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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:
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:
Not available
Cas Number:
2135769-54-7
Molecular formula:
NA
IUPAC Name:
Not available
Test material form:
liquid: viscous
Details on test material:
Vapour pressure: 3.9 x 10-3 Pa
Water solubility: TBC
Density: 1.0
Appearance: black viscous liquid
Melting point: -52 to -21 °C
Boiling point: no boiling and no decomposition up to 300°C
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
Identification: Tall Oil Pitch, reaction product with triethylene glycol
Batch: LABO 16-02
Physical state: Brown viscous liquid
Expiry date: 14/11/2018
Storage Conditions: room temperature in the dark under nitrogen

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V. Inc. Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15 - 23 g
- Housing: suspended solid floor polypropylene cages furnished with softwood woodflakes
- Diet (e.g. ad libitum): 2014C Teklad Global Rodent diet
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 -70
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 hr light / 12 hr nigh
- IN-LIFE DATES: From: To: 30 Nov 2017 - 19 Dec 2017

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Preliminary test: the mouse was treated by daily application of 25 µL of the test item at concentration of 50% w/w in acetone/olive oil 4:1
Main test: the mouse was treated by daily application of the test item at concetration of 50, 25 and 10% in acetone/olive oil 4:1
No. of animals per dose:
5
Details on study design:
TEST ITEM PREPARATION AND ANALYSYS
The test item was freshly prepared as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the most suitable formulation at the required concentration.
The test item was formulated within 2 hours of being applied to the test system. It is assumed that formulation was stable for this duration. no analysis was conducted to determine the homogeneity, concentration or stability of the test item formulation due to the short-term nature of the study. This is an exception with regards to GLP, which has been included in the GLP statement and is considered not to affect the integrity or the validity of the study.

PRE-SCREEN TESTS:
A preliminary test was done using one animal. The mouse was treated by daily application of 25 µL of the test item at concentration of 50% w/w in acetone/olive oil 4:1 to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Day 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily accordingly to the scale. Any clinical sign of toxicity, if present, were also recorded.
The body weight of the mouse was recorded on Day 1 and on Day 6.
The thickness of the ear was measured on pre-dose Day 1, post dose on Day 3 and 6. Any change was recorded.
Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.

MAIN STUDY
Groups of five mice were treated with the test item at concentrations of 50%, 25% or 10% w/w 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 concentrations. 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.

3H-Methyl Thymidine Administration
All mice were injected via the tail vein with 0.25 mL of PBS containing 3H-Methyl Thymidine ( total of 20 micro C1 to each mouse) five days following the first topical application of the test item or vehicle (Day 6).

A further group of five mice received the vehicle alone (control group).

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 values were evaluated for dose response relationships.
Data was first assessed for suitability by analysis of normality and homogeneity of variace.
Statistical model used:
- ANOVA
- Dunnett's test
- Kruskal-Wallis Rank Sum
- Mann-Whitney U test

P values were presented as follows:
p<0.001 ***
p<0.01 **
p<0.05 *
P>=0.05 (not significant)

Results and discussion

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Value:
1.07
Test group / Remarks:
Concentration (%w/w) in acetone/olive oil 4:1 = 10
Remarks on result:
other: NEGATIVE
Remarks:
NEGATIVE
Key result
Parameter:
SI
Value:
0.86
Test group / Remarks:
Concentration (%w/w) in acetone/olive oil 4:1 = 25
Remarks on result:
other: NEGATIVE
Remarks:
NEGATIVE
Key result
Parameter:
SI
Value:
1.29
Test group / Remarks:
Concentration (%w/w) in acetone/olive oil 4:1 = 50
Remarks on result:
other: NEGATIVE
Remarks:
NEGATIVE
Cellular proliferation data / Observations:
Clinical sign:
No signes 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.

Any other information on results incl. tables

The Stimulation Index is expressed as the mean radioactive incorporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group.

The results is as as follows:

 Concentration (%w/w) in acetone/olive oil 4:1)  Simulation Index (SI)  Results
 10  1.07 Negative
 25  0.86 Negative
 50  1.29 Negative

Details on the SI calculation and the decision process are available in the "background information box".

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The test material Tall Oil Pitch, reaction product with triethylene glycol is not considered to be a sensitiser, according to the conditions of this test.
Executive summary:

The sensitisation potential of the test material Tall Oil Pitch, reaction product with triethylene glycol was assessed following the OECD 429 Guideline and under GLP conditions.

Following a preliminary screening test in which no clinical signs of toxicity were noted at concentration of 50% w/w, 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 test item as a solution in acetone/olive 4:1 at concentrations of 50%, 25% or 10% w/w. A further group of five animals was treated with acetone/olive oil 4:1 alone.

The results of the study, expressed in Simulation Index, were as follows:

- Concentration (%w/w) in acetone/olive oil 4:1=10 --> SI: 1.07

- Concentration (%w/w) in acetone/olive oil 4:1=25 --> SI: 0.86

- Concentration (%w/w) in acetone/olive oil 4:1=50 --> SI: 1.29

The results of the study showed that test item is not considered to be a sensitizer. The test item does not meet the criteria for the classification according to the GHS system.