N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

10 October 2017 - 19 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was conducted in accordance with international guidelines and in accordance with 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:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

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:

Fatty Acids, C14 and C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C34H68N2O3

IUPAC Name:

Fatty Acids, C14 and C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 2

Reference substance name:

Fatty Acids, C15 and C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C35H70N2O3

IUPAC Name:

Fatty Acids, C15 and C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 3

Reference substance name:

Fatty Acids, C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C36H72N2O3

IUPAC Name:

Fatty Acids, C16 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 4

Reference substance name:

Fatty Acids, C15 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C37H74N2O3

IUPAC Name:

Fatty Acids, C15 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 5

Reference substance name:

Fatty Acids, C16 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C38H76N2O3

IUPAC Name:

Fatty Acids, C16 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 6

Reference substance name:

Fatty Acids, C17 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C39H78N2O3

IUPAC Name:

Fatty Acids, C17 and C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 7

Reference substance name:

Fatty Acids, C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Molecular formula:

C40H80N2O3

IUPAC Name:

Fatty Acids, C18 sat., bisamides with 2-[(2-aminoethyl)amino]ethanol

Constituent 8

Reference substance name:

Stearic acid

EC Number:

200-313-4

EC Name:

Stearic acid

Cas Number:

57-11-4

Molecular formula:

C18H36O2

IUPAC Name:

stearic acid

Constituent 9

Reference substance name:

Palmitic acid

EC Number:

200-312-9

EC Name:

Palmitic acid

Cas Number:

57-10-3

Molecular formula:

C16H32O2

IUPAC Name:

palmitic acid

Constituent 10

Reference substance name:

Myristic acid

EC Number:

208-875-2

EC Name:

Myristic acid

Cas Number:

544-63-8

Molecular formula:

C14H28O2

IUPAC Name:

myristic acid

Constituent 11

Reference substance name:

2-(2-aminoethylamino)ethanol

EC Number:

203-867-5

EC Name:

2-(2-aminoethylamino)ethanol

Cas Number:

111-41-1

Molecular formula:

C4H12N2O

IUPAC Name:

2-(2-aminoethylamino)ethanol

Test material form:

solid

Details on test material:

Identification: N-(2-{[C16-18 (EVEN NUMBERED) ALKANOYL]AMINO}ETHYL)-N-(2-HYDROXYETHYL)[C16-18 (EVEN NUMBERED) ALKYLAMIDE
Storage Conditions: Room temperature

Specific details on test material used for the study:

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: n/a
- Specific activity: n/a
- Locations of the label: n/a
- Expiration date of radiochemical substance: n/a

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature in the dark.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: soluble in propylene glycol
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No
- Preliminary purification step (if any): No
- Final dilution of a dissolved solid, stock liquid or gel: No
- Final preparation of a solid: No

FORM AS APPLIED IN THE TEST (if different from that of starting material): n/a

OTHER SPECIFICS: No

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
- Females (if applicable) nulliparous and non-pregnant: Yes
- Microbiological status of animals, when known: Not reported
- 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 woodflakes
- Diet (e.g. ad libitum): Free access to food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK)
- Water (e.g. ad libitum): Free access to mains tap water
- Acclimation period: At least 5 days
- Indication of any skin lesions: No

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

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

10, 5 and 2.5%

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Soluble
- Irritation: No
- Systemic toxicity: No
- Ear thickness measurements: < 25 % increase
- Erythema scores: No

Positive control substance(s):

other: Phenylacetaldehyde (Sigma-Aldrich: Batch number: MKCD0809, Expiry date 24 October 2022)freshly prepared as a 5% v/v dilution in propylene glycol.

Statistics:

Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non‑parametric Kruskal‑Wallis Rank Sum and Mann‑Whitney U test procedures were used.
Probability values (p) are presented as follows:
P < 0.001 ***
P < 0.01 **
P < 0.05 *
P > 0.05 (not significant)

Results and discussion

Positive control results:

Concurrent control was within the historical control data. The positive control Phenylacetaldehyde gave a Stimulation Index of greater than 3 (6.67) when tested at a concentration of 5% v/v in propylene glycol, thus, demonstrating the sensitivity and reliability of the test system.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 0.78

Test group / Remarks:

10%

Results 2

Key result

Parameter:

SI

Value:

ca. 0.94

Test group / Remarks:

5%

Results 3

Key result

Parameter:

SI

Value:

ca. 0.97

Test group / Remarks:

2.5%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: < 2.5 % at 10% concentration.

DETAILS ON STIMULATION INDEX CALCULATION: Mean treatment group dpm / mean vehicle dpm

EC3 CALCULATION: n/a

CLINICAL OBSERVATIONS: No signs of systemic toxicity or other clinical signs

BODY WEIGHTS: Within normal limits

Any other information on results incl. tables

Table 1       Stimulation index results.

Treatment Group	Animal Number	dpm/ Animal a	Mean dpm/Animal (Standard Deviation)	Stimulation Index b	Result
Vehicle propylene glycol	1-1	799.20	1036.20 (±290.69)	na	na
	1-2	777.68
	1-3	1369.28
	1-4	904.26
	1-5	1330.56
Test Item 2.5% w/w in propylene glycol	2-1	769.83	1006.88 (±323.24)	0.97	Negative
	2-2	1085.46
	2-3	614.54
	2-4	1124.26
	2-5	1440.32
Test item 5% w/w in propylene glycol	3-1	1084.52	972.65 (±306.17)	0.94	Negative
	3-2	694.01
	3-3	1382.03
	3-4	1060.84
	3-5	641.84
Test item 10% w/w in propylene glycol	4-1	1411.84	813.13 (±515.63)	0.78	Negative
	4-2	263.04·
	4-3	1305.24
	4-4	458.83
	4-5	626.71
Positive Control item 5% v/v in propylene glycol	5-1	7680.02	6907.32** (±1151.56)	6.67	Positive
	5-2	8091.08
	5-3	7218.07
	5-4	6339.07
	5-5	5208.35

dpm =   Disintegrations per minute

a =      Total number of lymph nodes per animal is 2

b =      Stimulation Index of 3.0 or greater indicates a positive result

**=     Significantly different from control group p<0.01

* =     Low dpm value possibly due to spillage during lymph node cell preparation

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item was considered to be a non-sensitiser under the conditions of the test. Based on the condition of this study, the test item does not meet the criteria for classification according to the Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances.

Executive summary:

OECD 429 (2017) - In a dermal sensitisation study with the test item,

N-(2-{[C16-18 (even numbered) alkanoyl]amino}ethyl)-N-(2-hydroxyethyl)[C16-18 (even numbered) alkylamide

in propylene glycol, young female adult mice (CBA/Ca (CBA/CaOlaHsd)) were tested using the Local Lymph Node Assay (LLNA).

Following a preliminary screening test in which no clinical signs of toxicity were noted at a maximum attainable concentration of 10% w/v, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay.  Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the test item as a suspension in propylene glycol at concentrations of 10%, 5% or 2.5% w/v. A further group of five animals was treated with propylene glycol alone.  A concurrent positive control test, using a group of five animals, was also performed with the known sensitiser, Phenylacetaldehyde at a concentration of 5% v/v in propylene glycol.

There were no signs of systemic toxicity, local skin irritation observed and ear thickness were within normal range. 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. Since there was no indication that the test item elicited a SI ≥ 3 when tested up to 10% the test item was considered to be a non-sensitiser under the conditions of the test.

Based on the condition of this study, the test item does not meet the criteria for classification according to the Globally Harmonized Classification System and to the Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances.