[No public or meaningful name is available]

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

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Test material form:

solid: flakes

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source of test material: Clariant Plastics & Coatings (Deutschland) GmbH, BU Additives

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories,UK,Ltd
- Females (if applicable) nulliparous and non-pregnant: [yes]

- Age at study initiation: 9 to 11 weeks
- Weight at study initiation: 19.0 to 22.4 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 (e.g. ad libitum): 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
- Water (e.g. ad libitum): 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: 04 October 2018 to 09 October 2018
- Indication of any skin lesions: No

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 23°C
- Humidity (%): 59 to 67 per centy
- Air changes (per hr): 13.9 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness
- IN-LIFE DATES: From: 10 October 2018 To: 15 October 2018

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

The required quantity of the test item was mixed with MEK to get stock concentration of 60% w/v. The required volume of stock formulation was mixed with MEK to get dose formulations concentrations of 15 and 30% w/v.

No. of animals per dose:

6 females per group

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The current Guideline recommends vehicles/solvents such as acetone/olive oil (4:1 v/v), N,N-dimethylformamide (DMF), methyl ethyl ketone (MEK), propylene glycol (PG), dimethyl sulfoxide (DMSO) or 1% Pluronic® L92.
The miscibility / solubility of the test item was tested using AOO, DMF, MEK, PG, DMSO and 1% L92.
The test item is solid. Based on the maximum soluble concentration, MEK was selected as the vehicle

- Irritation: A concentration of 60% w/v was the maximum soluble concentration in Methyl ethyl ketone (MEK). Hence the highest possible dose concentration of 60% w/v in MEK was selected as highest dose concentration for testing.
Prior to the LLNA main study, the vehicle MEK and concentrations of 5, 10, 30, 45 and 60% w/v of the test item in MEK were evaluated for irritation potential as measured by erythema of the ears.

- Ear thickness measurements: Yes
- Erythema scores: Yes

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: The purpose of this Local Lymph Node Assay (LLNA) was to assess the potential of the test item to cause contact sensitization via measurement of lymph node proliferation following topical application of the test item to the dermal surfaces of the mouse ear.
- Criteria used to consider a positive response: The sensitizing potential was subsequently determined by the magnitude of the lymphocyte proliferative response in the auricular lymph nodes draining the ears. A test item that elicits a stimulation index [SI of >3 (i.e., 3-fold greater proliferation than vehicle control treated animals)] should be considered positive for a dermal sensitization potential.

TREATMENT PREPARATION AND ADMINISTRATION:
The application of the test item (25 µL/ear) was made on the dorsum of both ears as described above. Six female mice/group received the vehicle (MEK, or the positive control substance (25% v/v α-hexylcinnamaldehyde), or 15, 30 and 60 w/v test item in MEK, once daily for three consecutive days. Ears were inspected and skin reactions were evaluated prior to each application of the test item and on day 6. All mice were weighed on days 1 and 6.

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

Mean 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 as stated below:
+/- : Significantly higher (+)/lower (-) than the vehicle control group

Results and discussion

Positive control results:

The sensitivity of this LLNA test was demonstrated via the response from the positive control (25% HCA in MEK), which elicited a stimulation index (SI) of 12.33, in comparison with the vehicle-treated mice.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.54

Test group / Remarks:

15 % test substance

Results 2

Key result

Parameter:

SI

Value:

1.68

Test group / Remarks:

30 % test substance

Results 3

Key result

Parameter:

SI

Value:

1.78

Test group / Remarks:

60 % test substance

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION : Since the test item Licocare RBW 300 FL TP produced Stimulation Index (SI) < 3, it is considered “Negative” for skin sensitization, and therefore an EC3 value was not determined.


