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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 June 2015 - 20 July 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

1
Chemical structure
Reference substance name:
2,2'-isopropylidenebis(p-phenyleneoxy)diethanol
EC Number:
212-985-6
EC Name:
2,2'-isopropylidenebis(p-phenyleneoxy)diethanol
Cas Number:
901-44-0
Molecular formula:
C19H24O4
IUPAC Name:
2,2'-isopropylidenebis(p-phenyleneoxy)diethanol
Test material form:
liquid: viscous

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France
- Age at study initiation: approximately 12 weeks old for the animals of the preliminary test and approximately 8 weeks old for the animals of the main test
- Mean body weight at study initiation: the animals of the preliminary test had a mean body weight of 22.1 g (range: 20.4 g to 23.6 g) and the animals of the main test had a mean body weight of 20.3 g (range: 18.0 g to 22.1 g).
- Fasting period before study: no
- Housing:
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 6 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 08 July 2015 to 20 July 2015.

Study design: in vivo (LLNA)

Vehicle:
acetone/olive oil (4:1 v/v)
Remarks:
used for the positive control
Concentration:
25%
No. of animals per dose:
4 per dose.
Details on study design:
RANGE FINDING TESTS:

To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was first performed in order to define the test item concentrations to be used in the main test. Two groups of two female mice received the test item, by topical route to the dorsal surface of both ears (one concentration per ear) on Days 1, 2 and 3 at concentrations of 5, 10, 25 or 50% under a dose-volume of 25 µL. From Day 1 to Day 3 then on Day 6, the thickness of both ears of each animal was measured and the local reactions were recorded. Each animal was observed once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on Days 1 and 6.On completion of the observation period, the animals were sacrificed then discarded without macroscopicpost-mortemexamination.

- Compound solubility: The solubility assay first started at the concentration of 75%, and according to the OECD guideline, the first choice vehicle was a mixture of acetone/olive oil (4/1; v/v) (AOO).

As unsatisfactory solubility of the test item was obtained in AOO (i.e. heterogeneous suspension to the naked eye at the concentration of 75% was obtained), another vehicle was chosen from the following organic solvents (in order of preference): dimethylformamide (DMF), Methyl Ethyl Ketone (MEK), Propylene Glycol (PG) and dimethyl sulfoxide (DMSO). As heterogeneous emulsions or suspensions to the naked eye at 75% were obtained in any of the proposed vehicles, the test item was tested at lower concentrations in the same vehicles.
A solution was obtained at the concentration of 50% in DMF.

- Irritation: the maximum concentration of the test item was selected to avoid overt systemic toxicity and excessive local skin irritation the latter being defined by an increase of the ear thickness = 25%.

MAIN STUDY
In the main test, five groups of four female mice received the test item by topical routeto the dorsal surface of both earson Days 1, 2 and 3 at concentrations of 2.5, 5, 10, 25 or 50% under a dose-volume of 25 µL. One negative control group of four females received the vehicle (dimethylformamide) under the same experimental conditions. Additionally, one positive control group of 4 females received the positive control, a-hexylcinnamaldehyde (HCA), at 25% in a mixture acetone/olive oil (4/1; v/v) under the same experimental conditions.
From Day 1 to Day 3 then on Day 6, the thickness of the left ear of each animal was measured, except in animals of the positive control group, and the local reactions were recorded. Each animal was observed once a day for mortality and clinical signs. Body weight was recordedonce during the acclimation period, and thenon Days 1 and 6.
After 2 days of resting, on Day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of3H-TdR.The results are expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI).

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: murine Local Lymph Node Assay
- Criteria used to consider a positive response: stimulation Index SI >= 3 and dose-relationship; additional consideration of ear thickness

TREATMENT PREPARATION AND ADMINISTRATION:
- Treatment preparation: The test item was prepared at the chosen concentrations in the vehicle.
The positive control was dissolved in AOO at the concentration of 25% (v/v).
- Administration:
On Days 1, 2 and 3, a dose-volume of 25 µL of the control or dosage form preparations was applied to the dorsal surface of both ears, using an adjustable pipette fitted with a plastic tip.
In order to avoid licking and to ensure an optimized application of the test materials, the animals were placed under light isoflurane anesthezia during the administration.
No massage was performed but the tip was used to spread the preparation over the application sites. No rinsing was performed between each application.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The threshold positive value of 3 for the SI was reached in the positive control group (see Executive summary).

