Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 to 12 March 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source of test material: Nitika Pharmaceutical Specialities Pvt. Ltd.
- Lot/batch No.of test material: IRST7H112A
- Expiration date of the lot/batch: May 2023
- Purity test (release) date: June 2018 (CoA)

RADIOLABELLING INFORMATION (if applicable)
- Radiolabelled material: 3H Methyl Thymidine
- Radiochemical purity: 99.12%
- Specific activity: 18000 mCi/mmole
- Locations of the label: not specified
- Expiration date of radiochemical substance: Not specified

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature (ambient), in original container as received from the Sponsor. Container kept tightly closed in a dry, cool and well ventilated place.
- Stability under test conditions: Assumed stable for the duration of the test
- Solubility and stability of the test substance in the solvent/vehicle: The test item was non soluble in the vehicles detailed in OECD 429 (Acetone : Olive oil (AOO), DMF, MEK, propylene glycol, DMSO and 1% pluronic L92). The test item formed a homogenous (dosable) suspension in Olive oil up to 75% (w/v). Therefore, Olive oil was selected as the vehicle.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Prepared as a homogenous (dosable) suspension in Corn oil.


In vivo test system

Test animals

Species:
mouse
Strain:
CBA:J
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Animal Breeding Facility, Jai Research Foundation
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 10 to 11 weeks
- Weight at study initiation: Minimum: 18.7g, Maximum: 26.1g
- Housing: Solid floor polypropylene mice cages (size: approx. 290 mm x 220 mm x 140 mm). Each cage was fitted with a top grill having provision for keeping rodent pellet feed and water bottles.
- Diet: Teklad Certified Global High Fiber Rat/Mice Feed manufactured by Envigo, USA, was provided ad libitum.
- Water: UV sterilised water (Reverse Osmosis water filtration system) was provided ad libitum.
- Acclimation period: 6 days
- Indication of any skin lesions: None. Mice received a veterinary health examination on arrival and were thereafter acclimatised prior to study use.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 23 °C
- Humidity (%): 57 to 66%
- Air changes (per hr): Minimum of 15 air changes/hour
- Photoperiod (hrs dark / hrs light): The photoperiod was 12 h artificial light and 12 h darkness, light hours being 06:00 – 18:00 h (photoperiod maintained through an automatic timer).
- IN-LIFE DATES: From: To: 23 January 2019 (Experimental Start) to 14 March 2019 (Experimental Completion)

Study design: in vivo (LLNA)

Vehicle:
other: Olive oil
Remarks:
Non-standard vehicle
Concentration:
Based on the preliminary assay, dose concentrations of 25%, 50% and 75% (w/v) Fatty acids, C16-18 (even numbered), iron(III) salts in Olive oil were evaluated in the main LLNA.
No. of animals per dose:
2 female mice/group (Preliminary assay)
5 female mice/group (Main LLNA)
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The test item was non soluble in the vehicles detailed in OECD 429 (Acetone : Olive oil (AOO), DMF, MEK, propylene glycol, DMSO and 1% pluronic L92). The solubility was therefore checked using Olive oil, a non-standard vehicle, in consultation with the Sponsor. The test item formed a homogenous (dosable) suspension in Olive oil up to 75% w/v. Therefore, Olive oil was selected as the vehicle.
- Irritation: none
- Systemic toxicity: none
- Ear thickness measurements: yes, all ear thickness increases were below 25% at all tested concentrations.
- Erythema scores: No erythema was observed at the site of application at any dose tested

In the preliminary assay, no erythema was observed at the site of application at dose levels of 10%, 25%, 50% and 75% (w/v) Fatty acids, C16-18 (even numbered), iron(III) salts in Olive oil. Ear thickness increases were below 25% at all tested concentrations. Therefore, dose concentrations of 25%, 50% and 75% (w/v) Fatty acids, C16-18 (even numbered), iron(III) salts in Olive oil were evaluated in the main LLNA.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Skin Sensitisation Study using Local Lymph Node Assay (LLNA) to OECD test guideline 429 and test method EC B.42.

- Criteria used to consider a positive response:
If the test item produces a SI ≥ 3 in the LLNA, it is considered positive for contact sensitisation potential and therefore an EC3 should be determined.
If the test item produces a SI < 3 in the LLNA, it is considered negative for contact sensitisation potential and therefore an EC3 is not determined.
If the EC3 value is ≤ 2%, the test item is classified under Sub-category 1A while if EC3 value is > 2% it is classified under sub-category 1B based on the Globally Harmonized System of Classification and Labelling of Chemicals (GHS 2017).

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was mixed with the vehicle to obtain the desired dose concentrations of 25%, 50% and 75% (w/v) Fatty acids, C16-18 (even numbered), iron(III) salts in Olive oil. Fresh doses were prepared prior to application on days 0, 1 and 2. Doses were not analytically verified.
Three groups (G2 to G4) were treated topically for three consecutive days (days 0, 1 and 2) on the dorsal surface of both ears (25 µL/ear) using a calibrated micropipette at test concentrations of 25%, 50% and 75% (w/v) Fatty acids, C16-18 (even numbered), iron(III) salts in Olive oil, respectively. Mice from the vehicle control group (G1), vehicle control for positive control (G5) and positive control group (G6) were handled in the same manner but received 25 µL/ear of Olive oil, AOO and 25% (v/v) a-Hexylcinnamaldehyde in AOO, respectively.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
In addition to an assessment of the magnitude of the SI, statistical analysis was carried out for the assessment of the dose response relationship and a pair-wise comparison made between the treatment and the solvent/vehicle control group. All parameters characterised by continuous data such as body weight and radioactive disintegrations per minute (DPM) were subjected to Bartlett’s test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA). To compare vehicle and positive control data, a Student’s t-test was performed to calculate significance.

Results and discussion

Positive control results:
The DPM value for positive control (25% α-Hexylcinnamaldehyde) was 2488.00.
A statistically significant increase in the DPM was observed in the positive control group (25% v/v α-Hexylcinnamaldehyde), when compared to vehicle control group values

The SI of 4.03 obtained for the positive control, α-Hexylcinnamaldehyde, showed a greater than a three-fold increase over the control value indicating a positive response in agreement with the historical control data for this known weak sensitiser.

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
G1 - Vehicle control (Olive oil)
Key result
Parameter:
SI
Value:
1.21
Test group / Remarks:
G2 - 25% w/v test substance
Key result
Parameter:
SI
Value:
1.26
Test group / Remarks:
G3 - 50% w/v test substance
Key result
Parameter:
SI
Value:
1.86
Test group / Remarks:
G4 - 75% w/v test substance
Key result
Parameter:
SI
Value:
1
Test group / Remarks:
G5 - vehicle control for positive control, acetone;olive oil
Key result
Parameter:
SI
Value:
4.03
Test group / Remarks:
G6 - 25% v/v HCA in acetone;olive oil
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
The proliferative response of lymph nodes from each mouse was expressed as the number of radioactive disintegrations per minute (DPM), calculated by subtracting the background DPM (measured in a 1 mL aliquot of 5% TCA).

DETAILS ON STIMULATION INDEX CALCULATION
The DPM/mouse, along with an appropriate measure of inter-animal variability (i.e., mean ± standard deviation), were calculated for each test group and vehicle and positive control groups. Final results were expressed as the Stimulation Index (SI) which is calculated as a ratio of the mean DPM of the test group divided by mean DPM of the vehicle control group.

EC3 CALCULATION
If the test item produces a SI ≥ 3 in the LLNA, it is considered positive for contact sensitisation potential and therefore an EC3 should be determined. EC3 value (theoretical concentration resulting in a SI value of 3) is determined by linear interpolation of points on the dose-response curve, immediately above and below the 3-fold threshold (Basketter et al., 1999). The equation used for calculation of EC3 was:
EC3 = c + [(3 - d)/(b - d)] x (a - c)
Where a = the lowest concentration giving stimulation index > 3; b = the actual stimulation index caused by a; c = the highest concentration failing to produce a stimulation index of 3; and d = the actual stimulation index caused by c.

Categorisation of contact allergens on the basis of relative skin sensitisation potency, uses the EC3 values derived from the LLNA (Kimber et al, 2003; ECETOC, 2003).
EC3 value Category
< 0.1% Extreme sensitiser
≥0.1 - < 1% Strong sensitiser
≥1 - < 10% Moderate sensitiser
≥ 10 - ≤100% Weak sensitiser

If the test item produces a SI < 3 in the LLNA, it is considered negative for contact sensitisation potential and therefore an EC3 is not determined. If the EC3 value is ≤ 2%, the test item is classified under Sub-category 1A while if EC3 value is > 2% it is classified under sub-category 1B based on the Globally Harmonized System of Classification and Labelling of Chemicals (GHS 2017).

CLINICAL OBSERVATIONS: No clinical sign was observed in any mouse from the vehicle controls, positive control, or any test item treated group at 25%, 50% or 75% (w/v) Fatty acids, C16-18 (even numbered), iron(III) salts in Olive oil.
No erythema was observed in any treated mouse at 25%, 50% and 75% (w/v) Fatty acids, C16-18 (even numbered), iron(III) salts in Olive oil on day 0 to day 5. Very slight erythema was observed in the positive control group treated with 25% HCA (during days 1 to 4) in all mice (5/5 mice).

BODY WEIGHTS
The mean body weight of positive control and Fatty acids, C16-18 (even numbered), iron(III) salts treated mice was comparable to that of the respective vehicle control group.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on results of this study, the classification for Fatty acids, C16-18 (even numbered), iron(III) salts is as follows:

Globally Harmonized System of Classification and Labelling of Chemicals (GHS 2017): Not Classified as a Skin Sensitiser