CLINICAL OBSERVATIONS: There were no clinical signs, no erythema at the site of application

BODY WEIGHTS There were no significant effect on body weight gains

Any other information on results incl. tables

 

TABLE 1.      Summary of Body Weight, body weight changesand clinical signs

Group and Dose concentration	No. of mice		Body weight (g)
			Day 1 (Initial)	Day 6	Weight change (day 6 – Initial)	Clinical signs
G1 Vehicle : MEK	6
		Mean	20.70	21.35	0.65	NAD
		SD	1.18	1.16	0.08
G2 25% v/v HCA	6
		Mean	20.70	21.33	0.63	NAD
		SD	1.03	0.97	0.08
G3 15% w/v test item	6
		Mean	20.72	21.33	0.62	NAD
		SD	1.02	1.06	0.08
G4 30% w/v test item	6
		Mean	20.65	21.25	0.60	NAD
		SD	0.87	0.82	0.06
G5 60% w/v test item	6
		Mean	20.78	21.40	0.62	NAD
		SD	0.96	0.99	0.04

MEK: Methyl ethyl ketone

NAD  : No Abnormality Detected

HCA  : α –Hexylcinnamaldehyde

 

 

 

 

TABLE 2.      Summaryof Local Reaction Scores at the Site of Application

                                                                                                          

Group and Dose concentration	No. of Mice		Erythema Score of both ears (Mean ± SD)
			Pre-treatment	Day 2	Day 3	Day 6
G1 Vehicle : MEK	6	Mean SD	0 0	0 0	0 0	0 0
G2 25% v/v HCA	6	Mean SD	0 0	0.83 0.41	1.00 0.00	1.00 0.00
G3 15% w/v test item	6	Mean SD	0 0	0 0	0 0	0 0
G4 30% w/v test item	6	Mean SD	0 0	0 0	0 0	0 0
G5 60% w/v test item	6	Mean SD	0 0	0 0	0 0	0 0

MEK: Methyl ethyl ketone

NAD: No Abnormality Detected

HCA :α–Hexylcinnamaldehyde   

 

TABLE 3.      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: MEK	6
		Mean	923.50	1.00
		SD	116.41	0.13
G2 25% v/v HCA	6		+
		Mean	11391.33	12.33
		SD	1832.36	1.98
G3 15% w/v test item	6		+
		Mean	1427.50	1.54
		SD	210.01	0.23
G4 30% w/v test item	6		+
		Mean	1547.50	1.68
		SD	360.28	0.39
G5 60% w/v test item	6		+
		Mean	1644.67	1.78
		SD	472.81	0.51

 

+: Significantly higher than the vehicle control group

 

MEK: Methyl ethyl ketone

HCA :α –Hexylcinnamaldehyde

 

 

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

A topical application with 15, 30 and 60% w/v Licocare RBW 300 FL TP in Methyl ethyl ketone elicited a stimulation index (SI) of 1.54, 1.68 and 1.78, respectively. The test item Licocare RBW 300 FL TP 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:

This Local Lymph Node Assay (LLNA) was conducted to evaluate the potential of the test item Licocare RBW 300 FL TP to cause contact sensitization by measuring the lymphocyte proliferative response from auricular lymph nodes following topical application to the female CBA/Ca mouse ear. 

Screening Study: Oncedaily topicalapplication of the vehicleMethyl ethyl ketone (MEK) and 5, 10, 30, 45 and 60% w/vLicocare RBW 300 FL TP  in MEKwere 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. The results of this screening test were used to determine the dosing concentration for the main study.  

LLNA main study: Six female CBA/Ca mice/group received the vehicle (MEK) or 25% α-hexylcinnamaldehyde (HCA: positive control in MEK) or15, 30 and 60% w/vLicocare RBW 300 FL TP in MEK on days 1 to 3. On day 6, the uptake of³H-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 12.33, in comparison to 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 Licocare RBW 300 FL TP at dose concentrations of15, 30 and 60% elicited proliferative response with SI of 1.54, 1.68 and 1.78, respectively in comparison with the vehicle-treated mice.    

The test item Licocare RBW 300 FL TP 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.