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.12
Test group / Remarks:
2.5%
Parameter:
SI
Value:
0.63
Test group / Remarks:
5%
Parameter:
SI
Value:
0.79
Test group / Remarks:
10%
Parameter:
SI
Value:
1.11
Test group / Remarks:
25%
Parameter:
SI
Value:
1.28
Test group / Remarks:
50%

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Conclusions:
The test item was tested as a solution after preparation at concentrations of 2.5, 5, 10, 25 and 50% in dimethylformamide.
Under the experimental conditions of this study, the test item gave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties. Therefore, the test item should not be classified as a skin sensitizer according to the criteria of CLP Regulation.
Executive summary:

The objective of this study was to evaluate the potential of the test item, 2,2'-isopropylidenebis(p-phenyleneoxy)diethanol,to induce contact hypersensitivity, using the murine Local Lymph Node Assay (LLNA).

 

Methods

To assess the irritant potential of the test item (through ear thickness measurement), a preliminary test was first performed in order to define the test item concentrations to be used in the main test. Two groups of two female mice received the test item, by topical route to the dorsal surface of both ears (one concentration per ear) on Days 1, 2 and 3 at concentrations of 5, 10, 25 or 50% under a dose-volume of 25 µL. From Day 1 to Day 3 then on Day 6, the thickness of both ears of each animal was measured and the local reactions were recorded. Each animal was observed once a day for mortality and clinical signs. Body weight was recorded once during the acclimation period, and then on Days 1 and 6.On completion of the observation period, the animals were sacrificed then discarded without macroscopic post-mortem examination.

In the main test, five groups of four female mice received the test item by topical routeto the dorsal surface of both earson Days 1, 2 and 3 at concentrations of 2.5, 5, 10, 25 or 50% under a dose-volume of 25 µL. One negative control group of four females received the vehicle (dimethylformamide) under the same experimental conditions. Additionally, one positive control group of 4 females received the positive control, a-hexylcinnamaldehyde (HCA), at 25% in a mixture acetone/olive oil (4/1; v/v) under the same experimental conditions.

From Day 1 to Day 3 then on Day 6, the thickness of the left ear of each animal was measured, except in animals of the positive control group, and the local reactions were recorded. Each animal was observed once a day for mortality and clinical signs. Body weight was recordedonce during the acclimation period, and thenon Days 1 and 6.

After 2 days of resting, on Day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3H-TdR). Approximately 5 hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of3H-TdR.

The results are expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI).

 

Results

- Solubility assays

A solution was obtained at the maximal concentration of 50% in dimethylformamide.

 

- Preliminary test

Mortality and clinical signs

No unscheduled deaths and no clinical signs were observed during the observation period.Body weight of animals was unaffected by the test item treatment.

 

Ear thickness measurements and local reactions

Residual of product was observed from Day 2 on right ear of both females treated at 50%.

No increase in ear thickness = 25% was noted at any concentration. The highest concentration retained for the main test was then 50%.


- Main test

Mortality and clinical signs

No unscheduled deaths and no clinical signs were observed during the observation period.Body weight of animals was unaffected by the test item treatment.

 

Ear thickness measurements and local reactions

Residual of product was observed on Day 3 to Day 5 on both ears of 2/4 females treated at 25% and from Day 3 on both ears of all females treated at 50%.

In all test item-treated groups, the increase in ear thickness was below 10%. The test item was therefore considered as non irritant. As the acceptance limit of 25% was not reached at any concentrations, no bias in the results of proliferation measurements linked to an excessive irritant potential was considered.

 

Proliferation assay

The SI of the positive control was > 3; this experiment was therefore considered valid.

No significant lymphoproliferation was noted with the test item at any tested concentrations as all SI values were below the cut-off value of 3.

 

Conclusion

The test item was tested as a solution after preparation at concentrations of 2.5, 5, 10, 25 and 50% in dimethylformamide.

Under the experimental conditions of this study, the test itemgave a negative result in the murine Local Lymph Node Assay, indicative of the absence of skin sensitization properties. Therefore, the test item should not be classified as a skin sensitizer according to the criteria of CLP Regulation.

 

Treatment

Concentration

(%)

Irritation level

Stimulation Index

(SI)

Test item

2.5

I

1.12

Test item

5

I

0.63

Test item

10

I

0.79

Test item

25

I

1.11

Test item

50

I

1.28

HCA

25

-

25.02

-: not recorded.

I: non-irritant (increase in ear thickness < 10%).

HCA: a-hexyl cinnamaldehyde.

 

No significant lymphoproliferation was noted with the test item at any tested concentrations as all SI values were below the cut-off value of 3.