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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:
19 September 2017 to 21 March 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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (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
Reference substance name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']dioxodi-.mu.-thioxodi-, (Mo-Mo)
EC Number:
615-708-0
Cas Number:
72030-25-2
Molecular formula:
N/A
IUPAC Name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']dioxodi-.mu.-thioxodi-, (Mo-Mo)
Constituent 2
Reference substance name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']-.mu.-oxodioxo-.mu.-thioxodi-
Cas Number:
153128-45-1
Molecular formula:
N/A
IUPAC Name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']-.mu.-oxodioxo-.mu.-thioxodi-
Constituent 3
Reference substance name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']oxodi-.mu.-thioxothioxodi-
Molecular formula:
N/A
IUPAC Name:
Molybdenum, bis[O,O-bis(2-ethylhexyl) phosphorodithioato-.kappa.S,.kappa.S']oxodi-.mu.-thioxothioxodi-
Constituent 4
Reference substance name:
Unspecified 2-ethylhexyl thiophosphate esters
Molecular formula:
N/A
IUPAC Name:
Unspecified 2-ethylhexyl thiophosphate esters
Constituent 5
Reference substance name:
Unspecified 2-ethylhexyl phosphate esters
Molecular formula:
N/A
IUPAC Name:
Unspecified 2-ethylhexyl phosphate esters
Constituent 6
Reference substance name:
Unspecified 2-ethylhexyl dithiophosphate esters
Molecular formula:
N/A
IUPAC Name:
Unspecified 2-ethylhexyl dithiophosphate esters
Test material form:
liquid
Specific details on test material used for the study:
Purity: UVCB
Description: Dark green liquid
Storage: Room temperature in the dark.

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Supplied by Envigo RMS B.V., Inc., Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: [yes/no/not specified] : Yes
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing:Group housed by dose level in suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet: Free access to food (2014C Teklad Global Rodent diet) throughout the study
- Water: Free access to mains tap water throughout the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70°C
- Air changes (per hr): Fifteen changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours continuous light and 12 hours darkness.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25%, 10% and 5%
No. of animals per dose:
Preliminary: 1 mouse per test item concentration, except for the 50% concentration where 2 mice were used.
Main test: 5 animals per dose
Details on study design:
Preliminary Screening Test
A preliminary screening test was performed using one mouse per dose, except two mice were used for the 50% concentration. The mouse was treated by daily application of 25 µL of the appropriate test item concentration 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 test item at concentrations of 25%, 10%, or 5% v/v in acetone/olive oil 4:1. The preliminary screening test suggested that the highest test item concentration that would not produce systemic toxicity or excessive local skin irritation was 25% v/v acetone/olive oil 4:1. 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).
- Termination: 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. 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 °C, 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".
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
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.

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.59
Test group / Remarks:
5% v/v in acetone/ olive oil 4:1
Key result
Parameter:
SI
Value:
3.61
Test group / Remarks:
10% v/v in acetone/ olive oil 4:1
Key result
Parameter:
SI
Value:
6.37
Test group / Remarks:
25% v/v in acetone/ olive oil 4:1

Any other information on results incl. tables

Preliminary Screening Test Results:

One mouse treated with the test item at 100% (undiluted) and one mouse treated with 50% v/v acetone/olive oil were killed due to the severity of clinical signs, including body weight losses of 15.3% and 17.0%, respectively on Day 5.

No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted in the animal treated with the test item at a concentration of 25% v/v in acetone/olive oil or the initial animal treated with the test item at a concentration of 50% v/v in acetone/olive oil but the initial animal treated at a concentration of 50% v/v showed a body weight loss of approximately 9.9%

The results indicated that the test item at concentrations of ≥ 50% v/v in acetone/olive oil 4:1 caused systemic toxicity (> 5% reduction in body weight) in mice. Based on this information the dose levels selected for the main test were 25%, 10% and 5% v/v in acetone/olive oil 4:1.

Main Test Results:

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

 5  1.59  Negative
 10  3.61 Positive 
 25  6.37  Positive

The EC3 was calculated to be 8.5%.

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.

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The test item is classified as a contact sensitizer (Category 1B) according to Regulation (EC) No. 1272/2008, relating to the Classification, Labelling and Packaging of Substances and Mixtures and the United Nations Globally Harmonized System of Classification and Labelling of Chemicals.
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 preliminary screening tests in mice at concentrations of 100%, 50% and 25% v/v in acetone/olive oil 4:1, in which the test item caused systemic toxicity at concentrations of ≥ 50% and no clinical signs of toxicity at a concentration of 25%, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five female animals, were treated with 50 μL (25 μL per ear) of the test item as a solution in acetone/olive oil 4:1 at concentrations of 25%, 10% or 5% v/v. A further group of five female animals was treated with acetone/olive oil 4:1 alone. The sensitivity of the test system was confirmed periodically using the positive control substance α-Hexylcinnamaldehyde.

 

The Stimulation Index expressed as the mean radioactive incorporation for the treatment group divided by the mean radioactive incorporation of the vehicle control group, was 6.37 at a concentration of 25%v/v test item in acetone/olive oil, the EC3was calculated to be 8.5%.

 

Under the conditions of the test, the test item was considered to be a moderate skin sensitizer with an EC3of 8.5%.

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