[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

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

Test material information

Constituent 1

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

Female CBAlJ mice were received from Jackson Laboratories (Bar Harbor, ME) on 05 Apr 2016 and 12 Apr 2016. Following an acclimation period of at least five days, thirty-six nulliparous, non-pregnant female mice were assigned to the test groups without conscious bias.
The animals were born on 16 Feb 2016, (± 3 days). The Day 1 body weight range for the screen animals was 19.8 - 22.7 grams. The Day 1 body weight range for the main test animals was 18.9 - 23.7 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 PMI Rodent Chow (Diet No. 5001) 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:

other: N,N-dimethylacetamide

Concentration:

Preliminary Dermal Irritation Screen: Three groups of CBA/J mice (two animals per group) were treated with increasing concentrations of the test article (10%, 25% and 50% (w/v)) in DMA.
Main Test: The test article concentrations used in the main LLNA study were chosen such that the maximum concentration tested was the highest achievable solution of the test article in the vehicle, while avoiding both overt systemic toxicity and excessive local dermal irritation. Concentrations of 10%, 25% and 50% (w/v) were chosen by the Study Director in consultation with the Sponsor.

No. of animals per dose:

5

Details on study design:

Preliminary Dermal Irritation Screen: 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 at 25 µl/ear.
Main test : Six groups of CBA/J mice (five animals per group) were treated by topical application of the test article concentrations, vehicle control or positive controls in the same manner as in the screen. DMA was chosen as the vehicle, based on solubility. Historically, at MB Research, the DMA vehicle has produced consistent results with the positive controls 25% HCA and 0.1% DNCB, as well as with other sensitizers and non-sensitizers.

Lymph node proliferation: single-cell suspensions of lymph node cells were prepared. The Stimulation Index (SI) was calculated by dividing the proliferative response (number of BrdU+ cells) of each animal by the mean proliferative response of the vehicle control group. The mean SI and standard deviation for each treatment group was then calculated. A test article is regarded as a sensitizer in the LLNA if at least one concentration results in a 3-fold or greater increase in LNC proliferation relative to that of vehicle control animals, as indicated by an SI >= 3.0.

Body weight: Body weights were recorded immediately prior to dosing on Day 1 and prior to euthanasia on Day 6.

Mortality and Dermal irritation: 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. 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 and 72 hours after the third 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.

Immunophenotyping and Activation Marker Determination: lymph node cells were stained with both fluorescently-labeled anti-mouse B220 antibody (B cells) and anti-mouse CD3 antibody (T cells), or early activation (anti-mouse CD69) and MHC class (anti-mouse I-Ak) antibodies in PBS-based buffer using an appropriate titer (BD Biosciences). Live cells were analyzed by flow cytometry to determine % B cells, % T cells, B:T cell ratio, %I-Ak cells and %I-Ak+CD69+ or "double positive" cells. For immunophenotyping, increases in the %B cells or B:T ratio of >= 25% over vehicle control are considered predictive of a true sensitizer. If immunophenotyping data are equivocal, increases
in %I-Ak or I-Ak+CD69+ cells of >= 25% over vehicle control are considered predictive of a true sensitizer.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

other: 1-Chloro-2.4-dinitrobenzene

Statistics:

For each test 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.

Results and discussion

Positive control results:

Both the 25% HCA and 0.1% DNCB positive controls were found to be sensitizing.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2

Variability:

0.4

Test group / Remarks:

10% of test item

Results 2

Key result

Parameter:

SI

Value:

2.9

Variability:

0.5

Test group / Remarks:

15% of test item

Results 3

Key result

Parameter:

SI

Value:

2.4

Variability:

0.7

Test group / Remarks:

50% of test item

Results 4

Parameter:

SI

Value:

1

Variability:

0.5

Test group / Remarks:

DMA (vehicle control)

Results 5

Parameter:

SI

Value:

4.4

Variability:

0.8

Test group / Remarks:

Positive control (25% HCA)

Results 6

Parameter:

SI

Value:

5.7

Variability:

1.9

Test group / Remarks:

Positive control (0.1% DNCB)

Any other information on results incl. tables

Body weight: In the main test, body weight losses were noted but were not significant (<2g).

Mortality: All animals survived the in-life phase of the study.

Dermal irritation: Ear thickness measurements and individual animal observations indicated that none of the test article treatments resulted in excessive local dermal irritation.

Immunophenotyping and Activation Marker Determination: In the main test, none of the test article concentrations resulted in a >= 25% increase in % B cells, % T cells, B:T cell ratio, % I-Ak+ cells or %I-Ak+CD69+ cells.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Topical application of the test item at 10%, 25% and 50% (w/v) in N,N-dimethylacetamide resulted in SI values less than 3 (SI < 3). Therefore, the test item is not a dermal sensitizer in the Local Lymph Node Assay.
Treatment at all concentrations tested resulted in less than a 25% fold increase compared to the vehicle control for all of the immunophenotyping markers tested. Immunophenotyping results confirmed that the test item is not a dermal sensitizer.
These results support non-classification.

Executive summary:

Skin sensitization by the text item was evaluated according to OECD Guideline 429 (Local Lymph Node Assay (LLNA)). The study was conducted using female mice (CBA/J strain). After a preliminary dermal irritation screen, it was decided that concentrations of 105, 255, and 50% (w/v) of the test item would be used in the main LLNA study. The Stimulation Index (SI) for the main LLNA study was below the SI = 3 threshold for skin sensitizer. Furthermore, immunophenotyping confirmed the lack of immune cell ratio changes that would indicate skin sensitization. This results in the test item being unclassified for skin sensitization, according to GHS criteria.