N-{[Benzyl(ethyl)amino]-[(chloro-cyano-nitrophenyl)substituted]phenyl}acetamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (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

Reference

Name:

Unnamed

Type:

Constituent

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Remarks:

/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo, The Netherlands
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: The animals were free from known disease /pathogen as per the health monitoring report based on FELASA recommendation.
- Age at study initiation: 10 to 11 weeks
- Weight at study initiation: 17.8 to 21.5 grams
- Housing: Animals were housed individually (to avoid licking of test item by cage mates) in solid floor standard polysulfone cages (Size: Approximately L 360 x B 205 x H 140 mm), with stainless steel top grill having facilities for providing pelletted food and drinking water in polycarbonate bottle with stainless steel sipper tubes. Steam sterilized corn cob was used as bedding and changed along with the cage once a week. Cages were placed on tiered racks. Mouse huts were provided in the cages as environment enrichment to minimize animal stress and promote overall well-being during the in-life phase of the study.
- Diet: The experimental animals were provided ad libitum Teklad Certified (2014C) Global 14% Protein Rodent Maintenance Diet-Pellet (Certified) manufactured by Envigo, P.O. Box 44220, Madison, WI 53744-4220. The food, water and corn cob provided to the animals were tested for
contaminants. Contaminant analysis reports of food, water and corn cob are documented in the study file.
- Water: Deep bore-well water passed through activated charcoal filter and exposed to UV rays in Aquaguard on-line water filter-cum-purifier (manufactured by Eureka Forbes Ltd., Mumbai - 400 001, India).
- Acclimation period: 6 days before start of the treatment.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23
- Humidity (%): 64 - 66
- Air changes (per hr): 13.2
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness, light hours being 06:00 to 18:00 hours approximately.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

Screening study: 0, 2.5, 5, 10, 20 and 40% w/v of test substance in DMSO.
LLNA main study: 10, 20 and 40% w/v FAT 41047/A TE in DMSO

No. of animals per dose:

6

Details on study design:

Irritation Screening Test : Screening test was performed on six female mice (one mouse/concentration) in dosing 6 groups viz Vehicle (DMSO) and concentration of 2.5, 5, 10, 20 and 40% w/v test item in DMSO. Both ears of six female mice (one mouse/concentration) were topically treated once daily for three consecutive days with one of the concentrations of the test item. The test item was stable in DMSO. Results from this screening study were used to determine the dosing concentrations of FAT 41047/A TE for the main LLNA study.

None of the tested concentrations elicited any irritation reaction, no increased ear thickness and ear punch weights.

MAIN STUDY :
The application of the test item (25 μL/ear) was made on the dorsum of both ears. Six female mice/group received the vehicle (DMSO) or the positive control substance (25% v/v α-hexylcinnamaldehyde), or 10, 20 and 40% w/v test item in DMSO, once daily for three consecutive days. Both ears were observed for skin reaction prior to application of the test item (on day 1, 2 and 3), and on day 6. All mice were weighed on days 1 and 6.

Preparation of 3H-Methyl Thymidine (3H -TdR):
A solution of 3H-Methyl Thymidine (3H -TdR) (80 μCi/mL)[specific activity 6.7 Ci/mmol; Perkin Elmer, USA] in sterile phosphate buffer saline (PBS) was prepared freshly. The prepared working solution of 3H-TdR was analyzed for radioactivity.

Injection of 3H-Methyl Thymidine (3H -TdR):
On day 6, a volume of 250 μL (20 μCi) of 3H-TdR in PBS was administered to each mouse via the lateral tail vein.

Collection of auricular lymph nodes:
Approximately five hours post injection of 3H-TdR, animals were euthanized using isoflurane anaesthesia. The auricular lymph nodes (bilateral) were excised and placed in PBS, processed and the radioactivity was counted.

Ear punch weight
The animals were euthanized using isoflurane anesthesia and both ears (left and right) of mice were punched at the apical area using a biopsy punch (Ø 6 mm). For each animal both punches were immediately weighed using an analytical
balance.

Processing of auricular lymph nodes
A single cell suspension of the lymph node cells (LNC) from each mouse was prepared by gentle mechanical disaggregation using a tissue homogeniser
(Stomacher 80 MicroBiomaster, Seward Ltd, United Kingdom) for 30 seconds at medium speed using PBS (approx. 10 mL).
After all the nodes had been processed, the tubes were centrifuged at 200 x g for 10 minutes at 4°C. The supernatant was poured into a container for radiolabel waste collection. 10 mL of PBS was added to each tube and inverted to resuspend the pellet. The tubes were centrifuged as described above and the supernatant was poured off. After the second wash, the cell pellet was suspended in 3.0 mL of 5% trichloroacetic acid (TCA) and stored overnight at 2-8°C for approximately 18 hours. Clumping of LNC was avoided by ensuring that the pellet was completely resuspended in a small volume of PBS before making up to the final volume. The suspended precipitates were centrifuged at 200 x g for 10 minutes at 4 °C and the supernatant was poured off into container for radiolabel waste collection. The pellet from each mouse was reconstituted in 1 mL of 5% TCA and subsequently transferred to a scintillation vial containing 10 mL of a scintillation cocktail (Insta-Gel Plus, PerkinElmer, USA). Two additional 2 mL aliquots of water were used to rinse the tubes and the rinses were added to the scintillation vial containing 1 mL of the pellet in TCA and cocktail. The samples were mixed using a snapping wrist action.
The radioactivity in each precipitate was measured for 5 minutes using a ßscintillation counter (Tricarb 2900-TR, Packard Instruments, USA) as
disintegrations per minute (dpm) per mouse.

