Poly(oxy-1,2-ethanediyl), .alpha.,.alpha.'-[(1-methylethylidene)di-4,1-phenylene]bis[.omega.-hydroxy-, mixed acrylates and isononanoates

Endpoint summary

Description of key information

The murine Local Lymph Node Assay (LLNA, OECD 429) was performed to evaluate the skin sensitization potential of 4,4'-lsopropylidenediphenol, ethoxylated, esters with acrylic acid and isononanoic acid . The substance gave a positive result in this test, indicative of skin sensitization properties.

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:

15 April 2013 -- 11 June 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

In absence of reliable in vitro tests to evaluate the skin sensitisation potential in 2012, an in vivo skin sensitisation was performed.

Qualifier:

according to guideline

Guideline:

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

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:

other: CBA/J

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 9 weeks old on the day of treatment, 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 21.6 g (range: 20.3 g to 23.5 g) and the animals of the main test had a mean body weight of 20.6 g (range: 19.0 g to 22.9 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: for a period of 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: 23 May 2013 to 10 June 2013

Vehicle:

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 dose formulation 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

Positive control results:

The threshold positive value of 3 for the SI was reached in the positive control group (see Executive summary).

Key result

Parameter:

EC3

Value:

19

Parameter:

SI

Remarks:

5%

Value:

2

Test group / Remarks:

test group 5%

Parameter:

SI

Remarks:

10%

Value:

1.69

Test group / Remarks:

test group 10%

Parameter:

SI

Remarks:

25%

Value:

3.78

Test group / Remarks:

test group 25%

Parameter:

SI

Remarks:

50%

Value:

5.04

Test group / Remarks:

test group 50%

Parameter:

SI

Remarks:

100%

Value:

5.68

Test group / Remarks:

test group 100%

Cellular proliferation data / Observations:

A significant dose-related lymphoproliferation (SI > 3) was noted at 25, 50 and 100%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity. The EC3 value is equal to 19%.

In the main test :
No unscheduled deaths occurred during the main test.
No clinical signs indicative of systemic toxicity were observed in any animals.
No local reactions were observed in any animals. No notable increase in ear thickness was observed at any tested concentrations.
The body weight change of test item-treated animals was similar to that of control animals.

Stimulation index :
Group 4: 5%: 2.00
Group 5: 10%: 1.69
Group 6: 25%: 3.78
Group 7: 50%: 5.04
Group 8: 100%: 5.68
Group 9: 25%: 4.17 (positive control)


Desintegration per minute:
Group 3: Vehicle: 1893 dpm
Group 4: 5%: 3795
Group 5: 10%: 3193
Group 6: 25%: 7157
Group 7: 50%: 9536
Group 8: 100%: 10746
Group 9: 25%: 7900 (positive control)

Treatment	Concentration (%)	Irritation level	Stimulation Index (SI)
Test item	5	I	2.00
Test item	10	I	1.69
Test item	25	I	3.78
Test item	50	I	5.04
Test item	100	I	5.68
HCA	25	-	4.17

-: not recorded.

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

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, 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 weak 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 Practices.

 

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 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 earson days 1, 2 and 3 at concentrations of 5, 10, 25, 50 or 100% 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 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 nodes 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

No unscheduled deaths and no clinical signs indicative of systemic toxicity 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. No notable increase in ear thickness was observed at any tested concentrations.

 

The threshold positive value of 3 for the SI was reached in the positive control group (SI = 4.17). The experiment was therefore considered valid.

A significant dose-related lymphoproliferation (SI > 3) was noted at 25, 50 and 100%. In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity. The EC3 value is equal to 19%.

 

Conclusion

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 weak 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:

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 Practices.

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.

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 5, 10, 25, 50 or 100% 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 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 nodes 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).

No unscheduled deaths and no clinical signs indicative of systemic toxicity 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.No notable increase in ear thickness was observed at any tested concentrations. The threshold positive value of 3 for the SI was reached in the positive control group (SI = 4.17). The experiment was therefore considered valid. A significant dose-related lymphoproliferation (SI > 3) was noted at 25, 50 and 100%.In the absence of local irritation, the significant lymphoproliferative responses observed were attributed to delayed contact hypersensitivity.The EC3 value is equal to 19%.

Under the experimental conditions of this study, the test itemgave 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 weak sensitizer.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

According to the criteria of CLP Regulation, 4,4'-lsopropylidenediphenol, ethoxylated, esters with acrylic acid and isononanoic acid 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".

Justification: Based on the results of the LLNA, the EC3 value is equal to 19% corresponding to the category 1B of CLP.