N,N-Dimethyl-9-dodecenamide

Endpoint summary

Description of key information

A Local Lymph Node Assay (OECD TG 429) demonstrated that N,N-dimethyldodec-9-enamide has the potential to cause skin sensitisation.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 May - 02 August 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

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)

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaCrl

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK) Ltd., Margate
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 9 to 10 weeks old on Day 1
- Weight at study initiation: Animals in the main studies were in a body weight range of 17 to 22 g on Day 1 of dosing. Individual body weights were within ±20% of the mean body weight for mice on the study.
- Housing: The animals were housed in groups of up to six during acclimatisation and housed in pairs from Day 1 in cages that conformed to the 'Code of Practice for the Housing and Care of Animals Bred, Supplied or Used for Scientific Purposes’ (Home Office, London, 2014).
Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding (Datesand Ltd., Manchester, UK). The bedding had been analysed for specific contaminants and the results retained on file at Labcorp. No contaminants were present in bedding at levels which might have interfered with achieving the objective of the study.
- Diet (e.g. ad libitum): 5LF2 EU Rodent Diet 14%, was freely available to the animals at all times. Each batch of diet had been analysed for specific constituents and contaminants by the manufacturer. No contaminants were present in diet at levels which might have interfered with achieving the objective of the study.
- Water (e.g. ad libitum): Mains water was provided, ad libitum, via cage-mounted water bottles. The water had been periodically analysed for specific contaminants. No contaminants were present in water at levels which might have interfered with achieving the objective of the study.
- Acclimation period: 8 to 15 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 40 to 70
- Air changes (per hr): minimum of 15
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was controlled automatically to give a cycle of 12 hours light and 12 hours dark.
- IN-LIFE DATES: From: To: 31 May - 02 August 2022

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

100 %v/v; 50 % v/v; 25 %v/v; 10 %v/v; 5 %v/v; 2.5 %v/v

No. of animals per dose:

Preliminary screening test: 1 animal
Main study: 4 animals per dose

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The vehicle for the test article was 80% v/v acetone in olive oil, supplied by Labcorp. The vehicle was chosen because it was the first of the vehicles listed in the protocol that produced an overtly stable solution, emulsion or dispersion incorporating 50% v/v of the test article.
- Irritation: None observed
- Systemic toxicity: No death. Greasy fur to back of ears and neck observed on days 1, 3, 4, 5 & 6; Greasy fur to back, back of ears and neck observed on day 2
- Ear thickness measurements: See Table 9.5 below
- Erythema scores: 0 on days 1, 2, 3, 4, 5 & 6

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
Mice were arbitrarily selected from the delivery boxes and allocated to the appropriate number of cages. The condition of the animals was assessed daily throughout the acclimatisation period of 8 to 15 days. A second inspection was performed prior to study commencement to ensure the animals were suitable for the study. Overtly healthy animals were arbitrarily allocated to the study groups on the day of commencement of treatment.
- Criteria used to consider a positive response: The test article is regarded as a sensitiser when the maximum value of the SI is 3.0 or above. The test article is classified as a non-sensitiser when the maximum value of the SI is
less than 3.0. (This result is unchanged by observations of irritation at sites of application of the test formulation).

TREATMENT PREPARATION AND ADMINISTRATION:
Test Article Formulation
Formulations were freshly prepared as required using 80% v/v acetone in olive oil on Days 1, 2 and 3. The formulations were stored at room temperature, in sealed, air-tight containers prior to dosing and were used within two hours of preparation.
The formulations were mixed by multiple inversion of the containers prior to administration to ensure homogeneity.
Concentrations of test article were expressed volumetrically and in terms of test article received (without regard to purity or active content).

Test Article Administration
Each mouse was manually restrained with both auditory pinnae left free. The outer aspect of both pinnae of each mouse was treated by direct application of the appropriate vehicle control or test formulation (0.025 mL/pinna) dispensed from an automatic micro pipette.

Treatment Regimen
The groups of four female mice were subjected to application of the vehicle control or one of the test formulations to the outer aspect of the auditory pinnae, once daily on Days 1, 2 and 3. On Day 6 the mice were placed in a thermacage in order to dilate the peripheral blood vasculature and thus facilitate intravenous dosing. Each mouse was transferred to a cylindrical restrainer. A plastic syringe and fine gauge hypodermic needle were used to administer 0.25 mL phosphate buffered saline incorporating 20 μCi of 3HTdR into a tail vein of each mouse by slow bolus injection. After this treatment, the mice were returned to their cages. Approximately five hours after intravenous injection of the 3HTdR, all mice were killed by exanguination under a deep plane of inhalation anasthesia. Killing was organised to minimise the interval between death and the recovery of the auricular lymph nodes to no more than fifteen minutes.

Clinical Signs
Treated mice were observed twice daily on Days 1 to 5 and once on Day 6 for clinical signs of reaction to treatment or for irritation or other changes at the sites of application of the test article.
Routine Health Checks
All animals were examined at the beginning and end of the working day throughout the acclimatisation and study periods to ensure they were in good health.
Body Weights
Mice were weighed on Day 1 (the first day of dosing) and on Day 6 prior to intravenous administration of 3HTdR.

Positive control substance(s):

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

Positive control results:

See attached historical positive control data. These data confirm adequate performance of the assay.

Parameter:

EC3

Value:

24.6

Parameter:

SI

Value:

5.59

Test group / Remarks:

100 %v/v

Parameter:

SI

Value:

3.65

Test group / Remarks:

50 %v/v

Parameter:

SI

Value:

3.04

Test group / Remarks:

25 %v/v

Parameter:

SI

Value:

1.47

Test group / Remarks:

10 %v/v

Parameter:

SI

Value:

0.81

Test group / Remarks:

5 %v/v

Parameter:

SI

Value:

0.47

Test group / Remarks:

2.5 %v/v

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation counts obtained from the test groups relative to the corresponding mean scintillation count obtained from the vehicle control (see Table 9.1 & 9.2 below).
The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.

EC3 CALCULATION
The EC3 value was calculated using the following equation:
EC3 = c+[(3-d)/(b-d)](a-c)
where
a = lowest concentration giving SI ≥ 3
b = actual SI caused by a
c = highest concentration failing to produce SI of 3
d = actual SI caused by c

The EC3 was calculated to be 24.6%.

CLINICAL OBSERVATIONS:
There were no clinical signs indicative of a systemic effect of treatment among mice treated with the vehicle or with 2.5%, 5%, 10%, 25%, 50% or 100% v/v formulations of the test article.
In the first main study, greasy wet fur on the back of the neck and ears was noted in all animals on Day 1. Greasy fur on the back of the neck and ears was noted in all animal from Days 2 to 4, persisting in animals in Groups 2, 3 and 4 on Days 5 and 6.
Incidents of greasy fur on the dorsal thoracic area and on the front legs were also noted on Days 4 and 5 in animals in Groups 3 and 4.
In the second main study, greasy fur on the back of the neck and ears was noted in all animals on Day 2, with greasy fur on the back of the neck and ears noted on Days 3 and 4 in animals treated at a concentration of 10% v/v.

BODY WEIGHTS
There was no indication of a treatment related effect on body weight (see Tables 9.6 & 9.7 below).

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness).
All animals survived treatment with N,N-dimethyldodec-9-enamide.
The vehicle and test formulation application sites remained free of irritation.

Table 9.1 Group DPMs and Stimulation Index (SI), Experiment 1

Concentration (% v/v in 80% v/v acetone in olive oil)	Group Number	Group DPM	Stimulation Index (SI)^a
Vehicle	1	726	NA
25	2	2207	3.04
50	3	2648	3.65
100	4	4060	5.59

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

NA = Not applicable

Table 9.2 Group DPMs and Stimulation Index (SI), Experiment 2

Concentration (% v/v in 80% v/v acetone in olive oil)	Group Number	Group DPM	Stimulation Index (SI)^a
Vehicle	5	707	NA
2.5	6	335	0.47
5	7	576	0.81
10	8	1040	1.47

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

NA = Not applicable

Table 9.5: Preliminary Screening Test - Ear Thickness Measurements

Concentration (% v/v)	Animal Number	Ear	Thickness (mm) on Day:
			1	3	6
100	257	Left	0.21	0.21	0.21
		Right	0.20	0.21	0.22

  

Table 9.6 Body Weights, Main Experiment 1

 

Concentration (% v/v in 80% v/v acetone in olive oil)	Group Number	Animal Number	Body Weights (g)
			Day 1	Day 6
Vehicle	1	258	20	21
		259	19	20
		260	19	20
		261	22	22
25	2	262	19	20
		263	20	22
		264	22	24
		265	21	20
50	3	266	20	21
		267	20	20
		268	20	21
		269	20	21
100	4	270	20	19
		271	22	19
		272	20	20
		273	22	19

 

 Table 9.7 Body Weights, Main Experiment 2

 

Concentration (% v/v in 80% v/v acetone in olive oil)	Group Number	Animal Number	Body Weights (g)
			Day 1	Day 6
Vehicle	5	318	19	20
		319	19	21
		320	17	18
		321	18	20
2.5	6	322	19	19
		323	17	21
		324	20	22
		325	17	18
5	7	326	19	21
		327	19	20
		328	17	19
		329	19	21
10	8	330	17	19
		331	19	21
		332	17	19
		333	19	21

 

 

Interpretation of results:

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

Executive summary:

This study was conducted to assess the potential of the test article, N,N-dimethyldodec-9-enamide, to cause skin sensitisation in the mouse.

Following a preliminary screening test using the undiluted test article, the main study was conducted using the undiluted test article and 50 and 25% v/v formulations in 80% v/v acetone in olive oil. In order to calculate an EC3 value, a second experiment was conducted using 2.5%, 5% and 10% v/v formulations.

Groups of four female CBA/CaCrl mice were subjected to topical applications of vehicle control or of one of the test formulations to the outer aspect of the auditory pinnae once daily on Days 1, 2 and 3. On Day 6 a 20 μCi dose of tritiated 3H-methyl thymidine was injected intravenously into each mouse. Approximately five hours later the auricular lymph nodes were recovered from each animal. The pairs of nodes from each animal were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter.

Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation counts obtained from the test groups relative to the corresponding mean scintillation count obtained from controls. The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.

	Concentration of Test Article in Applied Formulation (% v/v)
	2.5	5	10	25	50	100
Stimulation Index	0.47	0.81	1.47	3.04	3.65	5.59

The EC3 was calculated to be 24.6%.

The Local Lymph Node Assay demonstrated that N,N-dimethyldodec-9-enamide has the potential to cause skin sensitisation.

Reference 2

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

02 - 04 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation Assays addressing the Adverse Outcome Pathway key event on covalent binding to proteins)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptides following incubation with the test article. Relative peptide concentration is measured by high performance liquid chromatography (HPLC) with UV detection.
Cysteine and lysine peptide percent depletion (PPD) values are then calculated and used in a prediction model which allows assigning the test article to one of four reactivity classes used to support the discrimination between sensitisers and nonsensitisers.

Details of test system:

cysteine peptide, (Ac-RFAACAA-COOH)

lysine peptide (Ac-RFAAKAACOOH)

Details on the study design:

PREPARATION OF TEST SOLUTIONS
- Preparation of the peptide/derivative stock solutions :
A stock solution containing cysteine at approximately 0.667 mM was prepared in 100 mM Phosphate Buffer pH 7.5 and a stock solution containing lysine at approximately 0.667 mM was prepared in 100 mM ammonium acetate buffer pH 10.2.
- Preparation of the test chemical solutions :
The test article was dissolved in acetonitrile (batch numbers 2034317 and 2043930 expiry dates 13 August 2023 and 19 May 2024, respectively) supplied by Fisher. This was the first of the listed vehicles that produced a visually clear solution at a concentration of 100 mM.
- Preparation of the positive controls, reference controls and co-elution controls :
The positive control was dissolved in acetonitrile at a concentration of 100 mM.
Reference controls were prepared for each peptide. Reference Control A and B for each peptide were prepared by adding 750 μL of peptide stock solution to 250 μL of acetonitrile.
Reference Control C for each peptide were prepared by adding 750 μL of peptide stock solution to 250 μL of acetonitrile (vehicle).
Reference Control A (in triplicate) was used to verify the HPLC system suitability prior to the analysis. Reference Control B (six replicates) was used to verify the stability of the reference controls over time and Reference Control C (in triplicate) was used to verify that the vehicle did not impact the percent peptide depletion.
Co-elution controls were prepared to detect possible co-elution of the test article with the peptides. A mixture of 750 μL of 100 mM Phosphate Buffer pH 7.5, 200 μL of acetonitrile and 50 μL of test article solution was used to detect possible co-elution of the test article with cysteine. A mixture of 750 μL of 100 mM ammonium acetate buffer pH 10.2 and 250 μL of test article solution was used to detect possible co-elution of the test article with lysine.