Positive control substance(s):

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

Statistics:

A mean dpm value ± SD (standard deviation) was calculated for each group and the stimulation index (SI) was calculated using the absolute dpm value for each mouse as the numerator, and the mean dpm value from the vehicle-treated mice as the denominator.
1. The % increase in ear thickness was calculated for each ear using the following equation:
% Ear swelling = [(B – A)/A] x 100

Where, A = ear thickness measurement on Day 1 (μm)
B = ear thickness measurement on Day 3 or 6 (μm)

2. The SI was calculated for each mouse using the following equation:
SI = Disintegrations per minute (dpm) of individual mouse / Average dpm of the vehicle control mice

Means and SD were generated for body weight data (absolute and gain) and LLNA response (dpm and SI values). The body weight and dpm data were analysed by one-way analysis of variance. When the differences were indicated by the ANOVA, a comparison of treated vs. control groups was done using a Dunnett’s t-test (p<0.05).
Statistically significant differences (p<0.05), indicated by the aforementioned tests, are designated by the superscripts throughout the report.

Results and discussion

Positive control results:

Positive control : 25% HCA in DMSO elicited a stimulation index (SI) of 5.07.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

ca. 2.25

Test group / Remarks:

10% w/v test item

Results 2

Key result

Parameter:

SI

Value:

ca. 2.36

Test group / Remarks:

20% w/v test item

Results 3

Key result

Parameter:

SI

Value:

ca. 2.5

Test group / Remarks:

40% w/v test item

Cellular proliferation data / Observations:

The analyzed radioactivity of 3H-TdR working solution was 78.65 μCi/mL against the nominal concentration of 80 μCi/mL.
There were no clinical signs, no erythema at the site of application and no significant effect on body weight gains.
The test item FAT 41047/A TE produces a Stimulation Index (SI) < 3, it is considered “Negative” for sensitization, and therefore an EC3 was not determined.

Any other information on results incl. tables

Summary of Disintegrations Per Minute (DPM) for³H-Methyl Thymidine Incorporation in Auricular Lymph Nodes and Stimulation Index (SI)

Group and Dose concentration	No. of mice		DPM / Mouse	SI
G1 Vehicle: DMSO	6
		Mean SD	882.50 260.39	1.00 0.29
G2 25% v/v HCA	6		+
		Mean SD	4476.00 1145.33	5.07 1.30
G3 10% w/v test item	6		+
		Mean SD	1988.50 295.59	2.25 0.33
G4 20% w/v test item	6		+
		Mean SD	2079.67 325.10	2.36 0.37
G5 40% w/v test item	6		+
		Mean	2206.17	2.50
		SD	449.45	0.51

+: Significantly higher than the vehicle control group

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item FAT 41047/A TE did not demonstrate dermal sensitization potential in the mouse LLNA, as the lymph nodes draining the area of topical application did not elicit a proliferative response greater than the 3X threshold.

Executive summary:

A Local Lymph Node Assay (LLNA) was conducted by measuring the lymphocyte proliferative response from auricular lymph nodes following topical application to the female CBA/Ca mouse ear accroding to OECD test guideline no 429.

SCREENING STUDY:

Once daily topical application of the vehicle Dimethyl sulfoxide (DMSO) and 2.5, 5, 10, 20 and 40% w/v FAT 41047/A TE in DMSO were performed to one animal at each dose level. There were no clinical signs, no erythema at the site of application, no significant increase in the ear thickness and ear punch weights and no effect on body weight. Based on these findings, dose concentrations of 10, 20 and 40% w/v were selected for the main study.

LLNA MAIN STUDY:

Six female CBA/Ca mice/group received the vehicle (DMSO) or 25% α-hexylcinnamaldehyde (HCA: positive control in DMSO) or 10, 20 and 40% w/v FAT 41047/A TE in DMSO on days 1 to 3.

On day 6, the uptake of 3H-methyl thymidine into the auricular lymph nodes draining the site of test item application was measured five hours post administration. Proper conduct of the LLNA was confirmed via a positive response using 25% α-hexylcinnamaldehyde, a contact sensitizer, which elicited proliferation with a Stimulation Index (SI) value of 5.07, in comparison to vehicle-treated mice.

The test item FAT 41047/A TE at dose concentrations of 10, 20 and 40% w/v elicited proliferative response with SI of 2.25, 2.36 and 2.50, respectively in comparison with the vehicle-treated mice. There were no clinical signs, no local skin reactions at the tested concentrations and treatment had no significant effect on body weight gain.

The test item FAT 41047/A TE did not demonstrate dermal sensitization potential in the mouse LLNA, as the lymph nodes draining the area of topical application did not elicit a proliferative response greater than the 3X threshold, thus not meeting the classification criteria to be classified as skin sensitizer, as per Globally Harmonized System of Classification and Labelling of Chemicals (GHS).