Oils, fish, sulfated, ammonium salt

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, but concerns for applicability of the method with this class of substances.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

GLP compliance:

yes

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: Charles River France.
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: 18 - 21 g
- Housing: Group caged during acclimatisation period, singly caged during study period. Polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm
- Diet (e.g. ad libitum): 4RF21 Mucedela S.r.l, ad libitum
- Water (e.g. ad libitum): Municipal supply, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 deg C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2012-04-11 To: 2012-05-02

Vehicle:

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

Concentration:

1.0, 2.5 and 5.0% w/w

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Good
- Irritation: Well defined irritation observed at a concentration of 50% and above. Very slight erythema observed at 10 and 25% concentrations. Ear thickness measurement was acceptable (less than 25% increase) at 5 concentration. Biopsy (ear punch) weight acceptable (less than 25% increase) at all concentrations.
- Lymph node proliferation response: Not assessed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: BrdU-ELISA method
- Criteria used to consider a positive response: The substance was considered to induce sensitisation when the stimulation index (SI) for any single treatment dose group was => 1.6. It was not required that an increased response was observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6 ≤ SI ≤ 1.9).

TREATMENT PREPARATION AND ADMINISTRATION: Dermal application of test item, positive control or vehicle(s) 25µL/ear (50 µL/animal) on Days 1,2 and 3. Intraperitoneal administration of BrdU on Day 5. Sacrifice of animals on Day 6, processing of lymph nodes and determination of Cell Proliferation

Positive control substance(s):

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

Statistics:

Differences between each treated group and the control group (individual BrdU labelling indices) were assessed by Dunnett's test. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied

Positive control results:

SI = 2.74

Parameter:

SI

Value:

2.51

Test group / Remarks:

Low dose, 1.0%w/w

Parameter:

SI

Value:

1.38

Test group / Remarks:

Mid dose, 2.5% w/w

Parameter:

SI

Value:

1.81

Test group / Remarks:

High dose, 5.0% w/w

Cellular proliferation data / Observations:

Increases in cell proliferation were observed in the three treatment groups (1%w/w, 2.5%w/w, 5%w/w), being the calculated stimulation indices (SI) 2.51, 1.38 and 1.81. These increases were not statistically significant and no dose relationship was indicated. The response observed in the low dose group (SI = 2.51) was higher than the threshold value of 1.9 for a clear positive result. It should be noted that the observed increase in proliferative response by draining lymph nodes is attributable to a single animal, which showed an abnormally high response when compared to the groupmates. This outlier result cannot be excluded for stimulation index calculation since no technical artefact was detected. However it should be noted that the stimulation index at this dose level, calculated considering only the remaining three animals, is 1.38, equal to that obtained at the intermediate dose level (2.5% w/w). In the high dose group the stimulation index was increased to 1.81. This value, although not statistically significant, is comprised between 1.6 and 1.9, thus indicating a borderline result.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The calculated stimulation indices (SI) were 2.51, 1.38 and 1.81 respectively at low-, mid- and high dose levels and indicate that the test item may elicit a sensitisation response in mice . Anyway, no dose-response relationship has been indicated, the hghest SI value is due to a single animal and, at highest dose, a not statistically significant borderline result of SI 1.81 was found (between 1.6 and 1.9). There are some concerns about the applicability of the method with this class of substances.

Executive summary:

Dermal sensitisation has been evaluated using OECD test methods (OECD 442B - Skin sensitisation: Local lymph node assay: BrdU-ELISA). Treatment resulted in an increase in cell proliferation of draining lymph nodes in the group treated at the highest concentration investigated when compared to controls, indicating that the test item may elicit a sensitisation response in mice. However, no dose-response relationship has been indicated, the hghest SI value (at the lowest dose) is due to a single animal and, at the highest dose, a not statistically significant borderline result was found.

