5,7-Di-t-butyl-3-[3,5-dimethyl-4-[(1,3,7,9-tetra-t-butyl-5-methyl-5H-benzo[d][1,3,2]benzodioxaphosphocin-11-yl)oxy]phenyl]-3H-benzofuran-2-one

Endpoint summary

Description of key information

The test substance was found to have a skin sensitising potential.

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:

2016-06-20 to 2017-03-02

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:

2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, under light exclusion, no direct sunlight
- Stability under test conditions: stable
- Solubility and stability of the test substance in the solvent/vehicle: After stirring with a magnetic stirrer the test substance was soluble in the vehicle MEK. The stability of the test substance in the vehicle was determined indirectly by the concentration control analysis.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Stirring with a magnetic stirrer to solve test item in the vehicle. The test-substance preparations were produced on a weight per weight (w/w) basis shortly before each application.

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V.,Inc., Postbus 6174, 5960 AD Horst, The Netherlands
- Age at study initiation: 8 weeks
- Weight at study initiation: 16.5 g – 20.5 g
- Housing: single housed
- Diet: Kliba mouse/rat maintenance diet “GLP”, supplied by Provimi Kliba SA, Kaiseraugst, Basel, Switzerland, ad libitum.
- Water: Drinking water ad libitum
- Acclimation period: at least 5 days before the first test substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 – 24
- Humidity (%): 30 – 70
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 2016-07-19 To:2016-08-01

Vehicle:

methyl ethyl ketone

Concentration:

2.5 %, 10 % and 25 %

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The test item was soluble in the vehicle.
- Irritation: After application of the 50 % concentration the animals showed considerably increased ear weights (compared to current vehicle values) and increases in ear thickness as indication of excessive ear skin irritation (increase >25 %). At the tested 10% concentration no relevant signs of local irritation were observed. Scaling was noted in one animal treated with the 10 % concentration on study day 5.
- Systemic toxicity: No relevant signs of systemic toxicity were observed in the pretest
- Ear thickness measurements: After application of the 50 % concentration the animals showed considerably increased ear weights (compared to current vehicle values) and increases in ear thickness as indication of excessive ear skin irritation (increase >25 %).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
A test item is regarded as a sensitizer in the LLNA if exposure to at least one concentration of the test item results in an incorporation of 3H thymidine at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index (SI ≥ 3.0). However, the biological relevance of the obtained stimulation indices is considered in conjunction with the other assessed end points (i.e. cell counts, lymph node weights, ear weights). Hereby, the thresholds used for assessment of cell counts and ear weights are represented by stimulation indices (SI) of 1.5 and 1.25, respectively.
If applicable, the EC (estimated concentration) leading to the respective SI values were calculated by linear or semi-logarithmical regression between the data points directly below and above the SI if possible or using the two nearest points below or above the SI.

TREATMENT PREPARATION AND ADMINISTRATION:
After stirring with a magnetic stirrer the test substance was soluble in the vehicle and applied as a solution. 25 µL per ear was applied on the dorsal part of the ear on 3 consecutive applications (day 0 – day 2) to the same application site.

Positive control substance(s):

other: Alpha-Hexylcinnamaldehyde, techn. 85%

Statistics:

Mean values and standard deviations of the measured parameters were calculated for the test
and control groups from the individual values. The stimulation indices of 3H-thymidine
incorporation, cell count, lymph node weight and ear weight measurements were calculated as the ratio of the test group mean values for these parameters divided by those of the vehicle control group.
Statistical analysis of 3H-thymidine incorporation, cell count, lymph node weight and ear weight was performed using the WILCOXON - Test.

Positive control results:

The 15 % HCA preparation in MEK induced a biologically relevant and statistically significant response in 3H-thymidine incorporation (SI = 7.99) and lymph node cell counts (SI = 2.24) as well as statistically significant increased lymph node weights (SI = 2.05).

Key result

Parameter:

SI

Remarks:

for 3H-thymidine incorporation

Value:

4.17

Test group / Remarks:

25 % test material

Key result

Parameter:

SI

Remarks:

for 3H-thymidine incorporation

Value:

2.47

Test group / Remarks:

10 % test material

Key result

Parameter:

SI

Remarks:

for 3H-thymidine incorporation

Value:

1.49

Test group / Remarks:

2.5 % test material

Key result

Parameter:

SI

Remarks:

auricular lymph node cell counts

Value:

1.67

Test group / Remarks:

25 % test material

Key result

Parameter:

SI

Remarks:

auricular lymph node cell counts

Value:

1.52

Test group / Remarks:

10 % test material

Key result

Parameter:

SI

Remarks:

auricular lymph node cell counts

Value:

1.13

Test group / Remarks:

2.5 % test material

Key result

Parameter:

