Ceraphyl 55

Endpoint summary

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

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

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

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: Jackson Laboratories
- Received on: 05/18/04
- Age at study initiation: Approximately 7 weeks old
- Weight at study initiation: 20-24 g
- Housing: Animals were housed 1/cage in suspended wire cages. Bedding was placed beneath the cages and changed at least three times/week
- Diet (e.g. ad libitum): Fresh PMI (Diet #5001) freely available
- Water (e.g. ad libitum): Freely available
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature and Humidity: Controlled
- Photoperiod (hrs dark / hrs light): 12 hr light/12 hr dark

Positive control substance(s):

yes

Remarks:

hexylcinnamic aldhehyde (CAS No 101-86-0) 1.471 ml 25% HCA was added to 3.529 ml of AOO (Acetone: Olive Oil)

Vehicle:

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

Concentration:

Group 1: Naive
Group 2: AOO -Vehicle
Group 3: 25% HCA (Control)
Group 4: 5% Ceraphyl 55
Group 5: 10% Ceraphyl 55
Group 6: 25% Ceraphyl 55
Group 7: 50% Ceraphyl 55

No. of animals per dose:

Six groups of 5 CBA/J female mice

Details on study design:

The test material, vehicle or control, was applied and spread to the dorsum of each ear of the mice by topical application one time per day for three consecutive days. The naive group was not treated. A volume of 25 ul/ear of was the test material, vehicle or control was applied using a micro pipette. Animals were observed once daily for pharmacological effects and mortality. Bodyweights were recorded at the beginning of the study and prior to termination. Ears were observed at Day 1, 3 and 6.
Five days following the first dose mice were sacrificed. Five hours before the termination, mice were treated with 5-bromo-2'-deoxy-uridine at dose of 100 mg/kg by peritoneal injection using a 27 gauge needle. This is a thymidine analogue that becomes incorporated into DNA of proliferating cells including proliferating nodal lymphocytes. The auricular lymphocytes were isolated and single cell suspensions were generated.

Positive control substance(s):

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

Statistics:

The Stimulation Index (SI) was calculated for each group as follow:
For each animal, the number of BrdU+cell in the node was determined by cytometry and the mean ± SD was then calculated for each group. The mean BrdU+LNC in each test article treated group was then divided by that of the mean number of BrdU+LNC in the vehicle control group.

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

Group 1: Naive SI=159 S.D=±0.79 Group 2: AOO -Vehicle SI=1.00 S.D=±0.71 Group 3: 25% HCA (Control) SI=16.85 S.D=±11.77 Group 4: 5% Ceraphyl 55 SI=2.14 S.D=±0.44 Group 5: 10% Ceraphyl 55 SI=1.69 S.D=±0.81 Group 6: 25% Ceraphyl 55 SI=6.96 S.D=±7.71 Group 7: 50% Ceraphyl 55 SI=23.77 S.D=±3.25

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test article Ceraphyl 55 is a potential dermal sensitiser as indicated by a positive result (SI>3).

Executive summary:

A Local Lymph Node Assay (LLNA) study was conducted to determine the sensitising potential of the test material Ceraphyl 55. The mice were divided in 7 groups and treated as follows: Group 1: Naive; Group2: AOO -Vehicle; Group 3: 25% HCA (positive control); Group 4: 5% Ceraphyl 55; Group 5: 10% Ceraphyl 55; Group 6: 25% Ceraphyl 55; Group 7: 50% Ceraphyl 55. A volume of 25 ul was applied topically to the dorsum of each ear one time per day for three consecutive days. Animals were observed once daily through the study for pharmacological effect and mortality. Body weights were recorded before the test and prior to termination. Ears measurements were taken on Day 1, 3, and 6. Five day following the initial dose and 5 hours before the sacrifice, a dose of 100 mg/kg 5-bromo-2'-deoxy-uridine was administrated by peritoneal injection using a 27 gauge needle. This is a thymidine analogue that becomes incorporated into DNA of proliferating cells including proliferating nodal lymphocytes. The auricular lymphocytes were isolated and single cell suspensions were generated. The mean Stimulation Index (SI) was calculated for each group with the following results:

Group 1: Naive SI=159 S.D=±0.79

Group 2: AOO -Vehicle SI=1.00 S.D=±0.71

Group 3: 25% HCA (Control) SI=16.85 S.D=±11.77

Group 4: 5% Ceraphyl 55 SI=2.14 S.D=±0.44

Group 5: 10% Ceraphyl 55 SI=1.69 S.D=±0.81

Group 6: 25% Ceraphyl 55 SI=6.96 S.D=±7.71

Group 7: 50% Ceraphyl 55 SI=23.77 S.D=±3.25

Based on the criteria of this test, if the SI is≥3, the test article is considered to be a skin sensitiser. The result of the test shows that the SI of 25% Ceraphyl 55 (v/v) (Acetone/Olive oil 4:1) and 50% Ceraphyl 55 are 6.96 and 23.77, respectively. Therefore Ceraphyl 55 is considered to be a skin sensitiser.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Migrated from Short description of key information:
One guinea Pig Sensitization (Buehler) and three LLNA studies are available on Ceraphyl 55. The results confirm that the test material is a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Additional information:

Ceraphyl 55 was investigated for its sensitisation potential. One guinea Pig Sensitization (Buehler) and three LLNA studies are available.

A Guinea Pig Sensitization (Buehler) study was conducted by Consumer Product Testing (1985) on the material Ceraphyl 55, which was tested on 12 male Hartley-strain guinea pigs. In a pre-experiment, the highest concentration of the test material that produced no-irritation was selected for the induction phase. For induction, 0.5 ml of the test article (50% gravimetric corn oil suspension) was applied to the skin of the animal and occluded with a patch. Applications were done 3 times per week for three weeks. In addition, 2 weeks after the ninth treatment the test material was applied to the original test site and a virgin site to test local and systemic effects. Erythema and edema scores were recorded after 6 and 24 hours. No erythema, edema and gross internal changes were observed during the study; therefore the test material Ceraphyl 55 is not considered a potential sensitiser to guinea pig.

The test material Ceraphyl 55 was tested in a Murine Local Lymph Node Assay (Covance 2004). An irritation screening study was performed to determine the maximum concentration to be tested in the main test. Animals were dosed once with 25 ul at concentration of 0, (acetone/olive oil); 10; 25 and 50% Ceraphyl 55 in acetone/olive oil (v/v) or 100%. One animal treated with 100% Ceraphyl 55 was found recumbent, with few faces and showed irregular/laboured respiration. All remaining animals survived. No clinical observation, swelling or redness was recorded. The dose levels of 25, 50, and 100% Ceraphyl 55 were selected for the main test. In the main test, the animals were dosed once daily for three consecutively days using the doses selected in the preliminary test. The auricular lymph nodes were removed and a single cell suspension was prepared. The radiolabeled macromolecules were acid-prepared and the samples were counted using liquid scintillation spectrometry. Exposure to 25% hexylcinnamaldehyde in acetone/olive oil (4:1) resulted in a stimulation index of 5.7 and the mean was statistically significant (p=0.0163) when compared with the group treated with acetone/olive oil (4:1). The result of test is expressed is Simulation Index:

Naive Control: SI=NA

Control (Acetone/Olive oil 4:1): SI=1.0

25% Ceraphyl 55 (v/v) (Acetone/Olive oil 4:1): SI=2.8

50% Ceraphyl 55 (v/v) (Acetone/Olive oil 4:1): SI=6.9

100% Ceraphyl 55 (v/v) (Acetone/Olive oil 4:1): SI=17.3

Positive Control 25% Hexylcinnamaldehyde in Acetone/Olive oil 4:1): SI=5.7

Based on the criteria of this test, if the SI is≥3, the test article is considered to be a skin sensitiser.

The result of the test shows that the SI of 50% Ceraphyl 55 (v/v) (Acetone/Olive oil 4:1) and 100% Ceraphyl 55 is 6.9 and 17.3, therefore Ceraphyl 55 is considered to be a skin sensitiser.

 

In a third study conducted by Covance (2004) the test material Ceraphyl 55 was tested in a Murine Local Lymph Node Assay. Five animals per group were dosed once daily for three consecutively days using the following doses: 0% (acetone/olive oil), 0% (naive control), 10%; 25% and 50% Ceraphyl 55 in acetone/olive oil (v/v) , 100% Ceraphyl 55 and 25% (positive control) v/v. 25 microliters of the selected dose was applied to the dorsal aspect of each ear. The auricular lymph nodes were removed and a single cell suspension was prepared. The radiolabeled macromolecules were acid-prepared and the samples were counted using liquid scintillation spectrometry. Exposure to 25% hexylcinnamaldehyde in acetone/olive oil (4:1) resulted in a stimulation index of 2.4, which does not meet the general accepted criteria for classification as a skin sensitiser in the LLNA test.

The result of test is expressed is Simulation Index:

Naive Control: SI=NA

Control (Acetone/Olive oil 4:1): SI=1.0

10% Ceraphyl 55 (v/v) (Acetone/Olive oil 4:1): SI=1.4

25% Ceraphyl 55 (v/v) (Acetone/Olive oil 4:1): SI=1.2

50% Ceraphyl 55 (v/v) (Acetone/Olive oil 4:1): SI=3.6

100% Ceraphyl 55 (v/v) (Acetone/Olive oil 4:1): SI=5.8

Positive Control 25% Hexylcinnamaldehyde in Acetone/Olive oil 4:1): SI=2.4

Based on the criteria of this test, if the SI is≥3, the test article is considered to be a skin sensitiser.The result of the test shows that the SI of 50% Ceraphyl 55 (v/v) (Acetone/Olive oil 4:1) and 100% Ceraphyl 55 is 3.6 and 5.8, therefore Ceraphyl 55 is considered to be a skin sensitiser.

 

A forth Local Lymph Node Assay (LLNA) study was conducted to determine the sensitising potential of the test material Ceraphyl 55 (MB Reasearch, 2008). The mice were divided in 7 groups and treated as follow: Group 1: Naive; Group2: Acetone: Olive Oil -Vehicle; Group 3: 25% Hexylcinnamaldehyde ( positive control); Group 4: 5% Ceraphyl 55; Group 5: 10% Ceraphyl 55; Group 6: 25% Ceraphyl 55; Group 7: 50% Ceraphyl 55. A volume of 25 ul was applied topically to the dorsum of each ear one time per day for three consecutive days. Animals were observed once daily through the study for pharmacological effect and mortality. Body weights were recorded before the test and prior to termination. Ears measurements were taken on Day 1, 3, and 6. Five day following the initial dose and 5 hours before the sacrifice, a dose of 100 mg/kg 5-bromo-2'-deoxy-uridine was administrated by peritoneal injection using a 27 gauge needle. This is a thymidine analogue that becomes incorporated into DNA of proliferating cells including proliferating nodal lymphocytes. The auricular lymphocytes were isolated and single cell suspensions were generated. The mean Stimulation Index (SI) was calculated for each group with the following results:

Group 1: Naive SI=159 S.D=±0.79

Group 2: AOO -Vehicle SI=1.00 S.D=±0.71

Group 3: 25% HCA (Control) SI=16.85 S.D=±11.77

Group 4: 5% Ceraphyl 55 SI=2.14 S.D=±0.44

Group 5: 10% Ceraphyl 55 SI=1.69 S.D=±0.81

Group 6: 25% Ceraphyl 55 SI=6.96 S.D=±7.71

Group 7: 50% Ceraphyl 55 SI=23.77 S.D=±3.25

Based on the criteria of this test, if the SI is≥3, the test article is considered to be a skin sensitiser. The result of the test shows that the SI of 50% Ceraphyl 55 (v/v) (Acetone/Olive oil 4:1) and 100% Ceraphyl 55 are 6.96 and 23.77, therefore Ceraphyl 55 is considered to be a skin sensitiser. 

Justification for classification or non-classification

Based on the information presented above, Ceraphyl 55 is considered to be a skin sensitiser, therefore needs to be classified for skin sensitisation when considering the criteria outlined in Annex I of 1272/2008/EC and Annex VI of 67/548/EEC.