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, but concerns for applicability of the method with this class of substances.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

GLP compliance:

yes

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: Charles River France.
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: 16 - 20 g
- Housing: Group caged during acclimatisation period, singly caged during study period. Polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm
- Diet (e.g. ad libitum): 4RF21 Mucedela S.r.l, ad libitum
- Water (e.g. ad libitum): Municipal supply, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 deg C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2012-04-18 To: 2012-05-08

Vehicle:

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

Concentration:

5, 10 and 25% w/w

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Good
- Irritation: Well defined irritation observed at a concentration of 50% and above. Very slight erythema observed at 5, 10 and 25% concentrations. Ear thickness measurement was acceptable (less than 25% increase) at concentrations up to 25%. Biopsy (ear punch) weight acceptable (less than 25% increase) at all concentrations.
- Lymph node proliferation response: Not assessed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: BrdU-ELISA method
- Criteria used to consider a positive response: The substance was considered to induce sensitisation when the stimulation index (SI) for any single treatment dose group was => 1.6. It was not required that an increased response was observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6 ≤ SI ≤ 1.9).

TREATMENT PREPARATION AND ADMINISTRATION: Dermal application of test item, positive control or vehicle(s) 25µL/ear (50 µL/animal) on Days 1,2 and 3. Intraperitoneal administration of BrdU on Day 5. Sacrifice of animals on Day 6, processing of lymph nodes and determination of Cell Proliferation

Positive control substance(s):

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

Statistics:

Differences between each treated group and the control group (individual BrdU labelling indices) were assessed by Dunnett's test. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied

Positive control results:

SI = 3.07

Parameter:

SI

Value:

1.85

Test group / Remarks:

Low dose, 5% w/w

Parameter:

SI

Value:

2.53

Test group / Remarks:

Mid dose, 10% w/w

Parameter:

SI

Value:

4.16

Test group / Remarks:

high dose, 25% w/w

Cellular proliferation data / Observations:

Dose related increases in cell proliferation were observed in all test item treated groups being the calculated stimulation index equal to 1.85, 2.53 and 4.16 respectively at 5, 10 and 25% w/w dose levels. The response was statistically significant in the mid- and high dose groups.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The calculated stimulation indices (SI) were 1.85, 2.53 and 4.16 respectively at low-, mid- and high dose levels and the response was statistically significant in the mid- and high dose groups, indicating that the test item may elicit a sensitisation response in mice.

Executive summary:

Dermal sensitisation has been evaluated using OECD test methods (OECD 442B - Skin sensitisation: Local lymph node assay: BrdU-ELISA). Treatment resulted in a statistically significant increase in cell proliferation of draining lymph nodes in the group treated at the mid and high concentration investigated when compared to controls, indicating that the test item may elicit a sensitisation response in mice.

Cross-reference 3

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, but concerns for applicability of the method with this class of substances.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

GLP compliance:

yes

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: Charles River France.
- Age at study initiation: 9 - 10 weeks
- Weight at study initiation: 18 - 23 g
- Housing: Group caged during acclimatisation period, singly caged during study period. Polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm
- Diet (e.g. ad libitum): 4RF21 Mucedela S.r.l, ad libitum
- Water (e.g. ad libitum): Municipal supply, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 deg C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2012-04-05 To: 2012-04-19

Vehicle:

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

Concentration:

2.5, 5.0 and 10.0% w/w

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Good
- Irritation: Well defined irritation observed at a concentration of 50% and above. Very slight erythema observed at 10% and 25% concentration. No irritation observed at 5% concentration. Ear thickness measurement was acceptable (less than 25% increase) at 5 and 10% concentrations. Biopsy (ear punch) weight acceptable (less than 25% increase) at 5, 10 and 25% concentration.
- Lymph node proliferation response: Not assessed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: BrdU-ELISA method
- Criteria used to consider a positive response: The substance was considered to induce sensitisation when the stimulation index (SI) for any single treatment dose group was => 1.6. It was not required that an increased response was observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6 ≤ SI ≤ 1.9).

