concentrations of 50% or 10% v/v in acetone/olive oil 4:1, were humanely killed, on Days 4 or 5, due to the occurrence of clinical signs of toxicity that were considered to approach the moderate severity limit set forth in the UK Home Office Project License. Hunched posture and emaciation were noted in the animals treated with the test item at concentrations of 50% or 10% v/v in acetone/olive oil 4:1.
Main test = There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS:

Prelim test = Body weight loss was noted in the 100, 50 and 10 % dose groups.
Main test = Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

Table1       Prelim test – clinical observations, body weight and mortality data

 

Concentration (% v/v) in acetone/olive oil 4:1	Animal number	Body weight (g)		Day
				1		2		3		4	5	6
		Day 1	Day 6	Pre-dose	Post dose	Pre-dose	Post dose	Pre-dose	Post dose
100	S1	20.2	-	0	0	0	0	0	0	0	HEmK (16.5)	-
50	S2	20.3	-	0	0	0	0	0	0	0	HEmK (16.0)	-
10	S3	19.9	-	0	0	0	0	0	0	K (16.3)	-	-
5	S4	19.7	19.2	0	0	0	0	0	0	0	0	0
2.5	S5	19.4	19.2	0	0	0	0	0	0	0	0	0
1	S6	21.4	21.2	0	0	0	0	0	0	0	0	0

0 = no sign of systemic toxicity

Em = emaciation

H = hunched posture

K = animal humanely killed due to occurrence of clinical signs of toxicity that approached the moderate severity level

() = bodyweight at termination

- = no data, animal dead

 

Table 2       Prelim test – local skin irritation

 

Concentration (% v/v) in acetone/olive oil 4:1	Animal number	Local skin irritation
		Day 1		Day 2		Day 3		Day 4		Day 5		Day 6
		Left	Right	Left	Right	Left	Right	Left	Right	Left	Right	Left	Right
100	S1	0	0	0	0	0	0	0	0	0	0	-	-
50	S2	0	0	0	0	0	0	0	0	-	-	-	-
10	S3	0	0	0	0	0	0	0	0	-	-	-	-
5	S4	0	0	0	0	0	0	0	0	0	0	0	0
2.5	S5	0	0	0	0	0	0	0	0	0	0	0	0
1	S6	0	0	0	0	0	0	0	0	0	0	0	0

- no data, animal dead

Table 3       Prelim test – ear thickness

 

Concentration	Animal number	Ear thickness (mm)
		Day 1		Day 3		Day 6
		Pre-dose		Post dose
		Left	Right	Left	Right	Left	Right
100 %	S1	0.22	0.22	0.23	0.22	-	-
Overall thickness (mm)		0.220		0.225		-
Overall mean ear thickness change (%)		N/A		2.273		-
Concentration	Animal number	Ear thickness (mm)
		Day 1		Day 3		Day 6
		Pre-dose		Post dose
		Left	Right	Left	Right	Left	Right
50 %	S2	0.21	0.22	0.24	0.23	-	-
Overall thickness (mm)		0.215		0.235		-
Overall mean ear thickness change (%)		N/A		9.302		-
Concentration	Animal number	Ear thickness (mm)
		Day 1		Day 3		Day 6
		Pre-dose		Post dose
		Left	Right	Left	Right	Left	Right
10 %	S3	0.21	0.21	0.22	0.21	-	-
Overall thickness (mm)		0.210		0.215		-
Overall mean ear thickness change (%)		N/A		2.381		-
Concentration	Animal number	Ear thickness (mm)
		Day 1		Day 3		Day 6
		Pre-dose		Post dose
		Left	Right	Left	Right	Left	Right
5 %	S4	0.21	0.21	0.22	0.23	0.27	0.26
Overall thickness (mm)		0.210		0.225		0.265
Overall mean ear thickness change (%)		N/A		7.143		26.190
Concentration	Animal number	Ear thickness (mm)
		Day 1		Day 3		Day 6
		Pre-dose		Post dose
		Left	Right	Left	Right	Left	Right
2.5 %	S5	0.22	0.23	0.23	0.24	0.25	0.22
Overall thickness (mm)		0.225		0.235		0.235
Overall mean ear thickness change (%)		N/A		4.444		4.444
Concentration	Animal number	Ear thickness (mm)
		Day 1		Day 3		Day 6
		Pre-dose		Post dose
		Left	Right	Left	Right	Left	Right
1 %	S6	0.23	0.22	0.21	0.22	0.22	0.21
Overall thickness (mm)		0.225		0.215		0.215
Overall mean ear thickness change (%)		N/A		-4.444		-4.444

