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Description of key information

There is a fully reliable LLNA available. 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.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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
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)
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Clariant Plastics & Coatings (Deutschland) GmbH, BU Additives
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
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
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.
Key result
Parameter:
SI
Value:
1.54
Test group / Remarks:
15 % test substance
Key result
Parameter:
SI
Value:
1.68
Test group / Remarks:
30 % test substance
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

 

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 :αHexylcinnamaldehyde   

 


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

 

 

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.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test item did not show skin sensitizing properties in the Local Lymph Node Assay according to the current OECD guidelines. Therefore, the substance does not have to be classified as a skin sensitizer.