TREATMENT PREPARATION AND ADMINISTRATION: Dermal application of test item, positive control or vehicle(s) 25µL/ear (50 µL/animal) on Days 1,2 and 3. Intraperitoneal administration of BrdU on Day 5. Sacrifice of animals on Day 6, processing of lymph nodes and determination of Cell Proliferation

Positive control substance(s):

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

Statistics:

Differences between each treated group and the control group (individual BrdU labelling indices) were assessed by Dunnett's test. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied

Positive control results:

SI = 5.45

Parameter:

SI

Value:

0.84

Test group / Remarks:

low dose, 2.5% w/w

Parameter:

SI

Value:

1.14

Test group / Remarks:

mid dose, 5% w/w

Parameter:

SI

Value:

1.71

Test group / Remarks:

high dose, 10% w/w

Cellular proliferation data / Observations:

Increase of lymphocyte proliferation was observed at the highest concentration tested, 10% w/w. This increase was not statistically significant and the calculated stimulation index was 1.71.
No response was observed in the low and mid-dose groups treated at 2.5% and 5% w/w (stimulation indices of 0.84 and 1.14, respectively).

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The calculated stimulation indices (SI) were 0.84, 1.14 and 1.71 respectively at low-, mid- and high dose levels and indicate that the test item may elicit a sensitisation response in mice. However, no response was observed in the low and mid-dose groups; increases of lymphocyte proliferation observed were not statistically significant and the highest calculated stimulation index, for the high dose, falls in the borderline result range (1.6-1.9). There are some concerns on the applicability of the method with this class of substances.

Executive summary:

Dermal sensitisation has been evaluated using OECD test methods (OECD 442B - Skin sensitisation: Local lymph node assay: BrdU-ELISA). Treatment resulted in an increase in cell proliferation of draining lymph nodes in the group treated at the highest concentration investigated when compared to controls, indicating that the test item may elicit a sensitisation response in mice. However, no response was observed in the low and mid-dose groups; increases of lymphocyte proliferation observed were not statistically significant and the highest calculated stimulation index, for the high dose, falls in the borderline result range.

Cross-reference 4

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, but concerns for applicability of the method with this class of substances.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

GLP compliance:

yes

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: Charles River France.
- Age at study initiation: 8 - 9 weeks
- Weight at study initiation: 18 - 22 g
- Housing: Group caged during acclimatisation period, singly caged during study period. Polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm
- Diet (e.g. ad libitum): 4RF21 Mucedela S.r.l, ad libitum
- Water (e.g. ad libitum): Municipal supply, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 deg C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2012-04-05 To: 2012-04-16

Vehicle:

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

Concentration:

1.0, 2.5 and 5.0% w/w

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Good
- Irritation: Well defined irritation observed at a concentration of 25% and above. Very slight erythema observed at 10% concentration. No irritation observed at 5% concentration. Ear thickness measurement was acceptable (less than 25% increase) at 5 and 10% concentrations. Biopsy (ear punch) weight acceptable (less than 25% increase) at 5% concentration.
- Lymph node proliferation response: Not assessed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: BrdU-ELISA method
- Criteria used to consider a positive response: The substance was considered to induce sensitisation when the stimulation index (SI) for any single treatment dose group was => 1.6. It was not required that an increased response was observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6 ≤ SI ≤ 1.9).

TREATMENT PREPARATION AND ADMINISTRATION: Dermal application of test item, positive control or vehicle(s) 25µL/ear (50 µL/animal) on Days 1,2 and 3. Intraperitoneal administration of BrdU on Day 5. Sacrifice of animals on Day 6, processing of lymph nodes and determination of Cell Proliferation

Positive control substance(s):

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

Statistics:

Differences between each treated group and the control group (individual BrdU labelling indices) were assessed by Dunnett's test. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied

Positive control results:

SI = 4.74

Parameter:

SI

Value:

1.47

Test group / Remarks:

low dose, 1% w/w

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: BrdU labelling index/group Control - 0.061 +/- 0.015 Low dose (1.0%) - 0.089 +/- 0.030 Mid dose (2.5%) - 0.090 +/- 0.036 High dose (5.0%) - 0.150 +/- 0.044

