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Description of key information

A recent LLNA (2013) and two old studies performed according to the method of Magnusson and Kligman (GPMT) are available on Propoxylated neopentylglycol diacrylate. All these three studies showed the same results: Propoxylated neopentylglycol diacrylate is a skin sensitizer.

The most recent study was selected, and was chosen to classify the substance, because the composition of the tested substance is available.


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:
03 June 2013 - 26 June 2013
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)
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: 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
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
Positive control results:
The threshold positive value of 3 for the SI was reached in the positive control group.
Parameter:
SI
Remarks:
0.5%
Value:
1.33
Test group / Remarks:
test group 0.5%
Parameter:
SI
Remarks:
1%
Value:
1.47
Test group / Remarks:
test group 1%
Parameter:
SI
Remarks:
2.5%
Value:
1.74
Test group / Remarks:
test group 2.5%
Parameter:
SI
Remarks:
5%
Value:
3.19
Test group / Remarks:
test group 5%
Parameter:
SI
Remarks:
10%
Value:
7.89
Test group / Remarks:
test group 10%
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

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

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".

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Key study : LLNA performed on Propoxylated neopentylglycol diacrylate (composition similar to the substance registered).

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.

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 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).

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%.

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.

First supporting study : GPMT performed on Propoxylated neopentylglycol diacrylate (first batch).

Based on the results of a preliminary study and in compliance with the guideline (Method B6), the following dose levels were selected: 1% v/v in Alembicol D for intradermal injection, pure substance for topical application, 25% and 50% v/v in Alembicol D for challenge application. Ten test and five control guinea-pigs were used in this study.

No signs of ill health or toxicity were recorded. Bodyweight increases were recorded for all guinea-pigs over the period of the study.

After intradermal induction, necrosis was recorded at sites receiving Freund's Complete Adjuvant in test and control animals. Slight irritation was seen in test animals at sites receiving test substance, 1% v/v in Alembicol D and also in control animals receiving Alembicol D. After topical induction, moderate erythema was observed in test animals following topical application with test substance as supplied. No erythema was seen in control guinea-pigs. After the challenge, the dermal reactions seen in all of the test animals (100%) were more marked than in the controls. In this study, Propoxylated neopentylglycol diacrylate produced evidence of skin sensitisation (delayed contact hypersensitivity) in all of the test animals.

Second supporting study : GPMT performed on Propoxylated neopentylglycol diacrylate (second batch).

Based on the results of a preliminary study and in compliance with the guideline (B6 method), concentration of 1% v/v in Alembicol D was selected for intradermal injection, 50% v/v in Alembicol D for topical induction, 15% and 30% v/v in Alembicol D for challenge application. Ten tes and five control guinea pigs were used in this study.

No signs of ill health or toxicity were recorded. Bodyweight increases were recorded for all guinea-pigs over the period of the study.

After intradermal injections (induction), necrosis was recorded at Sites receiving Freund's complete adjuvant in test and control animals. Slight irritation was seen in test animals at sites receiving test substance, 1% v/v in Alembicol D and also in control animals receiving Alembicol D. After topical application (induction), moderate erythema was observed in test animals following topical application with test substance 50% v/v in Alembicol D. Very slight erythema was seen in the control guinea-pigs. After challenge application, the dermal reactions seen in all of the test animals were more marked than the controls. Propoxylated neopentylglycol diacrylate produced evidence of skin sensitisation (delayed contact hypersensitivity) in all of the test animals.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the recent LLNA performed, Propoxylated neopentylglycol diacrylate is a moderate skin sensitizer according to the Regulation (EC) No 1272/2008 : Skin Sens. 1B (May cause an allergic skin reaction, H317).


Justification : In the LLNA, the EC3 is 4.67% (>2); that justifies the category 1B.