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Description of key information

A skin sensitisation study, local lymph node assay in the mouse (individual method) was performed in accordance to the standardized guidelines OECD 429 and EU Method B.42, under GLP conditions. The study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The test item was considered to be a non-sensitizerunder the conditions of the test and does not meet the criteria for classification according to the Globally Harmonized Classification System.

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:
10 August 2016 to ****
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese MAFF, 2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
CAS RN: 15337-18-5
Purity: 96.6%
Physical state/Appearance: Yellow viscous liquid
Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- The animals were nulliparous and non pregnant
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: Suspended solid floor polypropylene cages with stainless steel mesh lids and furnished with softwood wood flake bedding
- Diet: Certified rodent feed, ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: At least five days
- No indication of any skin lesions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): At least 15
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
50 or 25 (%v/v)
No. of animals per dose:
One mouse for the preliminary test, followed by five animals per dose in the main test.
Details on study design:
Preliminary Screening Test
A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 µL of the undiluted test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed for local skin irritation, ear thickness, clinical signs of toxicity, and body weight.

Main Test
Test Item Administration
Groups of five mice were treated with the undiluted test item (100%) or the test item at concentrations of 50% or 25% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the test item would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of five mice received the vehicle alone in the same manner.

3H-Methyl Thymidine Administration
Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 250 µL of phosphate buffered saline (PBS) containing 3H methyl thymidine (3HTdR: 80 µCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 µCi to each mouse.

Observations
Clinical Observations: All animals were observed twice daily (pre- and post-dose) on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6 for any signs of toxicity or ill health.
Body Weights: The body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

Sample Preparation: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed.

Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200 mesh stainless steel gauze. The lymph node cells were rinsed with PBS into a petri dish labeled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube and cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re suspended in PBS and re pelleted. The pellet was re suspended in 5% Trichloroacetic acid (TCA).

Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 ºC, the precipitates were recovered by centrifugation, re suspended in TCA and transferred to scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting.

Data Evaluation
The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).
The test item was regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation was classified as a "non sensitizer".

Statistical Analysis
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non parametric Kruskal Wallis Rank Sum and Mann Whitney U test procedures were used.
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non parametric Kruskal Wallis Rank Sum and Mann Whitney U test procedures were used.
Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
25% v/v in acetone/olive oil 4:1
Key result
Parameter:
SI
Value:
1.65
Test group / Remarks:
50% v/v in acetone/olive oil 4:1
Key result
Parameter:
SI
Value:
2.36
Test group / Remarks:
100% v/v in acetone/olive oil 4:1

Preliminary Screening Test

No signs of systemic toxicity or visual local skin irritation were noted. A greater than 25% (95.24%) increase in mean ear thickness was noted. As no other signs of irritation were noted the greater than 25% increase was considered to be due to residual test item on the ears rather than excessive irritation. Based on this information the undiluted test item and the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main test.

 

Main Test

The Stimulation Index expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

 

Concentration (%v/v) in acetone/olive oil 4:1

Stimulation Index

Result

25

1.6

Negative

50

1.65

Negative

100

2.36

Negative

 

Clinical Observations and Mortality Data

Colorless, sticky residual test item on the ears and fur loss were noted in animals treated with the undiluted test item. There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test.

 

Body Weight

Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Interpretation of results:
GHS criteria not met
Conclusions:
The test item was considered to be a non-sensitizerunder the conditions of the test and does not meet the criteria for classification according to the Globally Harmonized Classification System.
Executive summary:

A study was performed to assess the skin sensitization potential of the test item in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear.

 

Following a preliminary screening test in which no clinical signs of toxicity were noted at a concentration of 100%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in acetone/olive oil 4:1 at concentrations of 50% or 25% v/v. A further group of five animals was treated with acetone/olive oil 4:1 alone.

 

The test item was considered to be a non-sensitizer under the conditions of the test and does not meet the criteria for classification according to the Globally Harmonized Classification System.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

The test item was considered to be anon-sensitizerunder the conditions of the test and does notmeet the criteria for classification according to the Globally Harmonized Classification System.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification