Poly[oxy(methyl-1,2-ethanediyl)], .alapha.,α'-(2,2-dimethyl-1,3-propanediyl)bis[ω-[(1-oxo-2-propenyl)oxy

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 June 2013 - 26 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2010

Deviations:

no

Test guideline 2

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

Test material information

Constituent 1

Reference substance name:

Esterification products of acrylic acid with reaction products of 2,2-dimethylpropane-1,3-diol and methyloxirane

EC Number:

701-440-2

Molecular formula:

(C3H6O)x (C3H6O)y C11H16O4

IUPAC Name:

Esterification products of acrylic acid with reaction products of 2,2-dimethylpropane-1,3-diol and methyloxirane

Constituent 2

Reference substance name:

Poly [oxy(methyl-1,2 ethanediyl)], a,a'-(2,2-dimethyl-1,3-propanediyl) bis [¿-[(1-oxo-2-propen-1-yl) oxy]-

IUPAC Name:

Poly [oxy(methyl-1,2 ethanediyl)], a,a'-(2,2-dimethyl-1,3-propanediyl) bis [¿-[(1-oxo-2-propen-1-yl) oxy]-

Test material form:

liquid

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: the animals of the preliminary test were approximately 11 weeks old and the animals of the main test were approximately 8 weeks old
- Mean body weight at study initiation: the animals of the preliminary test had a mean body weight of 21.3 g (range: 19.5 g to 22.4 g) and the animals of the main test and had a mean body weight of 21.3 g (range: 20.3 g to 22.3 g)
- Fasting period before study: no
- Housing: polycarbonate cages
- 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 study

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

IN-LIFE DATES: 12 June 2013 to 24 June 2013

Study design: in vivo (LLNA)

Vehicle:

other: acetone/olive oil (4/1; v/v)

Concentration:

The maximum concentration tested in the main test was selected according to the criteria specified in the OECD Guidelines and on the basis of the data that was obtained in the preliminary test:
- the vehicle should be selected on the basis of producing a homogeneous preparation suitable for application of the test item,
- the concentrations were selected from the concentration series 100% (when test item can be sampled by a pipette), 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, etc,
- 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 = 25% increase of the ear thickness.

No. of animals per dose:

- preliminary test: 4 nulliparous and non pregnant females,
- main test: 28 nulliparous and non pregnant females.

Details on study design:

RANGE FINDING TESTS:
The maximum concentration tested in the main test was selected according to the criteria specified in the OECD Guidelines and on the basis of the data that was obtained in the preliminary test:
- the vehicle should be selected on the basis of producing a homogeneous preparation suitable for application of the test item,
- the concentrations were selected from the concentration series 100% (when test item can be sampled by a pipette), 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5%, etc,
- 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 = 25% increase of the ear thickness.

MAIN STUDY
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, at approximately the same time each day, 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.
The dose formulations were stirred manually throughout the dosing procedure.

Positive control substance(s):

other: a-hexyl cinnamaldehyde (HCA)

Statistics:

no

Results and discussion

Positive control results:

The threshold positive value of 3 for the SI was reached in the positive control group.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks:

0.5%

Value:

1.33

Test group / Remarks:

test group 0.5%

Results 2

Parameter:

SI

Remarks:

1%

Value:

1.47

Test group / Remarks:

test group 1%

Results 3

Parameter:

SI

Remarks:

2.5%

Value:

1.74

Test group / Remarks:

test group 2.5%

Results 4

Parameter:

SI

Remarks:

5%

Value:

3.19

Test group / Remarks:

test group 5%

Results 5

Parameter:

SI

Remarks:

10%

Value:

7.89

Test group / Remarks:

test group 10%

Results 6

Key result

Parameter:

EC3

Value:

4.67

Cellular proliferation data / Observations:

The observed SI values were 1.33, 1.47, 1.74, 3.19 and 7.86 at concentrations of 0.5%, 1%, 2.5%, 5% and 10% (w/v), respectively. A dose-related increase in the SI was recorded and the threshold positive value of 3 was exceeded from the concentration of 5%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity. The EC3 value was equal to 4.67%.

DPM per group: Group 3: Vehicle: 1106 Group 4: 0.5 %: 1466 Group 5: 1 %: 1626 Group 6: 2.5 %: 1927 Group 7: 5 %: 3523 Group 8: 10%: 8694

Any other information on results incl. tables

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
Test item	0.5	I	1.33
Test item	1	I	1.47
Test item	2.5	I	1.74
Test item	5	I	3.19
Test item	10	II	7.86
HCA	25	-	12.77

-: not recorded

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

II: slightly irritant (increase in ear thickness = 10 to 25%)

HCA: a-hexyl cinnamaldehyde

Applicant's summary and conclusion

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

Under the experimental conditions of this study and at the tested concentrations (test item up to 10% in solution), the test item gave a positive result in the murine Local Lymph Node Assay, indicative of skin sensitization properties. According to the EC3 value obtained, the test item should be considered as a moderate sensitizer.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce contact hypersensitivity using the murine Local Lymph Node Assay (LLNA).

This study was conducted in compliance with OECD Guideline No. 429 and the principles of Good Laboratory Practice.

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 10, 25, 50 or 100% under a dose-volume of 25 µL. From days 1 to 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 at least 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 route to the dorsal surface of both earson days 1, 2 and 3 at concentrations of 0.5, 1, 2.5, 5 or 10% under a dose-volume of 25 µL. One negative control group of four females received the vehicle (acetone/olive oil(4/1; v/v)) under the same experimental conditions. Additionally, one positive control group of four 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 days 1 to 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 at least once a day for mortality and clinical signs. Body weight was recorded once 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

As a homogenous solution was obtained at the concentration of 50% in a mixture of Acetone/Olive Oil (4/1; v/v) (AOO), AOO was selected for preparation of the test item. In the assessment of local skin irritation performed in the preliminary test, the increase of the ear thickness was lower than the limit of 25% at the concentration of 10%. The highest concentration retained for the main test was therefore 10%.

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

At 10%, dryness of ear skin was noted in all females on day 6. A mean increase of ear thickness of 20.62% was also recorded between days 1 and 6, showing a slightly irritant effect of the test item. At the concentration of 5%, a slight increase in ear thickness of 3.96% was noted. As it was below 10%, this increase was considered meaningless.

The SI of the positive control was > 3 (SI = 12.77); this experiment was therefore considered valid.

The observed SI values were 1.33, 1.47, 1.74, 3.19 and 7.86 at concentrations of 0.5%, 1%, 2.5%, 5% and 10% (w/v), respectively. A dose-related increase in the SI was recorded and the threshold positive value of 3 was exceeded from the concentration of 5%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.

The EC3 value was equal to 4.67%.

Conclusion

Under the experimental conditions of this study and at the tested concentrations (test item up to 10% in solution), the test item gave a positive result in the murine Local Lymph Node Assay, indicative of skin sensitization properties.

According to the EC3 value obtained, the test item should be considered as a moderate sensitizer.

According to the criteria of CLP Regulation,the test item should be classified as skin sensitizer (category 1 and sub-category 1B) and assigned the signal word "warning" and the hazard statement "H317: May cause an allergic skin reaction".