SI

Remarks:

lymph node weight

Value:

1.68

Test group / Remarks:

25 % test material

Key result

Parameter:

SI

Remarks:

lymph node weight

Value:

1.45

Test group / Remarks:

10 % test material

Key result

Parameter:

SI

Remarks:

lymph node weight

Value:

1.22

Test group / Remarks:

2.5 % test material

Key result

Parameter:

SI

Remarks:

ear weight

Value:

1.17

Test group / Remarks:

25 % test material

Key result

Parameter:

SI

Remarks:

ear weight

Value:

1.05

Test group / Remarks:

10 % test material

Key result

Parameter:

SI

Remarks:

ear weight

Value:

1.03

Test group / Remarks:

2.5 % test material

Parameter:

SI

Remarks:

³H-thymidine incorporation

Value:

1

Test group / Remarks:

vehicle

Parameter:

SI

Remarks:

³H-thymidine incorporation

Value:

7.99

Test group / Remarks:

positive control

Parameter:

SI

Remarks:

auricular lymph node cell counts

Value:

1

Test group / Remarks:

vehicle

Parameter:

SI

Remarks:

auricular lymph node cell counts

Value:

2.24

Test group / Remarks:

positive control

Parameter:

SI

Remarks:

lymph node weight

Value:

1

Test group / Remarks:

vehicle

Parameter:

SI

Remarks:

lymph node weight

Value:

2.05

Test group / Remarks:

positive control

Parameter:

SI

Remarks:

ear weight

Value:

1

Test group / Remarks:

vehicle

Parameter:

SI

Remarks:

ear weight

Value:

1.1

Test group / Remarks:

positive control

Key result

Parameter:

EC3

Value:

14.7

Cellular proliferation data / Observations:

EC3 CALCULATION
The threshold concentration for sensitization induction was >10 % and <25 %. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation was calculated by linear regression from the results of these concentrations to be 14.7 %.

CLINICAL OBSERVATIONS:
No signs of systemic toxicity were noticed in all animals during general observation.
Very slight erythema was noted on the ear skin of all animals at 25% on study days 1 and 2 and at 10 % on study day 2. Slight swelling and residues of test substance were observed in all animals at all concentrations during the observation period. The animals treated with the 25 % concentration showed pull out of hair at the head on study days 2 and/or 5.
Very slight erythema and slight swelling were observed on the ear skin of the animals treated with a 15 % HCA preparation on study days 1 and 2.

BODY WEIGHTS
The expected body weight gain was generally observed in the course of the study.

When applied as 25% preparation in MEK, the test substance induced a statistically significant increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes, which was above the cut off value for biological relevance (increase above the cut off stimulation index (SI) of 3). The increase of the 10% test-substance preparation was statistically significant, but failed to reach the cut off.

Concomitantly, the increases in the auricular lymph node cell counts at 25% (SI = 1.67) and 10% (SI = 1.52) were statistically significant and biologically relevant (above the cut-off value (increase to 1.5 fold or above of control value = stimulation index (SI)≥1.5).

In addition, statistically significant increases in lymph node weights at 25% (Si = 1.68) and 10% (SI = 1.45) were noted.

No relevant increase in either of above endpoints were observed at 2.5% preparation.

The 15% HCA preparation in MEK induced a biologically relevant and statistically significant response in 3H-thymidine incorporation (SI = 7.99) and lymph node cell counts (SI = 2.24) as well as statistically significant increased lymph node weights (SI = 2.05).

The threshold concentration for sensitization induction was >10% <25%. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation was calculated by linear regression from the results of these concentrations to be 14.7%.

Interpretation of results:

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

Conclusions:

The test item exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.

Executive summary:

