3-methyl-1,5-pentanediyl diacrylate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 June 2013 - 31 July 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

see below

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Principles of method if other than guideline:

the following deviations from the agreed study plan:
. the animals of the second preliminary experiment had only 1 day of acclimation period instead of the 5 days specified in the study plan. Since the experimental conclusions are not based on these animals, this deviation is considered to not have any impact on the assay,
. at the beginning of the second preliminary test, the weight of the animal No. 303, is slightly higher than the range of 18 to 25 g (27.4 g),
. the ear thickness of the animal No. 17 was measured on the right ear from the day 1 since the left ear was too small for measuring ear thickness.
These deviations were not considered to have compromised the validity or integrity of the study.

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:

3-methyl-1,5-pentanediyl diacrylate

EC Number:

264-727-7

EC Name:

3-methyl-1,5-pentanediyl diacrylate

Cas Number:

64194-22-5

Molecular formula:

C12H18O4

IUPAC Name:

3-methyl-5-(prop-2-enoyloxy)pentyl prop-2-enoate

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 first preliminary test were approximately 12 weeks old on the day of treatment, the animals of the second preliminary test were approximately 8 weeks old, and the animals of the main test were approximately 10 weeks old.
- Mean body weight at study initiation: the animals of the first preliminary had a mean body weight of 22.5 g (range: 19.3 g to 27.4 g), the animals of the second preliminary test had a mean body weight of 20.8 g (range: 19.8 g to 22.0 g) and the animals of the main test had a mean body weight of 22.1 g (range: 19.7 g to 24.6 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 5 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: 03 July 2013 to 29 July 2013

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

First preliminary test: 10% ; 25% ; 50% and 100%
Second preliminary test: 0.05% ; 0.1% ; 0.25% ; 0.5% ; 1% and 2.5%
Main test : 0.25% ; 0.5% ; 1% ; 2.5% and 5%

No. of animals per dose:

- first preliminary test: 4 nulliparous and non pregnant females,
- second preliminary test: 6 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, 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):

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 = 6.75).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

EC3

Value:

0.9

Test group / Remarks:

test group

Results 2

Parameter:

SI

Value:

1.52

Test group / Remarks:

0.25%

Results 3

Parameter:

SI

Value:

1.32

Test group / Remarks:

0.5%

Results 4

Parameter:

SI

Value:

3.28

Test group / Remarks:

1%

Results 5

Parameter:

SI

Value:

16.13

Test group / Remarks:

2.5%

Results 6

Parameter:

SI

Value:

25.66

Test group / Remarks:

5%

Cellular proliferation data / Observations:

As a homogenous solution was obtained at the concentration of 50% ina 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 2.5%.
Since the concentration of 5% was not tested in both preliminary tests it was therefore selected as the highest concentration for the main test.
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.
 
Erythema was observed on days 3 and all females treated at the concentration of 5%. Dryness of ear skin was noted in all animals treated at concentrations of 2.5% and 5% on day 6.
At 5%, a mean increase in ear thickness of 62% was also recorded between days 1 and 6, showing an irritant effect of the test item.
At concentrations of 1 and 2.5%, slight increases in ear thickness of 4.44% and 9.90% were noted, respectively. As they were below 10%, these increases were considered meaningless.
 
The SI of the positive control was > 3 (SI = 6.75); this experiment was therefore considered valid.
The observed SI values were 1.52; 1.32; 3.28; 16.13 and 25.66 at concentrations of 0.25%, 0.5%, 1%, 2.5% and 5% (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 1%. In the absence of local irritation (except at the highest concentration), the significant lymphoproliferative responses observed at 1% and 2.5% were attributed to delayed contact hypersensitivity.
The EC3 value is equal to 0.9%.

Any other information on results incl. tables

Table of results

 

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
Vehicle	0	-	-
Test item	0.25	I	1.52
Test item	0.5	I	1.32
Test item	1	I	3.28
Test item	2.5	I	16.13
Test item	5	III	25.66
HCA	25	-	6.75

-: not recorded,

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

III: irritant (increase in ear thickness = 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 and at the tested concentrations, the test item gave a positive result in the murine Local Lymph Node Assay, indicative of skin sensitization properties.

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. 429and the principles of Good Laboratory Practices.

Methods

To assess the irritant potential of the test item (through ear thickness measurement), two preliminary tests were performed in order to define the test item concentrations to be used in the main test.

In the first preliminary 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 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.

As excessive local irritation (ear thickness > 25%) was noted in all concentrations tested during the first preliminary test, a second preliminary test was scheduled. In the second preliminary test, three 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 0.05%, 0.1%, 0.25%, 0.5%, 1% and 2.5% 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 route to the dorsal surface of both ears on days 1, 2 and 3 at concentrations of 0.25%, 0.5%, 1%, 2.5% and 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 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 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% ina 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 2.5%.

Since the concentration of 5% was not tested in both preliminary tests it was therefore selected as the highest concentration for the main test.

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.

 

Erythema was observed on days 3 and all females treated at the concentration of 5%. Dryness of ear skin was noted in all animals treated at concentrations of 2.5% and 5% on day 6.

At 5%, a mean increase in ear thickness of 62% was also recorded between days 1 and 6, showing an irritant effect of the test item.

At concentrations of 1 and 2.5%, slight increases in ear thickness of 4.44% and 9.90% were noted, respectively. As they were below 10%, these increases were considered meaningless.

 

The SI of the positive control was > 3 (SI = 6.75); this experiment was therefore considered valid.

The observed SI values were 1.52; 1.32; 3.28; 16.13 and 25.66 at concentrations of 0.25%, 0.5%, 1%, 2.5% and 5% (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 1%. In the absence of local irritation (except at the highest concentration), the significant lymphoproliferative responses observed at 1% and 2.5% were attributed to delayed contact hypersensitivity.

The EC₃value is equal to 0.9%.

Conclusion

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 strong sensitizer.

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