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Diss Factsheets

Administrative data

Description of key information

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:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD guideline 429 and in accordance with GLP
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Principles of method if other than guideline:
Guidelines followed
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
other: MICE
Strain:
other: CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd
- Age at study initiation:11 to 12 weeks
- Weight at study initiation:17.7 to 21.0 grams
- Housing: Animals were housed individually (to avoid licking of test item by cage mates)
- Diet (e.g. ad libitum): ad libitum standard pelletted food (Ssniff mice pellet food – maintenance manufactured by Ssniff Spezialdiäten GmbH., Ferdinand-Gabriel-Weg 16, D-59494 SÖest, Germany)
- 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: the animals were acclimatized for 5 days before start of the treatment. Animals were nulliparous and non-pregnant.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 24°C
- Humidity (%): 65 to 67 per cent
- Air changes (per hr): Adequate fresh air supply of 13.3 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness, light hours being 06.00 to 18.00 hours approximately.

IN-LIFE DATES: From: To:
Route:
epicutaneous, open
Vehicle:
other: N,N-dimethylformamide
Concentration / amount:
10, 20 and 40% w/v
Route:
other:
Vehicle:
other: N,N-dimethylformamide
Concentration / amount:
10, 20 and 40% w/v
No. of animals per dose:
6
Details on study design:
RANGE FINDING TESTS: Once daily topical applications of vehicle- N,N-dimethylformamide (DMF), 2.5, 5, 10, 20 and 40% w/v of test item in DMF were applied to one animal at each dose level for 3 days. 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. Results from this study were used to determine the dosing concentration for the main study.

MAIN STUDY: Six female CBA/Ca mice/group received the vehicle (DMF) or 25% α-hexylcinnamaldehyde (HCA: positive control in DMF) or 10, 20 and 40% w/v test item in DMF on days 1 to 3. On day 6, uptake of 3H-methyl thymidine into the auricular lymph nodes draining the site of test item application was measured approximately five hours post administration.
Challenge controls:
None
Positive control substance(s):
yes
Remarks:
25% α-hexylcinnamaldehyde
Vehicle:
dimethylformamide
Concentration:
No data
No. of animals per dose:
No data
Details on study design:
RANGE FINDING TESTS: Once daily topical applications of vehicle- N,N-dimethylformamide (DMF), 2.5, 5, 10, 20 and 40% w/v test item in DMF were applied to one animal at each dose level for 3 days. 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. Results from this study were used to determine the dosing concentration for the main study.

- Compound solubility:
The miscibility/solubility of the test item was tested using acetone/olive oil [AOO](4:1 v/v), N,N-dimethylformamide (DMF), methyl ethyl ketone (MEK), propylene glycol (PG), dimethyl sulfoxide (DMSO) and 1% Pluronic® L92 at the concentrations of 90, 75, 60, 50, 40, 25 and 10 % w/v. The test item formed solution/ suspension at ≤25% w/v dilutions with AOO, DMSO, PG, MEK, 1% Pluronic® L92 and ≤40% w/v dilutions with DMF. All the samples were sonicated for 10 minutes at 35ºC. The maximum test item solubility was observed in DMF, hence DMF was preferred and selected as the vehicle.

- Irritation:
Six female CBA/Ca mice/group received the vehicle (DMF) or 25% α-hexylcinnamaldehyde (HCA: positive control in DMF) or 10, 20 and 40% w/v test item in DMF on days 1 to 3. On day 6, uptake of 3H-methyl thymidine into the auricular lymph nodes draining the site of test item application was measured approximately five hours post administration. There were no clinical signs, no local skin reactions at the tested concentrations and treatment had no significant effect on body weight gain.


- Lymph node proliferation response: The test item at dose concentrations of 10, 20 and 40% w/v elicited proliferative response with SI of 2.57, 3.56 and 4.69, respectively in comparison with the vehicle-treated mice.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
Group Dose concentration Vehicle Volume Applied to each ear Sex No. of animals Animal Nos.
G1(Vehicle Control) 0 DMF 25 µL F 6 Mb4801 to Mb4806
G2(Positive Control) 25% v/v DMF 25 µL F 6 Mb4807 to Mb4812
G3 10% w/v DMF 25 µL F 6 Mb4813 to Mb4818
G4 20% w/v DMF 25 µL F 6 Mb4819 to Mb4824
G5 40% w/v DMF 25 µL F 6 Mb4825 to Mb4830

- Name of test method: LOCAL LYMPH NODE ASSAY

- Criteria used to consider a positive response: Stimulation Index (SI) >3 in the LLNA test is normally considered as “positive” for a dermal sensitization potential

TREATMENT PREPARATION AND ADMINISTRATION:
The required quantity of the test item was mixed with DMF and sonicated for 10 minutes at 35ºC to obtain stock solution of 40% w/v and the stock solution was diluted with DMF to obtain concentrations of 10 and 20% w/v. The test item solutions were prepared daily just prior to dosing. Preparation of the dosing materials was documented in the study file. The concentrations of the dosing solutions were not verified analytically.