The skin sensitizing potential of the test article was assessed using the radioactive Murine Local Lymph Node Assay. Groups of 5 female CBA/CaOlaHsd mice each were treated with 25 µl of 2.5%, 10% and 25% w/w preparations of the test substance in methyl ethyl ketone (MEK) or with the vehicle alone. The high concentration was selected based on the presence of ear skin irritation in a pretest using a 50% preparation. A concurrent positive control group with 5 female mice was treated with 25µl of 15% Alpha-Hexylcinnamaldehyde in MEK. Three days after the last application the mice were injected into the tail vein with 20 μCi of ³H-thymidine in 250 μL of sterile saline. About 5 hours after the ³H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring ³H-thymidine incorporation. Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition, the ear weights were determined in order to obtain an indication of possible skin irritation. No signs of systemic toxicity were noticed in all animals during general observation. When applied as 25% preparation in MEK, the test substance induced a statistically significant increase of ³H-thymidine incorporation into the cells from the auricular lymph nodes, which was above the cut off value for biological relevance (increase above the cut off stimulation index (SI) of 3). The increase of the 10% test-substance preparation was statistically significant, but failed to reach the cut off. Concomitantly, the increases in the auricular lymph node cell counts at 25% and 10% were statistically significant and biologically relevant (above the cut-off value (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5). In addition, statistically significant increases in lymph node weights at 25% and 10% were noted. No relevant increase in either of above endpoints were observed at 2.5% preparation. The ear weight stimulation indices did not indicate relevant increases in ear weights (SI ≥ 1.25), demonstrating the absence of excessive ear skin irritation. However, there was a statistically significant increase in ear weights at 25%. In addition, very slight erythema was noted on the ear skin of all animals at 25% on study days 1 and 2 and at 10% on study day 2. Slight swelling and residues of the test substance were observed in all animals at all test-substance concentrations during the observation period. The animals treated with the 25% concentration showed pull out of hair at the head on study days 2 and/or 5. The 15% HCA preparation in MEK induced a biologically relevant and statistically significant response in ³H-thymidine incorporation and lymph node cell counts as well as statistically significant increased lymph node and ear weights. In addition, very slight erythema and slight swelling were observed on the ear skin on study day 1 and 2. Thus, it is concluded that the test article exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen. The threshold concentration for sensitization induction was >10% <25%. The EC 3 (estimated concentration that leads to the SI of 3.0) for ³H-thymidine incorporation was calculated by linear regression from the results of these concentrations to be 14.7%.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

The skin sensitizing potential of the test item was assessed using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of cells in the auricular lymph nodes to repeated application of the test substance to the dorsal skin of the ears. Groups of 5 female CBA/CaOlaHsd mice each were treated with 2.5 %, 10 % and 25 % w/w preparations of the test substance in methyl ethyl ketone (MEK) or with the vehicle alone. The high concentration was selected based on the presence of ear skin irritation in a pretest using a 50 % preparation. A concurrent positive control group with 5 female CBA/CaOlaHsd mice was treated with 15 % Alpha-Hexylcinnamaldehyde, techn. 85 % (HCA) in MEK. The study used 3 test groups, 1 control group and 1 positive control group. Each test animal was treated with 25 μL per ear of the appropriate test-substance preparation, applied to the dorsal surfaces of both ears for three consecutive days. The control group was treated with 25 μL per ear of the vehicle alone. The animals of the positive control group were treated with 25 μL of a 15 % preparation of Alpha-Hexylcinnamaldehyde in MEK. Three days after the last application the mice were injected into the tail vein with 20 μCi of 3H-thymidine in 250 μL of sterile saline. About 5 hours after the 3H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring 3H-thymidine incorporation (indicator of cell proliferation). Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition, a 0.8 cm diameter sample was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined in order to obtain an indication of possible skin irritation. No signs of systemic toxicity were noticed in all animals during general observation.

When applied as 25 % preparation in MEK, the test substance induced a statistically significant increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes, which was above the cut off value for biological relevance (increase above the cut off stimulation index (SI) of 3). The increase of the 10 % test-substance preparation was statistically significant, but failed to reach the cut off. Concomitantly, the increases in the auricular lymph node cell counts at 25 % and 10 % were statistically significant and biologically relevant (above the cut-off value (increase to 1.5 fold or above of control value = stimulation index (SI) ≥ 1.5). In addition, statistically significant increases in lymph node weights at 25 % and 10 % were noted. No relevant increase in either of above endpoints were observed at 2.5 % preparation. The ear weight stimulation indices did not indicate relevant increases in ear weights (SI ≥ 1.25), demonstrating the absence of excessive ear skin irritation. However, there was a statistically significant increase in ear weights at 25 %. In addition, very slight erythema was noted on the ear skin of all animals at 25 % on study days 1 and 2 and at 10% on study day 2. Slight swelling and residues of the test substance were observed in all animals at all test-substance concentrations during the observation period. The animals treated with the 25 % concentration showed pull out of hair at the head on study days 2 and/or 5. The 15 % HCA preparation in MEK induced a biologically relevant and statistically significant response in 3H-thymidine incorporation and lymph node cell counts as well as statistically significant increased lymph node and ear weights. In addition, very slight erythema and slight swelling were observed on the ear skin on study day 1 and 2. Thus, it is concluded that the test substance exhibits a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen. The threshold concentration for sensitization induction was >10 % and <25 %. The EC 3 (estimated concentration that leads to the SI of 3.0) for 3H-thymidine incorporation was calculated by linear regression from the results of these concentrations to be 14.7 %.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. The test item was found to have a skin sensitising property in the Local Lymph Node Assay in mice and an EC 3 value of 14.7 % was calculated. As a result the substance is classified as skin sensitising (Cat 1B) under Regulation (EC) No 1272/2008, as amended for the tenth time in Regulation (EU) No 2017/776.