N/A = not applicable

 

Table 4       Main test – DPM, DPM/node and stimulation index

 

Concentration (% v/v) in acetone/olive oil 4:1	DPM	DPM/node	Stimulation index	Result
Vehicle	13624.59	1703.07	N/A	N/A
0.5	17500.67	2187.58	1.28	Negative
1	25163.78	3145.47	1.85	Negative
2.5	58049.35	7256.17	4.26	Positive

DPM = disintegrations per minute

DPM/node = Total DPM / 8 (i.e. no. of nodes = 8)

Stimulation index ≥ 3.0 = positive result

N/A = not applicable

 

Table 5       Main test – Individual clinical observations, mortality and body weight data

 

Concentration (% v/v) in acetone/olive oil 4:1	Animal number	Body weight (g)			Day
					1		2		3		4	5	6
		Day 1	Day 6	Change (g)	Pre-dose	Post dose	Pre-dose	Post dose	Pre-dose	Post dose
Vehicle	1-1	19.2	19.3	0.1	0	0	0	0	0	0	0	0	0
	1-2	19.3	21.2	1.9	0	0	0	0	0	0	0	0	0
	1-3	18.4	20.0	1.6	0	0	0	0	0	0	0	0	0
	1-4	18.5	18.6	0.1	0	0	0	0	0	0	0	0	0
0.5	2-1	20.3	20.6	0.3	0	0	0	0	0	0	0	0	0
	2-2	19.0	20.3	1.3	0	0	0	0	0	0	0	0	0
	2-3	18.9	21.0	2.1	0	0	0	0	0	0	0	0	0
	2-4	17.6	18.4	0.8	0	0	0	0	0	0	0	0	0
1	3-1	19.7	20.4	0.7	0	0	0	0	0	0	0	0	0
	3-2	21.1	20.9	-0.2	0	0	0	0	0	0	0	0	0
	3-3	19.3	18.9	-0.4	0	0	0	0	0	0	0	0	0
	3-4	17.6	18.6	1.0	0	0	0	0	0	0	0	0	0
2.5	4-1	18.6	19.2	0.6	0	0	0	0	0	0	0	0	0
	4-2	18.3	18.6	0.3	0	0	0	0	0	0	0	0	0
	4-3	19.2	21.1	2.0	0	0	0	0	0	0	0	0	0
	4-4	18.8	18.6	-0.2	0	0	0	0	0	0	0	0	0

0 = no sign of systemic toxicity

 

 

 

 

 

 

 

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

The test item was considered to be a sensitiser (Cat. 1A) under the conditions of the test according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008.

Executive summary:

OECD 429 (2018) - In a dermal sensitisation study, the test item was evaluated with young female adult mice (CBA/Ca) using the Local Lymph Node Assay (LLNA) in accordance with OECD 429 guidance and GLP.

 

In a preliminary screening test, conducted at test item concentrations of 100, 50, 10, 5, 2.5 and 1 % v/v, signs of clinical toxicity and body weight loss were noted at 100, 50 and 10 % v/v, resulting in the test animals being humanely killed. The mean ear thickness increased by >25 % during the study period (comparison of Day 1-6 ear thicknesses) in the 5 % v/v test group, indicating excessive irritation. The concentrations for the main test were therefore set to 0.5, 1 and 2.5 % v/v.

 

In the main test, three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item as a solution in the vehicle (acetone/ olive oil 4:1 v/v). at the aforementioned test concentrations. A further group of four animals was treated with the vehicle alone. A concurrent positive control test, using a group of four animals, was also performed with the known sensitiser, α-Hexylcinnamaldehyde tech., 85%, at a concentration of 25 % v/v in the vehicle.

 

There were no signs of systemic toxicity or local skin irritation observed. There were no clinical abnormalities or macroscopic abnormalities of the surrounding area were noted for any of the animals and no mortality reported during the study. The Stimulation Index, expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group, was determined for each treatment group.

 

The test item elicited a positive response (Stimulation Index ≥ 3) when tested at a concentration 2.5 % v/v. The test item was considered to be a sensitiser under the conditions of the test with a calculated EC3 value of 1.7. Both the vehicle and positive control group animals met the validity criteria of the OECD 429 guidance document.

 

Based on the condition of this study, the test item met the criteria for classification as a sensitiser (Category 1A) according to the Globally Harmonized Classification System and Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures.