N-(2-hydroxyethyl)dodecanamide

Endpoint summary

Description of key information

Skin Sensitisation:

OECD 442C (D. Fleet 2018) - Not sensitising: This study was disregarded due to the test item was identified as a potential pro-hapten as part of an in-silico profiling exercise that concluded following completion of the study.  

Furthermore the test item was identified as being outside of the applicability domain of the OECD 442E study.  The test item has an experimentally measured Log Pow of > 3.5.  

The Registrant is therefore not able to fulfil the requirement to conduct at least two out of three OECD 442 studies as outlined in ECHA’s Guidance on Information Requirements and Chemical Safety Assessment Chapter R.7a: Endpoint specific guidance (Version 6.0 - July 2017).  The Registrant was unable to provide a conclusive prediction on skin sensitisation using in-vitro methods and was required to proceed to in vivo testing.

OECD 429; (Anon., 2018) - Not sensitising.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

other information

Study period:

15 November 2017 - 08 May 2018

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other:

Remarks:

Whilst the study was conducted in accordance with international guidelines and in accordance with GLP, the test item was identified as a potential pro-hapten following conclusion of the study. The OECD 442C study is not valid for pro-haptens.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Version / remarks:

2017

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The in vitro testing was intitatiated before further data was generated from in silico analysis. However, the result of the in silico assessment was indicative that the test item was a know pro-hapten and as such in vitro sensitisation testing would not be applicable.

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: At room temperature.
- Stability under test conditions: Assumed stable
- Solubility and stability of the test substance in the solvent/vehicle: n/a
- 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

Details on the study design:

The positive control was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution in propan-2-ol of the test item was prepared.

The test solutions were incubated with the cysteine and lysine peptides, respectively, for 22 hours in glass vials at 25°C.

The remaining concentration of cysteine or lysine-containing peptides following the 22 hour incubation period was measured by high performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

Positive control results:

No

Key result

Parameter:

other: Peptide depletion

Value:

0.034

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

For each Analytical Run:
The standard calibration curve linearity should have a coefficient of determination (r2) not less than 0.99
The mean percentage peptide depletion for the cinnamic aldehyde positive control should be between:
60.8 % -100 % for the cysteine peptide
40.2 % -69.0 % for the lysine peptide
Individual values for the depleted cinnamic aldehyde positive control should be <14.9 % for the cysteine peptide and <11.6 % for the lysine peptide from mean values.
The mean peptide concentration of the reference controls should be between 0.45-0.55 mM with a precision of <15 %.
Individual values for the depleted test item should be <14.9 % for the cysteine peptide and <11.6% for the lysine peptide from mean values.

All analytical acceptance criteria for each peptide run were met:

	Peptide	Standard Linearity	Positive control depletion (%)	Reference controls	Test item
Acceptance criteria	Cysteine	r^2> 0.99	60.8-100 (SD < 14.9 %)	0.45-0.55 mM (CV < 15%)	SD < 14.9 %
	Lysine	r^2>0.99	40.2-69.0 (SD < 11.6 %)	0.45-0.5 mM (CV <15%)	SD < 11.6 %
Achieved results	Cysteine	r^2> 0.999	70.8 (SD, 0.45 %, n =3 )	B: 0.503 mM (CV 0.24 %, n = 6) C: 0.498 mM (CV 0.09 %, n = 3)	SD 0.12 % (n = 3)
	Lysine	r^2> 0.999	57.9 (SD, 0.69 %, n = 3)	B: 0.502 mM (CV 0.50 %, n = 6) C: 0.502 mM (CV 0.40 %, n = 3)	SD 0.49 % (n = 3)

CV       Coefficient of Variation

SD       Standard deviation

The depletion of peptide in the presence of the test item was:

Peptide	Mean peak area of reference control (µV.sec)	Mean peak area of peptide with test item (µV.sec)	Mean peptide depletion by N-(2-hydroxyethyl) dodecanamide (%)
Cysteine	Control B: 861710 (n = 6) Control C: 853410 (n = 3)	852830 (n = 3)	0.0680
Lysine	Control B: 766130 (n = 6) Control C: 755020 (n = 3)	758800 (n = 3)	-0.501

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the test article was considered to be negative in the Direct Peptide Reactivity Assay according to EU CLP and UN GHS.

Executive summary:

OECD 442C (2018): Solutions of N-(2-hydroxyethyl) dodecanamide were successfully analysed by the validated DPRA- OECD 442C analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides.  The overall mean depletion result of 0.0340% places the test item in the reactivity class of “no to minimal activity” and hence it is predicted by DPRA to be a potential non-skin sensitiser under the condition of this study.  

Under the conditions of this study, the test article was considered to be negative in the Direct Peptide Reactivity Assay according to EU CLP and UN GHS.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08 February - 04 May 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.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

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)

Specific details on test material used for the study:

ADIOLABELLING 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

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

Vehicle:

propylene glycol

Concentration:

5, 10 and 25 % (w/w)

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 (>90%) at a concentration of 5% v/v 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)

Positive control results:

Concurrent control was within the historical control data. The positive control Phenylacetaldehyde gave a Stimulation Index of greater than 3 (4.64) when tested at a concentration of 5% v/v in propylene glycol, thus, demonstrating the sensitivity and reliability of the test system.

Key result

Parameter:

SI

Value:

ca. 0.73

Test group / Remarks:

5% w/w in propylene glycol

Key result

Parameter:

SI

Value:

ca. 1.1

Test group / Remarks:

10% w/w in propylene glycol

Key result

Parameter:

SI

Value:

ca. 1.49

Test group / Remarks:

25%w/w in propylene glycol

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA: < 5 % at 25 % 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

Table 1:       Stimulation index results.

Treatment Group	Animal Number	dpm/ Animal^a	Mean dpm/Animal (Standard Deviation)	Stimulation Index^b	Result
Vehicle propyleneglycol	1-1	397.08	607.26 (±129.32)	na	na
	1-2	684.01
	1-3	727.01
	1-4	648.68
	1-5	579.53
Test Item 5% w/win propylene glycol	2-1	429.44	444.93 (±159.51)	0.73	Negative
	2-2	293.94
	2-3	640.42
	2-4	288.82
	2-5	572.02
Test Item 10% w/win propylene glycol	3-1	819.97	668.89 (±146.73)	1.10	Negative
	3-2	675.65
	3-3	797.10
	3-4	580.22
	3-5	471.53
Test Item 25% w/win propylene glycol	4-1	705.50	901.88** (±164.24)	1.49	Negative
	4-2	774.89
	4-3	922.07
	4-4	994.95
	4-5	1111.97
Positive Control Item 5% v/v in propylene glycol	5-1	2698.91	2815.84** (±637.00)	4.64	Positive
	5-2	3455.56
	5-3	3164.59
	5-4	1788.99
	5-5	2971.15

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 vehicle control group p < 0.01

na     =     Not applicable

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 (2018) - In a dermal sensitisation study with the test item, N-(2-hydroxyethyl) dodecanamide 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 25 % 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 25 %, 10 % or 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 25 % 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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

In an OECD 429 study (2018), the test item, N-(2-hydroxyethyl) dodecanamide 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 25 % 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 25%, 10 % or 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 25 % 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

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The substance does not meet the classification criteria for skin sensitisation according to Regulation (EC) No 1272/2008 (CLP).