INCUBATION
- Incubation conditions : Each test solution was prepared at ratios of 1:10 and 1:50 (based on molecular weight) with the cysteine and lysine stock solutions, respectively. The preparations were placed in an incubator set at 25˚C for 24±2 hours.
- Precipitation noted : Prior to, and at the end of the incubation period the samples were visually inspected for precipitate formation. Precipitation was observed pre and post-incubation in all cysteine and lysine test article and co-elution samples and post-incubation only in the cysteine and lysine positive control samples. Precipitate was removed from all affected post-incubation samples by centrifugation at 400 g for 5 minutes; and the remaining sample aspirated to a clean autosampler vial.

PREPARATION OF THE HPLC
- Standard calibration curve for both Cys and Lys
Calibration curves were prepared for each peptide using a range of concentrations from approximately 0.534 mM to 0.0167 mM (Standards 1 to 6).
Standard 1 was prepared at approximatively 0.534 mM by dilution of 1600 μL of the peptide stock solution (0.667 mM) with 400 μL of acetonitrile.
Standards 2 to 6 for cysteine were prepared by serial dilution using dilution buffer (20% acetonitrile in 100 mM Phosphate Buffer pH 7.5).
Standards 2 to 6 for lysine were prepared by serial dilution using dilution buffer (20% acetonitrile in 100 mM ammonium acetate buffer pH 10.2).
Samples of dilution buffer alone were also prepared.
- Verification of the suitability of the HPLC for test chemical and control substances

DATA EVALUATION
- Cys and Lys peptide detection wavelength: 220 nm

Vehicle / solvent:

acetonitrile

Positive control:

cinnamic aldehyde

Positive control results:

Cysteine depletion:
Mean PPD: 74.83
SD: 0.06
CV: 0.09
Lysine depletion:
Mean PPD: 59.36
SD: 1.35
CV: 2.27

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

mean cystein depletion

Value:

28.57 %

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

mean lysine depletion

Value:

0.83 %

Positive controls validity:

valid

Outcome of the prediction model:

low reactivity [in chemico]

Other effects / acceptance of results:

ACCEPTANCE OF RESULTS: (see tables below)
- Acceptance criteria met for positive control: yes
- Acceptance criteria met for reference controls A to C: yes
- Acceptance criteria met for co-elution controls (Lysine and Cysteine): yes
- Acceptance criteria met for variability between replicate measurements: yes

Cysteine Depletion

The percentage peptide depletion values were as follows:

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD	CV
Test Article	22.5 22.27 22.19	31.27	27.89 28.78 29.04	28.57	0.60	2.12
Positive Control	7.87 7.89 7.87	31.27	74.90 74.77 74.83	74.83	0.06	0.09

The r² value for the standard calibration curve was 0.99962.

The peptide concentrations for the Reference Controls A and C were as follows:

Reference Control	Peptide Concentration (mM
	Replicate 1	Replicate 2	Replicate 3	Mean	SD	CV
A	0.50	0.50	0.50	0.50	0.00	0.39
C	0.50	0.50	0.50	0.50	0.00	0.20

The peak area results for Reference Controls B and C (acetonitrile) were as follows:

Reference Control	Replicate	Peptide Peak Area
B	1 2 3 4 5 6	31.39 31.37 31.38 29.46 31.24 31.43
C	1 2 3	31.32 31.20 31.28
Mean SD CV		31.12 0.63 2.1

Lysine Depletion

The percentage peptide depletion values were as follows:

Substance	Replicate Peptide Peak Areas	Reference Control C Mean Peptide Peak Area	PPD	Mean PPD	SD	CV
Test Article	27.07 26.80 26.93	27.16	0.33  1.33 0.85	0.85	0.50	59.57
Positive Control	11.46 10.82 10.83	27.16	57.81 60.16 60.13	59.36	1.35	2.27

The r² value for the standard calibration curve was 1.00000.

The peptide concentrations for the Reference Controls A and C were as follows:

Reference Control	Peptide Concentration (mM
	Replicate 1	Replicate 2	Replicate 3	Mean	SD	CV
A	0.50	0.52	0.50	0.51	0.01	1.70
C	0.50	0.52	0.50	0.50	0.01	2.23

The peak area results for Reference Controls B and C (acetonitrile) were as follows:

Reference Control	Replicate	Peptide Peak Area
B	1 2 3 4 5 6	26.94 28.47 28.55 26.88 26.84 26.80
C	1 2 3	26.79 27.86 26.83
Mean SD CV		27.33 0.75 2.74

Executive summary:

The study was conducted to quantify the reactivity of N,N-dimethyldodec-9-enamide towards model synthetic peptides containing either lysine or cysteine. The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The test article was dissolved in acetonitrile at a concentration of 100 mM.

The test solutions were incubated at 1:10 and 1:50 ratios with the cysteine and lysine peptides, respectively, for 24±2 hours in glass autosampler vials, protected from light, in an incubator set to 25°C.

The remaining concentration of cysteine- or lysine-containing peptides following the 24-hour incubation period was measured by high performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm.

The mean of the cysteine and lysine depletion values for N,N-dimethyldodec-9 -enamide was 14.70% which provided a positive prediction with low reactivity. This was derived from the mean PPD values of 28.57% and 0.83% for cysteine and lysine, respectively.

Precipitate was observed immediately upon addition of the test article solution to the cysteine and lysine peptide solutions, there was therefore uncertainty as to how much test article initially remained in solution to react with the peptide. In addition, precipitation was also observed post-incubation which may result in an underestimation of peptide depletion.

The test article, N,N-dimethyldodec-9-enamide, was considered to be positive in the Direct Peptide Reactivity Assay.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

08 - 18 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 luciferase KeratinoSens™ test method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

ARE-Nrf2 luciferase KeratinoSens™ test method

Justification for non-LLNA method:

The ARE-Nrf2 luciferase test method utilises an immortalised adherent cell line derived from HaCaT human keratinocytes. The cell line is stably transfected with a plasmid containing a luciferase gene under the transcriptional control of the SV40 promoter fused with the ARE from a gene known to be up-regulated by contact sensitisers.
The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic substances.

Details of test system:

other: KeratinoSens™ cell line supplied by Givaudan Schweiz, Zurich, Switzerland.

Details on the study design:

TEST SYSTEM
Specifications
KeratinoSens™ cell line supplied by Givaudan Schweiz, Zurich, Switzerland.
Identification
The test system was appropriately labelled with the study number, assay type, experiment number and test/positive/negative control.
Preparation of Cultures
A fresh vial of cells was used for each experimental occasion and cultured using Dulbecco’s modified Eagle medium (DMEM) containing serum and Geneticin.

PREPARATION OF TEST SOLUTIONS
- Test article formulation: Test formulations were prepared using DMSO. This was the first of the listed vehicles that produced a visually clear solution at a concentration of 200 mM. Serial dilutions were made (from the 200 mM stock) using the vehicle to obtain 12 master concentrations ranging from 0.098 to 200 mM. The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations range from 0.98 to 2000 μM.
Formulations were prepared shortly before and on the same day as testing.
- Preparation of negative control: The negative control was diluted into culture medium containing serum so that the final concentration was 1%.
- Preparation of positive control: The positive control was prepared at a concentration of 6.4 mM in DMSO. Five master concentrations ranging from 0.4 to 6.4 mM were prepared in DMSO (from a 6.4 mM stock solution). The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4-fold dilution factor so that the final concentrations range from 4 to 64 μM.

APPLICATION OF THE TEST CHEMICAL AND CONTROL SUBSTANCES
- Treatment Plate Preparation: The cells were 80 - 90% confluent. On the day prior to treatment, cells were harvested and distributed into 96-well plates (10000 cells/well) and incubated for 24±1 hours in an incubator set to 37°C, 5% CO2. For each repetition, three replicates were used for the luciferase activity measurements and one parallel replicate used for the cell viability assay.
- Treatment: At the end of the 24-hour incubation period, the medium was removed and replaced with fresh culture medium (containing serum but without Geneticin) to which test article and control formulations were added. On each plate, one well per replicate was left empty (no cells and no treatment) to assess background values.
Aliquots of 50 μL of each of the final concentrations were transferred to give three luciferase replicates on a white-walled plate and a single viability replicate on a clear-walled plate. Each plate was sealed and incubated for 48±1 hours in a humidified incubator set to 37°C, 5% CO2.
For each test article and positive control, one experiment was needed to derive a prediction (positive or negative), consisting of two independent repetitions each containing three replicates of each concentration.
Each independent repetition was performed on a different day with fresh stock solutions of chemicals and independently harvested cells. The cells came from different cell batches.

LUCIFERASE ACTIVITY MEASUREMENTS
After the 48-hour exposure period, the cells were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plate was then incubated for 20 minutes in an incubator set to 25°C, loaded into the luminescence plate reader and read using the following parameters: 100 μL injection (Luciferase assay substrate), 15 second delay, 7 second luminescence integration time.

DATA EVALUATION
- Cytotoxicity assessment :
After the 48-hour exposure period, the medium was replaced with fresh medium containing MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). The plate was sealed and placed in an incubator set to 37°C, 5% CO2 for 4 hours. The MTT medium was removed and 200 μL SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator set to 37°C, 5% CO2, and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm.
- Analysis of results:
The following parameters were calculated:
• The maximal average fold induction of luciferase activity (Imax) value observed at any concentration of the test article and positive control;
• The EC1.5 value representing the concentration for which induction of luciferase activity is above the 1.5-fold threshold (i.e. 50% enhanced luciferase activity) was obtained; and
• The IC50 and IC30 concentration values for 50% and 30% reduction of cellular viability.
- Fold Luciferase Induction:
Fold luciferase activity induction at each concentration of test article and positive control was calculated as follows:
Fold induction = (Lsample - Lblank)/(Lsolvent - Lblank)
where
• Lsample is the luminescence reading in the test article well,
• Lblank is the luminescence reading in the blank well containing no cells and no treatment,
• Lsolvent is the average luminescence reading in the wells containing cells and solvent (negative control).
The overall maximal average fold induction (Imax) was calculated as the average of the individual repetitions.
- Luciferase Activity
EC1.5 was calculated by linear interpolation as follows:
EC1.5 = (Cb – Ca) x (1.5 – Ia)/(Ib-Ia) + Ca
where
• Ca is the lowest concentration in μM with >1.5-fold induction
• Cb is the highest concentration in μM with <1.5-fold induction
• Ia is the fold induction measured at the lowest concentration with >1.5-fold induction (mean of three replicate wells)
• Ib is the fold induction at the highest concentration with <1.5-fold induction (mean of three replicate wells).
The overall EC1.5 was calculated as the geometric mean of the individual repetitions.
- Viability:
Viability was calculated as follows:
Viability = (Vsample - Vblank) x 100/(Vsolvent - Vblank)
where
• Vsample is the MTT-absorbance reading in the test article well
• Vblank is the MTT-absorbance reading in the blank well containing no cells and no treatment
• Vsolvent is the MTT-absorbance reading in the wells containing cells and solvent (negative control).
- Reduction of Cell Viability:
IC50 and IC30 were calculated by linear interpolation as follows:
ICx = (Cb – Ca) x ((100 – x) –Va)/(Vb-Va) + Ca
where
• x is the % reduction at the concentration to be calculated (50 and 30 for IC50 and IC30)
• Ca is the lowest concentration in μM with >x% reduction in viability
• Cb is the highest concentration in μM with • Va is the % viability at the lowest concentration with >x% reduction in viability
• Vb is the % viability at the highest concentration with The overall IC50 and IC30 were calculated as the geometric mean of the individual repetitions.
For each concentration showing a luciferase activity induction equal or higher than 1.5-fold, statistical significance was determined by comparing the luminescence values of the three replicate samples with the luminescence values in the solvent/vehicle control wells to assess whether the luciferase activity induction was statistically significant (p<0.05).
- Assay Acceptance Criteria:
The following acceptance criteria should be met:
• The luciferase activity induction obtained with the positive control should be statistically significant above the threshold of 1.5 in at least one of the tested concentrations (from 4 to 64 μM)
• The EC1.5 value for the positive control should be within two standard deviations of the historical mean and the average induction in the three replicates for the positive control at 64 μM should be between 2 and 8. If the latter criterion is not fulfilled, the dose-response of cinnamic aldehyde will be evaluated to determine acceptability
• The average coefficient of variation of the luminescence reading for the negative control (DMSO) should be below 20% in each repetition which consists of 6 wells in triplicate.
- Prediction model used :
A KeratinoSens™ prediction is considered positive if the following 4 conditions are all met in 2 of 2 or in the same 2 of 3 repetitions, otherwise the KeratinoSens™ prediction is considered negative:
1. The Imax is >1.5-fold and statistically significantly different when compared to the solvent (negative) control (as determined by a two-tailed, unpaired Student’s T-test);
2. The cell viability is >70% at the lowest concentration with induction of luciferase activity above 1.5-fold (i.e. at the EC1.5 determining concentration);
3. The EC1.5 value is <1000 μM;
4. There is an apparent overall dose response for luciferase induction or if the dose response curve is biphasic.

Vehicle / solvent control:

DMSO

Positive control:

cinnamic aldehyde [442D]

Positive control results:

The EC1.5 value for the positive control was 7.45 and 8.38 μM in Experiments 1 and 2, respectively. The average induction in the three replicates for the positive control at 64 μM were 7.97 and 7.62 in Experiments 1 and 2, respectively. (see TAbles 9.1 - 9.3 attached).

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

2.2 µM

Cell viability:

101.86 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

1.86 µM

Cell viability:

102.44 %

Vehicle controls validity:

valid

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

Imax [442D]

Value:

7.57

At concentration:

62.5 other: μM

Vehicle controls validity:

valid

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

16.28

At concentration:

62.5 other: μM

Vehicle controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

Calculation of Maximal Average Fold Induction and Determination of Luciferase Activity:
Luminescence readings and fold increases are given in Table 9.1 and Table 9.2. (attached)
The Imax and EC1.5 values were calculated as follows:

Parameter Experiment 1 Experiment 2 Mean*
Imax 7.57# 16.28# 11.93
EC1.5 2.20 μM 1.86 μM 2.03 μM
* Mean EC1.5 is the geometric mean
# Luciferase activity induction statistically significant above the threshold of 1.5 In both experiments, there was an apparent dose response observed for luciferase induction up to concentrations of 62.5 μM. A reduction in luciferase induction was observed at the higher test concentrations (125 to 2000 μM), however this was considered to be due to the increased cytotoxicity at these doses (viability fell to 0.28 and 0.16 at 2000 μM, in Experiments 1 and 2, respectively).

Viability:
MTT-absorbance readings are given in Table 9.3 (attached)
No significant cytotoxic effects occurred at the lowest concentration leading to >1.5-fold luciferase induction as cell viability at the EC1.5 determining concentrations was 101.86% and 102.44% in Experiments 1 and 2, respectively.
The cell viability was above 70% at concentrations of 0.98 to 62.5 μM in both experiments.
IC50 and IC30 values were calculated as follows:
Parameter Experiment 1 Experiment 2 Geometric Mean
IC50 98.53 μM 93.27 μM 95.86 μM
IC30 87.39 μM 80.31 μM 83.78 μM
The EC1.5 value was therefore less than the IC30 value in both Experiment 1 and 2.

Assay Acceptance:
Luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 at concentrations of 8 to 64 μM in Experiment 1 and at concentrations of 16 to 64 μM in Experiment 2.
The EC1.5 value for the positive control was 7.45 and 8.38 μM in Experiments 1 and 2, respectively. The average induction in the three replicates for the positive control at 64 μM were 7.97 and 7.62 in Experiments 1 and 2, respectively.
The average coefficient of variation of the luminescence reading for the negative control (DMSO) was 10.98% and 9.91% in Experiments 1 and 2, respectively.

A summary of historical control data for the KeratinoSens™ assay is attached.

Executive summary:

The study was conducted to investigate the potential of N,N-dimethyldodec-9 -enamide to induce genes that are regulated by the antioxidant response element (ARE). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and nonsensitisers for the purpose of hazard classification and labelling.

The test article was dissolved in dimethyl sulfoxide (DMSO) to the final stock concentration (200 mM). Serial dilutions were then made using DMSO to obtain 12 master concentrations of the test article in the range 0.098 to 200 mM. The master concentrations were then further diluted 25-fold into culture medium containing serum and finally used for treatment with a further 4 -fold dilution factor so that the final concentrations ranged from 0.98 to 2000 μM.

Aliquots of 50 μL of each of the final concentrations were transferred to give three luciferase replicates on a white-walled plate and a single viability replicate on a clear-walled plate. Each plate was sealed using a plate sealer and then incubated for 48±1 hours in a humidified incubator set to 37°C, 5% CO2.

After the 48-hour exposure period, the medium in the viability plate was replaced with fresh medium containing 3 -(4,5 -dimethylthiazol-2 -yl)-2,5 -diphenyltetrazolium bromide (MTT). The plate was sealed and incubated for 4 hours in an incubator set to 37°C, 5% CO2. The MTT medium was then removed and 200 μL SDS (at 10% w/v) added per well. The plate was sealed and placed into an incubator set to 37°C, 5% CO2, and left overnight. After the overnight incubation, the plate was shaken to ensure homogeneity of the solution in the wells and then absorption read at 600 nm.

After the 48-hour exposure period, the cells in the luciferase plate were washed with phosphate buffered saline and lysis buffer for luminescence readings was added to each well. The plate was then incubated for 20 minutes in an incubator set to 25°C, loaded into the luminescence plate reader and read.

The results are summarised as follows:

Criteria	Experiment 1	Experiment 2
Imax	7.57	16.28
Cell Viability	101.86%	102.44%
EC1.5	2.20 μM	1.86 μM
Dose Response	Yes	Yes
Prediction	Positive	Positive

The average coefficient of variation of the luminescence reading for the negative control (DMSO) was <20% in each experiment; luciferase activity induction obtained with the positive control was statistically significant above the threshold of 1.5 in at least one tested concentration in each experiment and a clear positive dose response was observed for luciferase induction for positive control treatments, thereby confirming assay validity.

All four conditions required for a positive prediction were met in both experiments, therefore N,N-dimethyldodec-9-enamide was considered to be positive in the ARENrf2 Luciferase Test.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

31 January - 15 February 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442E (In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT))

Version / remarks:

June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

human Cell Line Activation Test (h-CLAT)

Justification for non-LLNA method:

The human Cell Line Activation Test (h-CLAT) has been evaluated in a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM)-coordinated validation study and subsequent independent peer review by the EURL ECVAM Scientific Advisory Committee (ESAC) and has been recommended to be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

Details of test system:

THP-1 cell line [442E]

Details on the study design:

TEST SYSTEM
- Specifications: Human monocytic leukaemia cell line, THP-1 (ATCC® TIB-202™, an immortalised human monocytic leukaemia cell line, used as a surrogate for dendritic cells), was supplied by American Type Culture Collection (ATCC), Manassas, VA 20110 USA.

- Identification: The test system was suitably labelled to clearly identify the study number, test article, test article concentration, positive and solvent/vehicle controls.

- Cell Culture Maintenance: THP-1 cells were cultured in a humidified incubator set to 37ºC, 5% CO2, in RPMI-1640 medium supplemented with 10% heat inactivated (HI) foetal bovine serum (FBS), 0.05 mM 2-mercaptoethanol, 100 units/mL penicillin and 100 μg/mL streptomycin. The cells were passaged every 2-3 days at a density of 0.1 to 0.2 x 106 cells/mL and maintained at a density from 0.1 x 106 to 0.8 x 106 cells/mL. Cell density did not exceed 1 x 106 cells/mL, and cells did not exceed 30 passages.

- Reactivity Check: This was performed using DNCB (CAS no. 97-00-7, ≥99% purity), nickel sulphate (CAS no. 10101-97-0, ≥99% purity) and lactic acid (CAS no. 50-21-5, ≥85% purity) two weeks after thawing. DNCB and nickel sulphate should produce a positive response of both CD86 and CD54 and lactic acid should produce a negative response of both CD86 and CD54. Only cells which passed the reactivity check were used for the assay.

- Plate Preparation: THP-1 cells were pre-cultured in culture flasks either at a density of 0.2 x 106 cells/mL for 48 hours or at a density of 0.1 x 106 cells/ml for 72 hours. On the days of testing, cells were harvested from the flasks and were resuspended with fresh culture medium at 2 x 106 cells/mL. The cells were then distributed into a 24 well flat-bottom plate (500 μL/well) for the expression assay, or into a 96-well flat-bottomed plate (80 μL/1.6 x 105 cells per well) for the DRF assay.

TEST ARTICLE FORMULATION
- Dose Finding Assay:
A dose finding assay was performed to determine the CV75, being the test item concentration that results in 75% cell viability (CV) compared to the solvent/vehicle control.
The test article was formulated at 500 mg/mL in DMSO then eight stock solutions were prepared by 2-fold serial dilutions using the corresponding solvent. The stock solutions were then further diluted 250-fold in culture medium (working solutions). The working solutions were used for exposure by adding an equal volume of working solution to the volume of THP-1 cell suspension in the plate to obtain a final range of concentrations in the plate of 7.8-1000 μg/mL.
The test article was prepared shortly before and on the same day as testing. Preparation was conducted under subdued lighting with the aid of vortex mixing.

- CD86/CD54 Expression Measurement
The test article was formulated at 11.214 mg/mL in DMSO then eight stock solutions of each test article were prepared by 1.2-fold serial dilutions using the corresponding solvent, and then further diluted 250-fold into the culture medium to give eight
working solutions ranging from 0.335 x CV75 to 1.2 x CV75. The working solutions were used for exposure by adding an equal volume of working solution to the volume of THP-1 cell suspension in the plate to obtain a final range of concentrations in the plate of 6.26-22.43 μg/mL.
The test article was prepared shortly before and on the same day as testing. Preparation was conducted under subdued lighting.

STUDY DESIGN
- Dose Finding Assay:
The test article working solutions or solvent controls were mixed 1:1 (v/v) with the cell suspensions in the 96-well plates. The plates were sealed and then incubated for 24±0.5 hours (incubator set to 37ºC, 5% CO2).
After the 24-hour incubation period, all cells from the 96-well flat-bottomed plate were transferred into a 96-well round-bottomed plate. The cells were washed at least twice in 200 μL of phosphate buffered saline containing 0.1% bovine serum albumin
(FACS buffer) and re-suspended in 190 μL of FACS buffer. 10 μL of propidium iodide solution (PI) was added just before FACS analysis (final concentration of PI = 0.625 μg/mL). PI uptake was analysed using flow cytometry with the acquisition channel FL-3. A total of 10,000 viable cells were acquired.
Cell viability was calculated using the following equation:
Cell Viability = 100x number of living cells/ total number of acquired cells
The CV75 value, i.e. a concentration showing 75% of THP-1 cell survival (25% cytotoxicity), was calculated by log-linear interpolation using the following equation:
Log CV75 = ((75 - c) x Log (b) - (75-a) x Log (d))/(a - c)
Where:
a was the minimum value of cell viability over 75% in testing groups
c was the maximum value of cell viability below 75% in testing groups
b and d were the concentrations showing the value of cell viability a and c respectively.

- CD86/CD54 Expression Measurement:
Two independent runs (experiments) were needed to drive a prediction. Each independent run was performed on the same day provided that for each run:
a) Independent fresh stock solutions and working solutions of the test article and antibody solutions were prepared and
b) Independently harvested cells were used (i.e. cells were collected from different culture flasks). The cells may have come from the same passage.
On the days of testing, cells harvested from the flasks were resuspended with fresh culture medium at 2 x 106 cells/mL. The cells were then distributed into a 24-well plate (500 μL/1 x 106 cells per well). The test article working solution or solvent control was mixed 1:1 (v/v) with the cell suspensions in the 24-well plates. The plates were sealed and then incubated for 24±0.5 hours (incubator set to 37ºC, 5% CO2).
After the 24-hour incubation period, the cells were transferred into sample tubes, collected by centrifugation (approximately 250 g, 5 minutes) and washed twice with 1 mL of FACS buffer. After washing, the cells were blocked with 600 μL of blocking
solution (FACS buffer containing 0.01% (w/v) globulin) on ice for 15 minutes.
After blocking, the cells were split into three aliquots of 180 μL into a 96-well plate and centrifuged (approximately 250 g, 3 minutes). After centrifugation, the cells were stained with 50 μL of FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes. The stained cells were washed three times with an excess of FACS buffer, re-suspended in FACS buffer and 12.5 μg/mL PI solution was added (to give a final PI concentration of 0.625 μg/mL). The expression levels of CD86 and CD54 and cell viability were analysed using flow cytometry.

DATA EVALUATION
- Analysis of Results:
Based on the geometric mean fluorescence intensity (MFI), the relative fluorescence intensity (RFI) of CD86 and CD54 for the positive control cells and test article-treated cells were calculated according to the following equation:
RFI = 100x(MFI of chemical-treated cells – MFI of chemical-treated isotype control cells)/( MFI of solvent/vehicle-treated cells – MFI of solvent/vehicle-treated isotype control cells)
The cell viability from the isotype control cells (stained with mouse IgG1 antibodies) was calculated using the following equation:
Cell Viability = (living cells/total number of acquired cells)x100

- Prediction Model:
An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs, otherwise the h-CLAT prediction is considered NEGATIVE:
• The RFI of CD86 is ≥150% at any tested concentration (with cell viability ≥50%)
• The RFI of CD54 is ≥200% at any tested concentration (with cell viability ≥50%).

- Calculation of Effective Concentration (EC) Values:
For test articles predicted as POSITIVE, two EC values, the EC150 for CD86 and the EC200 for CD54, are calculated as follows:
EC150 (for CD86) = Bconcentration + [(150 – BRFI) / (ARFI – BRFI) x (Aconcentration – Bconcentration)]
EC200 (for CD54) = Bconcentration + [(200 – BRFI) / (ARFI – BRFI) x (Aconcentration– Bconcentration)]
Where:
Aconcentration is the lowest concentration in μg/mL with RFI >150 (CD86) or 200 (CD54)
Bconcentration is the highest concentration in μg/mL with RFI <150 (CD86) or 200 (CD54)
ARFI is the RFI at the lowest concentration with RFI >150 (CD86) or 200 (CD54)
BRFI is the RFI at the highest concentration with RFI <150 (CD86) or 200 (CD54).

- Assay Acceptance Criteria:
• The cell viabilities of medium and solvent control should be higher than 90%
• In the solvent/vehicle control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%)
• For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control should be >105%
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%) and cell viability should be more than 50%
• For the test article, the cell viability should be more than 50% in at least four tested doses in each run.

Vehicle / solvent control:

DMSO

Positive control:

dinitrochlorobenzene (DNCB) [442E]

Positive control results:

For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54, and cell viability was >50% in each independent run.

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

other: RFI (CD54)

Value:

227

At concentration:

22.43 other: μg/mL

Cell viability:

>50%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

other: RFI (CD54)

Value:

216

At concentration:

22.43 other: μg/mL

Cell viability:

>50%

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Group:

test chemical

Run / experiment:

mean

Parameter:

EC200, CD54 [442E]

Value:

21.39 µg/mL

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Outcome of the prediction model:

positive [in vitro/in chemico]

Other effects / acceptance of results:

Dose Finding Assay
The cell viability results are given in Table 7.1 below
The CV75 value for the test article was 18.69 μg/mL.

CD86/CD54 Expression Results
Geometric mean fluorescence intensity (MFI) and viability results are given in Table 7.2. below.
The relative fluorescence intensity (RFI) values for the test article are shown in the table below.
In Experiment 1, the RFI values for CD86 were <150% at all tested concentrations. The RFI values for CD54 were >200% with cell viability >50% at the highest tested concentration only (22.43 μg/mL). In Experiment 2, the RFI values for CD86 were <150% at all tested concentrations. The RFI values for CD54 were >200% with cell viability >50% at the highest tested concentration only (22.43 μg/mL).
The EC200 value for CD54 calculated by linear regression of endpoint assay data was 21.39 μg/mL. No EC150 value was calculated for CD86 as this marker was negative in both experiments.

Assay Acceptance Criteria Results
All assay acceptance criteria were met.
The cell viabilities of medium and solvent/vehicle control were higher than 90% in each independent run.
In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%).
For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.
For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54, and cell viability was >50% in each independent run.
For the test article, the cell viability was more than 50% in all tested concentrations in each independent run.

Table 7.1 Dose Finding Assay: Cell Viability

 

	Viability (%) at Concentration (μg/mL)
Run	7.81	15.63	31.25	62.5	125	250	500	1000
1	98.3	93.4	26.1	0.6	0.1	0.1	0.1	0.1
2	98.4	93.2	18.1	0.4	0.1	0.1	0.2	0.1

Table 7.2 Expression Assay: MFI and Cell Viability Values

Experiment 1

	MFI (Geo Mean)			Corrected MFI		Viability
Concentration (μg/mL)	CD86	CD54	Isotype	CD86	CD54	IgG	CD86	CD54
6.26	1137	610	458	679	152	98.3	98.2	98.3
7.51	1191	609	459	732	150	98.3	98.0	97.9
9.01	1215	626	466	749	160	98.2	98.3	97.9
10.82	1168	648	461	707	187	98.2	98.0	98.1
12.98	1237	684	475	762	209	97.5	97.4	97.4
15.58	1345	745	535	810	210	96.5	96.6	96.6
18.69	1187	796	509	678	287	95.2	94.6	94.8
22.43	1500	836	495	1005	341	91.0	90.3	90.2
Culture medium	1248	634	500	748	134	98.6	98.9	98.8
DMSO 0.2%	1265	635	485	780	150	99.1	99.0	98.8
DNCB 4 μg/mL	3126	1630	550	2576	1080	91.1	88.8	90.3

Experiment 2

	MFI (Geo Mean)			Corrected MFI		Viability
Concentration (μg/mL)	CD86	CD54	Isotype	CD86	CD54	IgG	CD86	CD54
6.26	1457	619	471	986	148	98.3	98.2	98.2
7.51	1314	618	458	856	160	98.6	98.3	98.6
9.01	1378	643	464	914	179	98.3	98.0	97.7
10.82	1299	649	463	836	186	98.1	97.9	97.8
12.98	1372	677	483	889	194	97.8	97.1	97.1
15.58	1316	704	485	831	219	97.2	97.2	97.2
18.69	1292	745	488	804	257	96.8	96.0	96.4
22.43	1294	839	492	802	347	91.6	90.9	90.8
Cullture medium	1714	654	511	1203	143	98.8	98.5	98.8
DMSO 0.2%	1786	671	510	1276	161	98.8	98.3	98.6
DNCB 4 μg/mL	4852	1697	593	4259	1104	90.0	88.5	89.0

Relative fluorescence intensity (RFI) values for the test article

Concentration     (μg/mL)	RFI (CD86)		RFI (CD54)
	Exp 1	Exp 2	Exp 1	Exp 2
6.26	87	77	101	92
7.51	94	67	100	99
9.01	96	72	107	111
10.82	91	66	125	116
12.98	98	70	139	121
15.58	104	65	140	136
18.69	87	63	191	160
22.43	129	63	227	216
Solvent/vehicle control (DMSO)	104	106	112	113
Positive control (DNCB)	330	334	720	686

Assay Acceptance Criteria Results

All assay acceptance criteria were met.

The cell viabilities of medium and solvent/vehicle control were higher than 90% in each independent run.

In the solvent control, RFI values of both CD86 and CD54 did not exceed the positive criteria (CD86 RFI ≥150% and CD54 RFI ≥200%).

For both medium and solvent/vehicle controls, the MFI ratio of both CD86 and CD54 to isotype control was >105% on all occasions.

For the positive control, RFI values were ≥150% for CD86 and ≥200% for CD54, and cell viability was >50% in each independent run.

For the test article, the cell viability was more than 50% in all tested concentrations in each independent run.

Executive summary:

The study was conducted to investigate the potential of N,N-dimethyldodec-9 -enamide to activate monocytes and dendritic cells in the human monocytic leukaemia cell line THP-1, by quantifying changes in the expression of cell surface markers (CD86 and CD54). The data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling.

The human Cell Line Activation Test (h-CLAT) was performed according to OECD TG 442E.

For the dose finding assay, the test article was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 500 mg/mL giving a maximum test concentration of 1000 μg/mL. A reduction in viability was noted at the highest six concentrations resulting in mean CV75 of 18.69 μg/mL.

For the expression measurements, test concentrations in a range from 6.26 to 22.43 μg/mL (after dilution in medium) were used. Aliquots of 500 μL of each of the working solutions were mixed 1:1 with cell suspensions at 1 x 106 cells per well. After blocking, the cells were stained with FITC-labelled anti-CD86, anti-CD54 or mouse IgG1 antibodies on ice for 30 minutes. The stained cells were washed, re-suspended in FACS buffer and propidium iodide solution was added. The expression levels of CD86 and CD54 and cell viability were

analysed using flow cytometry.

In Experiment 1, the RFI values for CD86 were <150% at all tested concentrations. The RFI values for CD54 were >200% with cell viability >50% at the highest tested concentration only (22.43 μg/mL). In Experiment 2, the RFI values for CD86 were <150% at all tested concentrations. The RFI values for CD54 were >200% with cell viability >50% at the highest tested concentration only (22.43 μg/mL). The EC200 value for CD54 was calculated to be 21.39 μg/mL. No EC150 value was calculated for CD86 as this marker was negative in both experiments.

All acceptance criteria of the h-CLAT assay parameters were met in each experiment.

The test article, N,N-dimethyldodec-9 -enamide, was considered to be positive in the human Cell Line Activation Test.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

A study was performed according to OECD TG 429 to assess the potential of the test article, N,N-dimethyldodec-9-enamide, to cause skin sensitisation in the mouse.

Following a preliminary screening test using the undiluted test article, the main study was conducted using the undiluted test article and 50 and 25% v/v formulations in 80% v/v acetone in olive oil. In order to calculate an EC3 value, a second experiment was conducted using 2.5%, 5% and 10% v/v formulations.

Groups of four female CBA/CaCrl mice were subjected to topical applications of vehicle control or of one of the test formulations to the outer aspect of the auditory pinnae once daily on Days 1, 2 and 3. On Day 6 a 20 μCi dose of tritiated 3H-methyl thymidine was injected intravenously into each mouse. Approximately five hours later the auricular lymph nodes were recovered from each animal. The pairs of nodes from each animal were pooled and suspensions of the cellular components of the lymph nodes were prepared in 5% w/v trichloroacetic acid and processed through a scintillation counter.

Test results are expressed in terms of Stimulation Indices, the ratios of the mean scintillation counts obtained from the test groups relative to the corresponding mean scintillation count obtained from controls. The threshold level for the Stimulation Index to be considered a positive indicator of the potential to cause skin sensitisation is 3.0.

	Concentration of Test Article in Applied Formulation (% v/v)
	2.5	5	10	25	50	100
Stimulation Index	0.47	0.81	1.47	3.04	3.65	5.59

The EC3 was calculated to be 24.6%.

The Local Lymph Node Assay demonstrated that N,N-dimethyldodec-9-enamide has the potential to cause skin sensitisation.

This study is supported by in vitro/in chemico studies performed according to OECD TG 442C, 442D and 442E which identified the substances as a skin sensitiser in a WoE approach.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The EC3 was calculated to be 24.6% in a mouse lymphoma assay performed according to OECD TG 429. On this basis the substance is classified for Skin sensitisation, sub-category 1B.