Six female mice/group received the vehicle (DMF, or the positive control substance (25% α-hexylcinnamaldehyde), or 10, 20 and 40% w/v test item in DMF, once daily for three consecutive days. The test item was administered using an adjustable micropipette with disposable tips. All mice received 25 µL of one concentration of the test item, spread over the dorsal surface of each ear in a manner to prevent test item loss (50 µL total/mouse). Similarly the vehicle was applied to the ears of one animal.
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.
Positive control results:
25% α-hexylcinnamaldehyde, elicited proliferation with a Mean Stimulation Index (SI) value of 6.08, in comparison to vehicle-treated mice.
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
Group and Dose concentration Stimulation Index G1(Vehicle : DMF) 1.00 G2(25% HCA in DMF) 6.08 G3(10% FAT 40866/A TE in DMF) 2.57 G4(20% FAT 40866/A TE in DMF) 3.56 G5(40% FAT 40866/A TE in DMF) 4.69
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Group and Dose concentration Mean DPM/Mouse G1(Vehicle : DMF) 644.00 G2(25% HCA in DMF) 3914.83 G3(10% FAT 40866/A TE in DMF) 1656.17 G4(20% FAT 40866/A TE in DMF) 2290.50 G5(40% FAT 40866/A TE in DMF) 3020.00

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

Values: Mean ± SD                                         

Group andDose concentration

 

No. of mice

DPM/Mouse

 

SI

 

G1(Vehicle : DMF)

6

 

 

644.00

1.00

200.67

0.31

 

 

 

G2(25% HCA in DMF)

 

6

+

 

3914.83

6.08

313.28

0.49

 

 

 

G3(10% FAT 40866/A TE in DMF)

6

+

 

1656.17

2.57

141.20

0.22

 

 

 

G4(20% FAT 40866/A TE in DMF)

6

+

 

2290.50

3.56

550.18

0.86

 

 

 

G5(40% FAT 40866/A TE in DMF)

 

6

+

 

3020.00

4.69

586.11

0.91

 

 

+: Significantly higher than the vehicle control group

DMF:N,N-dimethylformamide

HCA : α – Hexylcinnamaldehyde

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: other: ECETOC categorisation for relative skin sensitization potency
Conclusions:
A topical application with 10, 20 and 40% w/v FAT 40866/A TE in dimethylformamide elicited a Mean stimulation index (SI) of 2.57, 3.56 and 4.69, respectively. The EC3 value was 14.34%. The test item FAT 40866/A TE is categorised as ‘Weak sensitizer’, as per the ECETOC categorisation for relative skin sensitization potency.
Executive summary:

This Local Lymph Node Assay (LLNA) was conducted to evaluate the potential of FAT 40866/A TE to cause contact sensitization by measuring lymphocyte proliferative response from auricular lymph nodes following topical application of the test item to the female CBA/Ca mouse ear. Six female CBA/Ca mice/group received the vehicle  (DMF) or 25% α-hexylcinnamaldehyde (HCA:positive control in DMF) or  10, 20 and 40%w/v test item in DMF on days 1 to 3. On day 6, uptake of3H-methyl thymidine into the auricular lymph nodes draining the site of test item application was measured approximately 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 Mean Stimulation Index (SI) value of 6.08, in comparison to vehicle-treated mice.

There were no clinical signs, no local skin reactionsat the tested concentrations and treatment had no significant effect on body weight gain. The test item FAT 40866/A TE at dose concentrations of 10, 20 and 40% w/v elicited proliferative response with SI of 2.57, 3.56 and 4.69, respectively in comparison with the vehicle-treated mice. 

The EC3 value was 14.34%. The test item FAT 40866/A TE is categorised as ‘Weaksensitizer’, as per the ECETOC categorisation for relative skin sensitization potency.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

This Local Lymph Node Assay (LLNA) was conducted to evaluate the potential of FAT 40866/A TE to cause contact sensitisation by measuring lymphocyte proliferative response from auricular lymph nodes. Six female CBA/Ca mice/group received the vehicle (DMF) or 25% α-hexylcinnamaldehyde (HCA: positive control in DMF) or 10, 20 and 40% w/v test item in DMF on days 1 to 3. On day 6, uptake of 3H-methyl thymidine into the auricular lymph nodes draining the site of test item application was measured approximately five hours post administration. 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 40866/A TE at dose concentrations of 10, 20 and 40% w/v elicited proliferative response with SI of 2.57, 3.56 and 4.69, respectively in comparison with the vehicle-treated mice. The EC3 value was 14.34%.

The test item FAT 40866/A TE is categorised as ‘Weak sensitiser’, as per the ECETOC categorisation for relative skin sensitisation potency.


Migrated from Short description of key information:
The test item FAT 40866/A TE is categorised as ‘Weak sensitiser’, as per the ECETOC categorisation for relative skin sensitisation potency.

Justification for selection of skin sensitisation endpoint:
OECD guideline and GLP study

Justification for classification or non-classification

The test item FAT 40866/A TE is categorised as ‘Weak sensitiser’, as per the ECETOC categorisation for relative skin sensitisation potency.