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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:
10 Oct 2017- 22 Nov 2017
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:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
additive
Type:
impurity
Test material form:
liquid: viscous
Details on test material:
- Other: R-9817

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Remarks:
J strain
Sex:
male/female
Details on test animals and environmental conditions:
Female CBA/J mice were received and following an acclimation period of at least five days, thirty-seven nulliparous, non-pregnant female mice were assigned to the test groups without conscious bias. The Day 1 body weight range for the QIT animals was 17.7 - 24.0 grams. The Day 1 body weight range for the main test animals was 19.3 - 23.0 grams. The weight variation of the animals at study start did not exceed ± 20% of the mean body weight. The animals were observed prior to the study start to ensure that no skin lesions were present on the ears.

The animals were identified by cage notation and indelible tail marks, and housed one per cage in suspended wire-bottom cages. Bedding was placed beneath the cages and changed at least three times per week. Fresh Rodent Chow and water were available ad libitum. The animal room, reserved exclusively for mice on acute tests, was temperature-controlled, had a 12-hour light/dark cycle, and was kept clean and vermin free. Temperature and humidity were continuously monitored using automatic recording devices.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Concentrations of 5%, 10%, 25%, and 50% (v/v) of the test article were chosen for the main Local Lymph Node study.Two positive control groups were also tested, alpha-hexylcinnamaldehyde in AOO (25% HCA) and 1-chloro-2,4-dinitrobenzene in AOO (0.25% DNCB)
No. of animals per dose:
The main test was conducted with seven separate groups of healthy female CBA/J mice (five animals per group)
Details on study design:
The test article was tested for solubility in acetone:olive oil (4:1, AOO) and found to be soluble at a concentration of 50% (v/v). A preliminary dermal irritation screen was conducted with three groups (two animals per group) of healthy female CBA/J mice to determine the concentrations of the test article to be used in the main Local Lymph Node study. Three concentrations of the test article were tested: 10%, 25% and 50% (v/v). The results of this preliminary screen indicated that the 50% (v/v) test article treatment resulted in significant dermal irritation. Based on these results, concentrations of 5%, 10%, 25%, and 50% (v/v) of the test article were chosen for the main Local Lymph Node study.

Dosing
Preliminary Dermal Irritation Screen: Three groups of CBA/J mice (two animals per group) were treated with increasing concentrations of R-9818 (10%, 25% and 50% [v/v]) in AOO. Treatment was made by topical application of the test article concentrations to the dorsum of each ear once daily for three consecutive days. The test article was spread over the entire dorsal surface of the ear using a micropipette to deliver 25 μl/ear.

Type and Frequency of Observations
All animals in the study were observed once daily throughout the study for clinical signs, either of local irritation at the application site or systemic toxicity, and for mortality.

Measurements
Body weights were recorded on Day 1, immediately prior to dosing, and on Day 6 (prior to euthanasia).

Ear thickness measurements were performed on Day 1 prior to dosing, on Day 3 before the third test article application (approximately 48 hours after the first test article application), and on Day 6 before euthanasia (approximately 120 hours after the first dose). Changes in ear thickness on Day 3 and Day 6 relative to Day 1 were expressed as a percent of the Day 1 pre-dose values. Ear thickness increases of 25% or more were considered biologically significant (based on the scientific literature and historical laboratory data) and deemed indicative of a greater than moderate local dermal irritation response.

Lymph Node Isolation and Processing
On Day 6 of the preliminary dermal irritation screen, each mouse was euthanized using CO2 asphyxiation, and the jugular vein was opened for complete exsanguination. Gross observations of the auricular lymph nodes were made at euthanasia on Day 6. The lymph nodes were not collected. On Day 5 of the main test, approximately 96 hours following the initial dose, and approximately 24 hours prior to euthanasia, the mice were injected with BrdU, in Dulbecco's phosphate-buffered saline (DPBS) at a dose of 500 μl per mouse (10 mg/ml). The BrdU was administered by intraperitoneal injection using a 26-gauge needle. This thymidine analog becomes incorporated into the DNA of proliferating cells, including proliferating nodal lymphocytes. On Day 6, each mouse was euthanized using CO2 asphyxiation, and the jugular vein was opened for complete exsanguination. Gross observations of the auricular lymph nodes were made, and the lymph nodes were collected. The auricular lymph nodes were combined for each animal and single-cell suspensions were generated in RPMI-10 medium.

Determination of BrdU Incorporation
A BrdU-specific Enzyme-Linked Immunosorbent Assay (ELISA) kit, Lot No. 24890800, was obtained from Roche Applied Science, Indianapolis, IN, on 29 Sep 2017 (expiration date Mar 2019). The kit was used to
measure the amount of BrdU incorporation in the LNC suspensions. An aliquot of 100 μl of the LNC preparation was added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the LNC, anti-BrdU antibody was added to each well and allowed to bind. Excess unbound anti-BrdU antibody solution was then removed by washing and the substrate solution was added and allowed to
generate chromogen. Absorbance of each well was measured using a MicroQuant plate reader (Bio-Tek Instruments) at 370 nm with a reference wavelength of 492 nm. The mean optical density (OD) values of three “blank” wells
(containing PBS) were used as a reference. Calculations were performed using MS Excel as describe in Analysis of Stimulation Index Data.

Analysis of Stimulation Index Data
Calculation of Stimulation Index (SI): For each animal, lymph node cell proliferation was determined by BrdU-ELISA and the mean Optical Density (OD) and SD were then calculated for each group. The mean OD for each animal was then divided by the mean number of proliferating lymphocytes in the vehicle control group. This “Test / Control Ratio” is the “Stimulation Index” (SI) and was calculated for each
animal as follows:
Mean ODtest article/Mean ODvehicle = Stimulation Index (SI) per animal
The mean SI and S.D. were calculated for each group.

EC1.6 calculation
The concentration at which the test article would produce a positive sensitization response (EC1.6) could not be calculated because no concentration produced an SI value less than 1.6
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
other: 1-chloro-2,4-dinitrobenzene in AOO (0.25% DNCB)
Statistics:
For each dose group, the individual animal SI values along with the mean group SI and standard deviation were calculated, and ANOVA followed by the Students’ t-Test was run to statistically compare each test article dose group to the vehicle control group. Although specified in the test guidelines, these calculations and results were not incorporated into the interpretation of the data. The EC1.6 is the sole determinant for a positive sensitization response.

Results and discussion

Positive control results:
Positive control results within historical laboratory data

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Remarks:
SI ≥ 1.6 indicates a sensitizing response
Value:
1
Variability:
S.D.= 0.5
Test group / Remarks:
Vehicle Control
Key result
Parameter:
SI
Remarks:
SI ≥ 1.6 indicates a sensitizing response
Value:
2.3
Variability:
S.D= 1.3
Test group / Remarks:
5% (v/v) Benzene sulfonic acid, di-C10-14-alkyl derivs., sodium salts
Key result
Parameter:
SI
Remarks:
SI ≥ 1.6 indicates a sensitizing response
Value:
2.3
Variability:
S.D.= 0.6
Test group / Remarks:
10% (v/v) Benzene sulfonic acid, di-C10-14-alkyl derivs., sodium salts
Key result
Parameter:
SI
Remarks:
SI ≥ 1.6 indicates a sensitizing response
Value:
2.8
Variability:
S.D= 0.8
Test group / Remarks:
25% (v/v) Benzene sulfonic acid, di-C10-14-alkyl derivs., sodium salts
Key result
Parameter:
SI
Remarks:
SI ≥ 1.6 indicates a sensitizing response
Value:
4.3
Variability:
S.D.= 1.7
Test group / Remarks:
50% (v/v) Benzene sulfonic acid, di-C10-14-alkyl derivs., sodium salts
Key result
Parameter:
SI
Remarks:
SI ≥ 1.6 indicates a sensitizing response
Value:
2.7
Variability:
S.D.= 1.6
Test group / Remarks:
25% HCA (Positive Control)
Key result
Parameter:
SI
Remarks:
SI ≥ 1.6 indicates a sensitizing response
Value:
5.7
Variability:
S.D. = 1.4
Test group / Remarks:
0.25% DNCB (Positive Control)
Cellular proliferation data / Observations:
A preliminary dermal irritation screen was performed and ear thickness measurements were used to assess irritation potential. Ear thickness measurements were performed on Day 1 prior to dosing, on Day 3 before the third test article application, and on Day 6 before euthanasia. The 50% (v/v) test article treatment resulted in increases in ear thickness of more than 25%, and therefore the test article was considered irritating at this concentration. Based on these screen results, test article concentrations of 5%, 10%, 25%, and 50% (v/v) were chosen for the main Local Lymph Node Assay. In the main test, the 10%, 25%, and 50% (v/v) test article treatments resulted in excessive dermal irritation. The 25% HCA positive control also had a greater than 25% increase in ear thickness on Day 6, indicating that 25% HCA is an irritant as well as a sensitizer, but the 0.25% DNCB positive control had no significant increase in ear thickness.
Stimulation Indices were calculated by dividing the mean optical density value (a representation of the total amount of LNC proliferation) for each animal by the mean optical density value for the vehicle control group. The mean SI and S.D. for each treatment group was then calculated. A result is regarded as a positive response in the LLNA if at least one concentration results in a 1.6-fold or greater increase in LNC proliferation relative to that of vehicle control animals, as indicated by an SI of 1.6 or more.

Test Article Dosing
No difficulties were experienced with the application of the test article to the ears or with the retention of test article by the ear surface.

Body Weights
Body weight losses were noted but were not significant (less than 2 grams). See Table 1 for individual body weight data.

Mortality and Systemic Observations
All animals survived the in-life phase of the study. Prior to the study start, all animals were observed to ensure that no skin lesions were present on the ears

Erythema Scores
The 25% and 50% (v/v) test article treatments in the screen, and the 5%, 10%, 25%, and 50% test article treatments in the main test produced no more than very slight erythema (barely perceptible). The 25% HCA and 0.25% DNCB positive controls also produced no more than very slight erythema

Ear Thickness as a Measure of Dermal Irritation
A preliminary dermal irritation screen was performed and ear thickness measurements were used to assess irritation potential. Ear thickness measurements were performed on Day 1 prior to dosing, on Day 3 before the third test article application, and on Day 6 before euthanasia. The 50% (v/v) test article treatment resulted in increases in ear thickness of more than 25%, and therefore the test article was considered irritating at this concentration. Based on these screen results, test article concentrations of 5%, 10%, 25%, and 50% (v/v) were chosen for the main Local Lymph Node Assay. In the main test, the 10%, 25%, and 50% (v/v) test article treatments resulted in excessive dermal irritation. The 25% HCA positive control also had a greater than 25% increase in ear thickness on Day 6, indicating that 25% HCA is an irritant as well as a sensitizer, but the 0.25% DNCB positive control had no significant increase in ear thickness.

Determination of the Stimulation Index (SI)
Stimulation Indices were calculated by dividing the mean optical density value (a representation of the total amount of LNC proliferation) for each animal by the mean optical density value for the vehicle control group. The mean SI and S.D. for each treatment group was then calculated. A result is regarded as a positive response in the LLNA if at least one concentration results in a 1.6-fold or greater increase in LNC proliferation relative to that of vehicle control animals, as indicated by an SI of 1.6 or more.

EC1.6 calculation
The EC1.6 was estimated to be 2.3% (v/v).

Any other information on results incl. tables

The %B cells, %T cells, %I-Ak + cells, and %I-Aκ +CD69+ cells were determined by flow cytometry as described in the Experimental Design section of this report. B:T ratios were then calculated. Group means and standard deviations are tabulated below. Test groups that show an increase of 25% or more (≥1.25 fold) when compared to the vehicle control in these LNC surface markers and have an SI value of 1.6 or more are considered dermal sensitizers. All four concentrations of the test article had a positive response, indicating that this test article is a true sensitizer. The positive controls 25% HCA and 0.25% DNCB were also found to be sensitizing.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Topical application of test article R-9817 at 5%, 10%, 25%, and 50% (v/v) in AOO resulted in SI values greater than 1.6 (SI > 1.6). Therefore, this test article is a dermal sensitizer in the Local Lymph Node Assay. In addition, all four concentrations of the test article yielded a positive response when analyzed by immunophenotyping, confirming that this test article is a sensitizer. The EC1.6 value was estimated to be 2.3% (v/v)