Parameter:

SI

Value:

1.48

Test group / Remarks:

mid dose, 2.5% w/w

Parameter:

SI

Value:

2.46

Test group / Remarks:

high dose, 5.0% w/w

Cellular proliferation data / Observations:

Statistically significant increase of lymphocyte proliferation was observed at the highest concentration tested, 5% w/w, with a calculated stimulation index of 2.46. SIs were lower than the "borderline range" (1.6-1.9) and only a slight, but not statistically significant, response was observed in the low and mid-dose groups treated at 1 and 2.5% w/w (SIs of 1.47 and 1.48).

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The calculated stimulation indices (SI) were 1.47, 1.48 and 2.46 respectively at low-, mid- and high dose levels and indicate that the test item may elicit a sensitisation response in mice, since in at least the high dose group the stimulation index is superior to the defined threshold of 1.9.

Executive summary:

Dermal sensitisation has been evaluated using OECD test methods (OECD 442B - Skin sensitisation: Local lymph node assay: BrdU-ELISA). Treatment resulted in a statistically significant increase in cell proliferation of draining lymph nodes in the group treated at the highest concentration investigated when compared to controls, indicating that the test item may elicit a sensitisation response in mice.

Cross-reference 5

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, but concerns for applicability of the method with this class of substances.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

GLP compliance:

yes

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: Charles River France.
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: 18 - 20 g
- Housing: Group caged during acclimatisation period, singly caged during study period. Polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm
- Diet (e.g. ad libitum): 4RF21 Mucedela S.r.l, ad libitum
- Water (e.g. ad libitum): Municipal supply, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 deg C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2012-04-11 To: 2012-04-24

Vehicle:

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

Concentration:

2.5, 5.0 and 10.0 % w/w

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Good
- Irritation: Well defined irritation observed at a concentration of 50% and above. Very slight erythema observed at 10 and 25% concentrations. No irritation observed at 5% concentration. Ear thickness measurement was acceptable (less than 25% increase) at concentrations up to 50%. Biopsy (ear punch) weight acceptable (less than 25% increase) at all concentrations.
- Lymph node proliferation response: Not assessed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: BrdU-ELISA method
- Criteria used to consider a positive response: The substance was considered to induce sensitisation when the stimulation index (SI) for any single treatment dose group was => 1.6. It was not required that an increased response was observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6 ≤ SI ≤ 1.9).

TREATMENT PREPARATION AND ADMINISTRATION: Dermal application of test item, positive control or vehicle(s) 25µL/ear (50 µL/animal) on Days 1,2 and 3. Intraperitoneal administration of BrdU on Day 5. Sacrifice of animals on Day 6, processing of lymph nodes and determination of Cell Proliferation

Positive control substance(s):

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

Statistics:

Differences between each treated group and the control group (individual BrdU labelling indices) were assessed by Dunnett's test. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied

Positive control results:

SI = 12.9

Parameter:

SI

Value:

1.59

Test group / Remarks:

low dose, 2.5% w/w

Parameter:

SI

Value:

3.87

Test group / Remarks:

mid dose, 5% w/w

Parameter:

SI

Value:

6.27

Test group / Remarks:

high dose, 10% w/w

Cellular proliferation data / Observations:

Increase in cell proliferation was observed at all dose levels tested, with a dose-related trend. The calculated stimulation indices were 1.59, 3.87 and 6.27 at the dosing concentrations of 2.5, 5 and 10% w/w respectively. The increment at the high dose was statistically significant when compared to the negative control group. In the group treated with the positive control item, a stimulation index of 12.90 was calculated. As it was greater than 2, the test system was regarded as valid.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The calculated stimulation indices (SI) were 1.59, 3.87 and 6.27 respectively at low-, mid- and high dose levels and indicate that the test item may elicit a sensitisation response in mice, since at mid and high dose groups the SIs were greater than the the defined threshold value of 1.9.

Executive summary:

Dermal sensitisation has been evaluated using OECD test methods (OECD 442B - Skin sensitisation: Local lymph node assay: BrdU-ELISA). Treatment resulted in a statistically significant increase in cell proliferation of draining lymph nodes in the group treated at the highest concentration investigated when compared to controls, indicating that the test item may elicit a sensitisation response in mice.

Cross-reference 6

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, but concerns for applicability of the method with this class of substances.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

GLP compliance:

yes

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: Charles River France.
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: 18 - 21 g
- Housing: Group caged during acclimatisation period, singly caged during study period. Polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm
- Diet (e.g. ad libitum): 4RF21 Mucedela S.r.l, ad libitum
- Water (e.g. ad libitum): Municipal supply, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 deg C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2012-04-11 To: 2012-04-27

Vehicle:

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

Concentration:

1.0, 2.5 and 5.0% w/w

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Good
- Irritation: Well defined irritation observed at a concentration of 25% and above. Very slight erythema observed at 5 and 10% concentrations. Ear thickness measurement was acceptable (less than 25% increase) at 5% concentration. Biopsy (ear punch) weight acceptable (less than 25% increase) at 10% concentration.
- Lymph node proliferation response: Not assessed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: BrdU-ELISA method
- Criteria used to consider a positive response: The substance was considered to induce sensitisation when the stimulation index (SI) for any single treatment dose group was => 1.6. It was not required that an increased response was observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6 ≤ SI ≤ 1.9).

TREATMENT PREPARATION AND ADMINISTRATION: Dermal application of test item, positive control or vehicle(s) 25µL/ear (50 µL/animal) on Days 1,2 and 3. Intraperitoneal administration of BrdU on Day 5. Sacrifice of animals on Day 6, processing of lymph nodes and determination of Cell Proliferation

Positive control substance(s):

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

Statistics:

Differences between each treated group and the control group (individual BrdU labelling indices) were assessed by Dunnett's test. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied

Positive control results:

SI = 3.53

Parameter:

SI

Value:

1.22

Test group / Remarks:

low dose, 1% w/w

Parameter:

SI

Value:

1.49

Test group / Remarks:

mid dose, 2.5% w/w

Parameter:

SI

Value:

1.79

Test group / Remarks:

high dose, 5% w/w

Cellular proliferation data / Observations:

Increases in cell proliferation were observed in a dose proportional manner in the three test item treatment groups and the calculated stimulation indices resulted to be 1.22, 1.49 and 1.79 respectively. These increments were not statistically significant when compared to the negative control group. The proliferative response observed at the high dose level (SI = 1.79) was comprised between 1.6 and 1.9, thus indicating a borderline result.

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The calculated stimulation indices (SI) were 1.22, 1.49 and 1.79 respectively at low-, mid- and high dose levels and indicate that the test item may elicit a sensitisation response in mice. However, increases in cell proliferation were not statistically significant when compared to the negative control group and the proliferative response observed at the high dose level (SI = 1.79) was comprised between 1.6 and 1.9, thus indicating a borderline result.There are some concerns about the applicability of the method with this class of substances.

Executive summary:

Dermal sensitisation has been evaluated using OECD test methods (OECD 442B - Skin sensitisation: Local lymph node assay: BrdU-ELISA). Treatment resulted in an increase in cell proliferation of draining lymph nodes in the group treated at the highest concentration investigated when compared to controls, indicating that the test item may elicit a sensitisation response in mice. However, increases in cell proliferation were not statistically significant when compared to the negative control group and the proliferative response observed at the high dose level (SI = 1.79) indicates a borderline result. There are some concerns about the applicability of the method with this class of substances.

Cross-reference 7

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, but concerns for applicability of the method with this class of substances.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

GLP compliance:

yes

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: Charles River France.
- Age at study initiation: 8 weeks
- Weight at study initiation: 17 - 20 g
- Housing: Group caged during acclimatisation period, singly caged during study period. Polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm
- Diet (e.g. ad libitum): 4RF21 Mucedela S.r.l, ad libitum
- Water (e.g. ad libitum): Municipal supply, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 deg C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2012-04-11 To: 2012-04-23

Vehicle:

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

Concentration:

2.5, 5.0 and 10.0% w/w

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Good
- Irritation: Well defined irritation observed at a concentration of 50% and above. Very slight erythema observed at 10 and 25% concentrations. No irritation observed at 5% concentration. Ear thickness measurement was acceptable (less than 25% increase) at 5 and 10% concentrations. Biopsy (ear punch) weight acceptable (less than 25% increase) at all concentrations.
- Lymph node proliferation response: Not assessed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: BrdU-ELISA method
- Criteria used to consider a positive response: The substance was considered to induce sensitisation when the stimulation index (SI) for any single treatment dose group was => 1.6. It was not required that an increased response was observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6 ≤ SI ≤ 1.9).

TREATMENT PREPARATION AND ADMINISTRATION: Dermal application of test item, positive control or vehicle(s) 25µL/ear (50 µL/animal) on Days 1,2 and 3. Intraperitoneal administration of BrdU on Day 5. Sacrifice of animals on Day 6, processing of lymph nodes and determination of Cell Proliferation

Positive control substance(s):

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

Statistics:

Differences between each treated group and the control group (individual BrdU labelling indices) were assessed by Dunnett's test. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied

Positive control results:

SI = 2.64

Parameter:

SI

Value:

1.13

Test group / Remarks:

low dose, 2.5% w/w

Parameter:

SI

Value:

1.17

Test group / Remarks:

mid dose, 5% w/w

Parameter:

SI

Value:

1.72

Test group / Remarks:

high dose, 10% w/w

Cellular proliferation data / Observations:

Increase of lymphocyte proliferation was observed at the highest concentration tested, 10% w/w, and was not statistically significant; the calculated stimulation index was 1.72. No response was observed in the low and mid-dose groups treated at 2.5% and 5% w/w (stimulation indices of 1.13 and 1.17 in Groups 2 and 3 respectively).

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The calculated stimulation indices (SI) were 1.13, 1.17 and 1.72 respectively at low-, mid- and high dose levels and indicate that the test item may elicit a sensitisation response in mice. However, no response was observed in the low and mid-dose groups treated at low and mid concentrations and increase of lymphocyte proliferation observed was not statistically significant: the highest calculated stimulation index was 1.72, for the high dose, falling in the borderline result range (1.6-1.9) . There are some concerns about the applicability of the method with this class of substances.

Executive summary:

Dermal sensitisation has been evaluated using OECD test methods (OECD 442B - Skin sensitisation: Local lymph node assay: BrdU-ELISA). Treatment resulted in an increase in cell proliferation of draining lymph nodes in the group treated at the highest concentration investigated when compared to controls, indicating that the test item may elicit a sensitisation response in mice. However, increase of lymphocyte proliferation observed was not statistically significant: and the highest calculated stimulation index was 1.72, for the high dose, falling in the borderline result range.

Cross-reference 8

Reason / purpose for cross-reference:

reference to other study

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012

Reliability:

3 (not reliable)

Rationale for reliability incl. deficiencies:

other: GLP guideline study, but concerns for applicability of the method with this class of substances.

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

GLP compliance:

yes

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: Charles River France.
- Age at study initiation: 6 - 8 weeks
- Weight at study initiation: 17 - 20 g
- Housing: Group caged during acclimatisation period, singly caged during study period. Polysulphone solid bottomed cages measuring 35.5 x 23.5 x 19 cm
- Diet (e.g. ad libitum): 4RF21 Mucedela S.r.l, ad libitum
- Water (e.g. ad libitum): Municipal supply, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 deg C
- Humidity (%): 40 - 70 %
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 2012-04-18 To: 2012-05-03

Vehicle:

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

Concentration:

2.5, 5.0 and 10.0% w/w

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Good
- Irritation: Well defined irritation observed at a concentration of 50% and above. Very slight erythema observed at 5, 10 and 25% concentrations. Ear thickness measurement was acceptable (less than 25% increase) at concentrations below 50%. Biopsy (ear punch) weight acceptable (less than 25% increase) at 5% concentration.
- Lymph node proliferation response: Not assessed

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: BrdU-ELISA method
- Criteria used to consider a positive response: The substance was considered to induce sensitisation when the stimulation index (SI) for any single treatment dose group was => 1.6. It was not required that an increased response was observed at increasing dose levels, but dose-related activity and/or statistical significance may be taken as further evidence of a sensitisation effect (i.e. in case of borderline results with 1.6 ≤ SI ≤ 1.9).

TREATMENT PREPARATION AND ADMINISTRATION: Dermal application of test item, positive control or vehicle(s) 25µL/ear (50 µL/animal) on Days 1,2 and 3. Intraperitoneal administration of BrdU on Day 5. Sacrifice of animals on Day 6, processing of lymph nodes and determination of Cell Proliferation

Positive control substance(s):

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

Statistics:

Differences between each treated group and the control group (individual BrdU labelling indices) were assessed by Dunnett's test. The homogeneity of the data was verified by Bartlett's test before Dunnett's test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied

Positive control results:

SI = 2.30

Parameter:

SI

Value:

1

Test group / Remarks:

low dose, 2.5% w/w

Parameter:

SI

Value:

0.93

Test group / Remarks:

mid dose, 5% w/w

Parameter:

SI

Value:

1.6

Test group / Remarks:

high dose, 10% w/w

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

The calculated stimulation indices (SI) were 1.00, 0.93 and 1.60 respectively at low-, mid- and high dose levels and indicate that the test item may elicit a sensitisation response in mice. However, neither statistically significant increases in cell proliferation at all doses nor a dose-response relationship were observed; in the highest calculated stimulation index was equal to 1.60, falling in the borderline result range (1.6-1.9). There are some concerns about the applicability of the method for this class of substances.

Executive summary:

Dermal sensitisation has been evaluated using OECD test methods (OECD 442B - Skin sensitisation: Local lymph node assay: BrdU-ELISA). Treatment resulted in an increase in cell proliferation of draining lymph nodes in the group treated at the highest concentration investigated when compared to controls, indicating that the test item may elicit a sensitisation response in mice. However, neither statistically significant incdreases in cell proliferation at all doses nor a dose-response relationship were observed and the maximum calculated SI was 1.60, the lowest limit of the range of borderline results.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A screening testing programme was carried out on seven sulfated oils representative samples using the Local Lymph Node Assay (LLNA: BrdU-ELISA method). Ambiguous results were found, suggesting weakly "positive" outcomes in the absence of QSAR flags for protein-binding (OECD toolbox) implying a false positive response. Thus, a confirmatory assay was undertaken using the GPMT on the substance for which the highest SI value was fouind in the LLNA testing programme (CSO3, SI = 6.27).

Test material

Test material information

Constituent 1

Reference substance name:

Rape oil, sulfated, sodium salt

EC Number:

281-978-8

EC Name:

Rape oil, sulfated, sodium salt

Cas Number:

84082-30-4

Molecular formula:

not available (substance is a UVCB)

IUPAC Name:

Not applicable

Test material form:

liquid

Details on test material:

Name of test material: CSO3
Lot/batch No.: 0008
Storage condition of test material: Room temperature

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: 403-480 g
- Housing: Up to 5 animals/cage noryl cages measuring 74.3 x 54.3 x 25 cm
- Diet (e.g. ad libitum): 8GP17 (Mucedola Srl) diet available ad libitum
- Water (e.g. ad libitum): tap water available ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2012-08-27 To: 2012-10-06

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

corn oil

Induction 2

Concentration / amount:

