Propylidynetrimethanol, ethoxylated, esters with acrylic acid, reaction products with diethylamine

Endpoint summary

Description of key information

The murine Local Lymph Node Assay (LLNA, OECD 429) was performed to evaluate the skin sensitization potential of Diethylamine modified ethoxylated trimethylolpropane triacrylate. The substance gave a positive result in this test, indicative of a strong potential of skin sensitization.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 June 2012 - 20 July 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

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: on the beginning of the treatment period, the animals of the preliminary test were approximately 8 (first assay) or 9 (complementary assay) weeks old and the animals of the main test were approximately 9 weeks old
- Mean body weight at study initiation: the animals of the preliminary test had a mean body weight of 19.8 g (range: 19.0 g to 21.1 g) and the animals of the main test had a mean body weight of 20.7 g (range: 18.6 g to 22.4 g)
- Fasting period before study: no
- Housing: the animals were housed by group of two (preliminary test) or four (main test) in 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: the animals were acclimated to the study conditions for a period of 6 (first preliminary assay) or 13 (main test) days.

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: 27 June 2012 to 16 July 2012.

Vehicle:

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

Concentration:

0.25, 0.5, 1, 2.5 and 5%.

No. of animals per dose:

4 per dose.

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):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

no

Positive control results:

The threshold positive value of 3 for the SI was reached in the positive control group (SI = 20.99). The experiment was therefore considered valid.

Parameter:

SI

Value:

6.15

Test group / Remarks:

Dose 0.25%

Parameter:

SI

Value:

3.23

Test group / Remarks:

Dose 0.5%

Parameter:

SI

Value:

6.73

Test group / Remarks:

Dose 1%

Parameter:

SI

Value:

72.65

Test group / Remarks:

Dose 2.5%

Parameter:

SI

Value:

128.99

Test group / Remarks:

Dose 5%

Key result

Parameter:

EC3

Value:

< 0.25

Test group / Remarks:

treated group

Cellular proliferation data / Observations:

Dose of 0.25%: SI = 6.15 Dose of 0.5%: SI= 3.23 Dose of 1%: SI = 6.73 Dose of 2.5%: SI=72.65 Dose of 5%: SI = 128.99
DPM per group: Group 5: Vehicle: 47 Group 6: 0.25%: 289.13 Group 7: 0.5%: 152 Group 8: 1%: 316.13 Group 9: 2.5%: 3414.50 Group 10: 5%: 6062.75

Table of results :

 

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
Test item	0.25	I	6.15
Test item	0.5	I	3.23
Test item	1	I	6.73
Test item	2.5	I	72.65
Test item	5	I	128.99
HCA	25	-	20.99

-: not recorded.

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

Mortality and clinical signs

No unscheduled deaths and no clinical signs were observed during the observation period.

 

Local irritation (main test)

No local reactions were observed in any animals.

No notable increase in ear thickness was observed at any tested concentrations.

Proliferation assay

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

 

A dose-related increase in the SI was recorded at concentrations = 0.5%, and the threshold positive value of 3 was exceeded at the concentration of 0.25%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.

 

The EC3 value is considered to be < 0.25%

Interpretation of results:

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

Conclusions:

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 or extreme sensitizer.

Executive summary:

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

This study was performed according to the international guidelines (OECD No. 429 and Council Regulation No. 440/2008 of 30 May 2008, Part B.42) andin compliance with 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. Four groups of two male 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 0.5, 1, 2.5, 5, 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 at least once a day for mortality and clinical signs. Body weight was recordedonce during the acclimation period, and thenon days 1 and 6.On completion of the observation period, the animals were sacrificedthen 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. 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 thicknessof 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 were expressed as disintegrations per minute (dpm) per group and as dpm/node. The obtained values were used to calculate Stimulation Indices (SI).

 

Results

No unscheduled deaths and no clinical signs were observed during the observation period.  

No local reactions were observed in any animals. No notable increase in ear thickness was observed at any tested concentrations.
The SI of the positive control was > 3; this experiment was therefore considered valid.   A dose-related increase in the SI was recorded at concentrations = 0.5%, and the threshold positive value of 3 was exceeded at the concentration of 0.25%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.   The EC3 value is considered to be < 0.25%.

 

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 or extreme sensitizer.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitisation study :

The objective of this study was to evaluate the potential of the test item to induce delayed contact hypersensitivity using the murine Local Lymph Node Assay (LLNA, OECD 429). To assess the irritant potential of the test item (through ear thickness measurement), five groups of four female mice (main test) 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. 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.

No unscheduled deaths and no clinical signs were observed during the observation period. No local reactions were observed in any animals.No notable increase in ear thickness was observed at any tested concentrations. The SI of the positive control was > 3; this experiment was therefore considered valid. A dose-related increase in the SI was recorded at concentrations = 0.5%, and the threshold positive value of 3 was exceeded at the concentration of 0.25%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity. The EC3 value is considered to be < 0.25%.

Diethylamine modified ethoxylated trimethylolpropane triacrylate gave a positive result in the murine Local Lymph Node Assay, indicative of skin sensitization properties. According to the EC3 value obtained (< 0.25%), the test item should be considered as a strong or extreme sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Diethylamine modified ethoxylated trimethylolpropane triacrylate showed a strong skin sensitization potential in the LLNA. A classification is expected for skin sensitization endpoint, according to the Regulation EC n°1272/2008 : Skin Sens. 1A (May cause an allergic skin reaction, H317).

Justification : In the LLNA, the EC3 is considered to be smaller than 0.25%. It is a criteria to classify the test substance in the category 1A (because EC3< 2%).