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Toxicological information

Skin sensitisation

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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

Constituent 1
Test material form:
solid
Details on test material:
Test item : FAT 40875/A TE
Physical appearance : Dark red powder
Purity as per CoA : 86.4% all organic components
Batch No. : BOP 05-17 (BS-ROE 1550 NIM 25+26+27)
Manufactured date : 20 December 2017
Expiry date : 11 December 2022
pH : 4.3 (aq. soln. 2 %w/w at room-temperature)
Recommended storage condition: Refrigeration (+2 to +8 °C) and protection from light
Specific details on test material used for the study:
Test Item: FAT 40875/A TE
Physical Appearance: Dark red powder
Purity as per Certificate of Analysis (Content): 86.4% all organic components
Batch No.: BOP 05-17 (BS-ROE 1550 NIM 25+26+27)
Manufactured Date: December 20, 2017
Expiry Date: December 11, 2022
Recommended Storage Condition : Ambient (+2 to +8 °C), Protect from light
pH: 4.3 (aq. soln.(2% (w/w) at room-temperature).

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo, The Netherlands
- Females (if applicable) 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: 9 to 10 weeks
- Weight at study initiation: 18.3 to 21.8 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 (manufactured by Eureka Forbes Ltd., Mumbai 400001, India).

- Acclimation period: After physical examination for good health and the suitability for the study, the animals were acclimatized for 6 days before start of the treatment.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 to 24
- Humidity (%): 59 to 67
- 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.

Inlife dates: 28 March 2018 to 18 April 2018

Study design: in vivo (LLNA)

Vehicle:
other: 1% Pluronic L92
Concentration:
Screening study: 0, 2.5, 5, 10, 20 and 40% w/v test item in 1% Pluronic L92
LLNA main study: 0, 10, 20 and 40% w/v test item in 1% Pluronic L92
No. of animals per dose:
6
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The miscibility/solubility of the test item was tested using Acetone/olive oil, N,N-dimethylformamide (DMF), methyl ethyl ketone (MEK), propylene glycol (PG), dimethyl sulfoxide (DMSO) and 1% L92 at various concentrations. Based on the maximum soluble concentration, 1% L92 was selected as the vehicle (Soluble - formed suspension at ≤40 % w/v dilutions).

- Irritation: None of the tested concentrations elicited any irritation reaction.
- Ear thickness measurements: No increase in ear thickness and ear punch weights.
- Erythema scores: zero (no erythema)


MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA study
- Criteria used to consider a positive response: Categorisation of contact allergens on the basis of EC3 values derived from the local lymph node assay was performed according to ECETOC (2003).

Category EC3 (%)
Extreme <0.1
Strong ≥0.1 - < 1
Moderate ≥1 - < 10
Weak ≥ 10 - < 100


TREATMENT PREPARATION AND ADMINISTRATION:
Dose preparation:
Concentrations tested for the irritancy screen were selected based upon miscibility / solubility in an appropriate LLNA vehicle. The toxicity data regarding the irritation potential was also taken into consideration.
The required quantity of the test item was mixed with 1% L92 to obtain a stock formulation with concentration of 40% w/v. The required volume of stock formulation was mixed with 1% L92 to get dose formulations concentrations of 10 and 20% w/v. The dose formulations were prepared daily just prior to dosing. Preparation of the dosing materials was documented in the study file. The concentrations of the dose formulations were not verified analytically.

Main Study, Dermal Sensitization
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 (1% L92, or the positive control substance (25% v/v α-hexylcinnamaldehyde), or 10, 20 and 40% w/v test item in 1% L92, 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.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
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).

Results and discussion

Positive control results:
The positive control (25% HCA in 1% L92), elicited a stimulation index (SI) of 6.91.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
10% w/v test item
Key result
Parameter:
SI
Value:
2.84
Test group / Remarks:
20% w/v test item
Key result
Parameter:
SI
Value:
4.18
Test group / Remarks:
40% w/v test item
Key result
Parameter:
EC3
Value:
22.39
Remarks on result:
other: 22.39%
Cellular proliferation data / Observations:
Calculation and 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

3) EC3 calculation:
EC 3 = XL + [(3-YL)/ (Yh-YL)](Xh-XL)
Where, YL = SI value below 3
XL = chemical concentration that elicits YL
Yh = SI value above 3
Xh = chemical concentration that elicits Yh

EC3: Estimated concentration of the chemical necessary to give a 3-fold increase in the lymph node cell proliferative activity compared to vehicle- treated group (SI ≥ 3).

BODY WEIGHTS: No abnormality in weight gain was observed.

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: 1% L92

 

6

 

Mean

 

728.00

 

1.00

SD

187.60

0.26

 

G2 25% v/v HCA

 

6

 

+

 

Mean

5032.67

6.91

SD

887.74

1.22

 

G3 10% w/v test item

 

6

 

+

 

Mean

1453.17

2.00

SD

186.01

0.26

 

G4 20% w/v test item

 

6

 

+

 

Mean

2069.33

2.84

SD

205.37

0.28

 

G5 40% w/v test item

 

6

 

+

 

Mean

3046.67

4.18

SD

1136.53

1.56

+: Significantly higher than the vehicle control group

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test item FAT 40875/A TE is categorized as ‘Weak sensitizer’, as per the ECETOC categorisation for relative skin sensitization potency.
Executive summary:

Local Lymph Node Assay (LLNA) was conducted to evaluate the potential of the test item FAT 40875/A TE to cause contact sensitization by measuring the lymphocyte proliferative response from auricular lymph nodes following topical application to the female CBA/Ca mouse ear. This test was conducted in accordance with OECD test guideline 429 in a GLP certified laboratory.

Screening Study: Once daily topical application of the vehicle 1% Pluronic L92 (1% L92) and 2.5, 5, 10, 20 and 40% w/v FAT 40875/A TE in 1% L92 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. 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 (1% L92) or 25% α-hexylcinnamaldehyde (HCA: positive control in 1% L92) or 10, 20 and 40% w/v FAT 40875/A TE in 1% L92 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 6.91, 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 FAT 40875/A TE at dose concentrations of 10, 20 and 40% w/v elicited proliferative response with SI of 2.00, 2.84 and 4.18 respectively incomparison with the vehicle-treated mice. The EC3 value was 22.39%.

The test item FAT 40875/A TE is categorized as ‘Weak sensitizer’, as per the ECETOC categorisation for relative skin sensitization potency.