Glycerol, propoxylated, esters with acrylic acid, reaction products with diethylamine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

06 January 2015 -- 09 February 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 guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

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:

Glycerol, propoxylated, esters with acrylic acid, reaction products with diethylamine

EC Number:

500-744-0

EC Name:

Glycerol, propoxylated, esters with acrylic acid, reaction products with diethylamine

Cas Number:

162492-10-6

Molecular formula:

(C3H8O3) (C3H6O)m(C3H2O)n ( C4H11N)o ; m (1-6) ; n (1-3) , o (1-3)

IUPAC Name:

Glycerol, propoxylated, esters with acrylic acid, reaction products with diethylamine

Test material form:

liquid

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France.
- Age/weight: at the beginning of the treatment period, the animals of the preliminary tests were approximately 8 to 10 weeks old and had a mean body weight of 20.3 g (range: 19.2 g to 22.0 g) and the animals of the main test were approximately 9 weeks old and had a mean body weight of 21.0 g (range: 18.8 g to 25.6 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: at least 6 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 30 to 70%
- 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: 08 January 2015 to 09 February 2015

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

main study: 0.25%, 0.5%, 1%, 2.5%, 5%, 25%
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 tests:
- the vehicle was 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 increase of the ear thickness = 25%.

No. of animals per dose:

Number:
- preliminary tests: 8 nulliparous and non pregnant females
- main test: 28 nulliparous and non pregnant females : 4/doses

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 tests:
- the vehicle was 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 increase of the ear thickness = 25%.

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, chemical toxicity, and radioactivity levels.

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.

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: SI = 20,01.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

1.49

Results 2

Parameter:

SI

Value:

0.78

Test group / Remarks:

0.25%

Results 3

Parameter:

SI

Value:

0.45

Test group / Remarks:

0.5%

Results 4

Parameter:

SI

Value:

1.33

Test group / Remarks:

1%

Results 5

Parameter:

SI

Value:

6.42

Test group / Remarks:

1.25%

Results 6

Parameter:

SI

Value:

19.06

Test group / Remarks:

5%

Any other information on results incl. tables

Table of results :

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
Test item	0.25	I	0.78
Test item	0.5	I	0.45
Test item	1	I	1.33
Test item	2.5	I	6.42
Test item	5	II	19.06
HCA	25	-	20.01

-: not recorded.

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

II: slightly irritant (increase in ear thickness=10 but <25%).

HCA: a-hexyl cinnamaldehyde.

Applicant's summary and conclusion

Interpretation of results:

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

Conclusions:

Under the experimental conditions of this study, 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 (1.49), 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 the principles of Good Laboratory Practice.

 

Methods

 To assess the irritant potential of the test item (through ear thickness measurement), two preliminary tests were first performed in order to define the test item concentrations to be used in the main test. Two groups of two female mice (for each preliminary test) 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% in the first preliminary test and at concentrations of 0.5, 1, 2.5 or 5% in the second preliminary test 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 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 ears on Days 1, 2 and 3 at concentrations of 0.25, 0.5, 1, 2.5 or 5% 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 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 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.

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 of 3H-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 irritation performed in the preliminary test, the increase in ear thickness between Days 1 and 6 was higher than the limit of 25% at concentrations of 10, 25, 50 and 100% and dryness of ear skin was observed at 10, 25, 50 and 100% together with erythema at 25% and 100%.

Since the increase in ear thickness was below the limit of 25% in 1/2 mice given the concentration of 5% (+20.83%) and since no local reactions were noted in both animals, the highest concentration retained for the main test was 5%.

 

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.

 

No local reactions were observed in any animals, however the test item was considered as slightly irritant at the concentration of 5% since an increase in ear thickness of 11.09% was noted.

 

The SI of the positive control was > 3 (SI = 20.01); this experiment was therefore considered valid.

 

The observed SI values were 0.78, 0.45, 1.33, 6.42 and 19.06 at concentrations of 0.25, 0.5, 1, 2.5 and 5% (v/v), respectively.

A dose-related increase in the SI was recorded and the threshold positive value of 3 was exceeded at tested concentrations of 2.5 and 5%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.

 

The EC3 value was equal to 1.5%.

 

Conclusion

The test item was tested as a solution after preparation at concentrations of 0.25, 0.5, 1, 2.5 and 5% in Acetone/Olive Oil (4/1; v/v).

Under the experimental conditions of this study, 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.