- Concentrations used for induction: intradermal treatment: 0.5% in water; dermal treatment: 5 % in water
- Concentration in Freunds Complete Adjuvant (FCA): 0.5% test substance in a mixture of FCA and water (1:1)
- Concentrations used for challenge: 1 %

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

corn oil

Challenge 2

Concentration / amount:

- Concentrations used for induction: intradermal treatment: 0.5% in water; dermal treatment: 5 % in water
- Concentration in Freunds Complete Adjuvant (FCA): 0.5% test substance in a mixture of FCA and water (1:1)
- Concentrations used for challenge: 1 %

No. of animals per dose:

test group: 20 animals
control group: 10 animals

Details on study design:

RANGE FINDING TESTS:
A dose range finding was performed in a preliminary study. 0.1 ml of test substance in water at various concentrations were injected intracutaneously into the skin of the scapula region. 24 hours and 7 days after application skin reaction was assessed.
For dermal applications various concentrations of the substance in water were applied on patches of 2x2 cm to the clipped skin. The patches were removed after 24 hours and dermal reactions were read immediately, 24 and 48 hours after patch removal.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2
- Exposure period: 1st exposure: intracutaneous injections (0.1 cm³); 2nd exposure: epicutaneous for 48 hours; evaluation after 24 hours each
- Concentration in Freunds Complete Adjuvants (FCA): test substance in vehicle at 0.5% concentration and in a mixture of FCA and vehicle (1:1)
- Test group: dermal: 5 % test substance in water (2 x 4 cm patch)
- Control group: vehicle (2 x 4 cm patch)
- Frequency of applications: day 1: intradermal treatment, day 8: dermal treatment
- Duration: 3 weeks


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: on day 22
- Exposure period: dermal application for 24 hours
- Test groups: test substance (2 x 2 cm patch)
- Control group: test substance (2 x 2 cm patch)
- Site: left flanks
- Concentrations: 1 %
- Evaluation: 24 and 48 hours after removal of dressings

Challenge controls:

see above B. Challenge exposure

Positive control substance(s):

yes

Remarks:

alfa-HEXYLCINNAMALDEHYDE

Results and discussion

Positive control results:

REFERENCE SUBSTANCE: alfa-HEXYLCINNAMALDEHYDE

CONCENTRATION: INDUCTION (INJECTION) - 20% in corn oil
(TOPICAL) - 50% in corn oil
CHALLENGE - 10% in acetone
CRITICAL DATES: INDUCTION (INJECTION) - 21 August 2012
(TOPICAL) - 28 August 2012
CHALLENGE - 11 September 2012

RESULTS: 70% response in test group and 0% response in control group at challenge.

INTERPRETATION: Incidence at challenge acceptable. Test system regarded as valid.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

1 %

No. with + reactions:

0

Total no. in group:

9

Clinical observations:

no visible change

Remarks on result:

no indication of skin sensitisation

Remarks:

Responses observed 24 h after challange by 24 h topical exposure to test item at 1% concentration and vehicle alone

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

1 %

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no visible change

Remarks on result:

no indication of skin sensitisation

Remarks:

Responses observed 24 h after challange by 24 h topical exposure to test item at 1% concentration and vehicle alone

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

1 %

No. with + reactions:

0

Total no. in group:

9

Clinical observations:

no visible change

Remarks on result:

no indication of skin sensitisation

Remarks:

Responses observed 48 h after challange by 24 h topical exposure to test item at 1% concentration and vehicle alone

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1 %

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

no visible change

Remarks on result:

no indication of skin sensitisation

Remarks:

Responses observed 48 h after challange by 24 h topical exposure to test item at 1% concentration and vehicle alone

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Remarks:

not sensitising

Conclusions:

Delayed dermal sensitisation has been investigated using the maximisation test according to OECD/EU test guideline No. 406. The substance did not induce delayed dermal sensitisation in the guinea pig.

Executive summary:

Delayed dermal sensitisation has been investigated using the maximisation test according to OECD/EU test guideline No. 406. The substance did not induce delayed dermal sensitisation in the guinea pig.