Cyclohexane, 1-methyl-4-(1-methylethyl)-, tetradehydro deriv.

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

Study period:

June 27, 2012- July 5, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted according to GLP and valid methods, therefore it is considered relevant, reliable and adequate for classification.

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: TOXI-COOP ZRT., H-1103, Budapest, Crezrkesz u. 90
- Age at study initiation: Young adult mice, 12 week old
- Weight at study initiation: 17.5-22.6 g ; the weight variation in animals involved in the study did not exceed ±20% of the mean weight
- Fasting period before study: no data
- Housing: 4 animals/cage, Type II Polypropylene/polycarbonate cages; deep wood sawdust bedding: Lignocel® Hygienic Animal Bedding ( J. Rettenmaier & Söhne GmbH +CO KG ,D-73494 Rosenberg (Germany) Holzmühle 1)
- Diet (e.g. ad libitum): sniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany, ad libitum
- Water (e.g. ad libitum): Tap water from municipal supply as for human consumption, from a bottle, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3°C
- Humidity (%): 30-70%
- Air changes (per hr): Not provided
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: June 20 to July 02, 2012.

Vehicle:

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

Remarks:

(AOO)

Concentration:

50%, 25%, 10% and 5% (w/v)

No. of animals per dose:

4

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The test item was a liquid,hence applicability of the 100 % concentration (the undiluted test item) was evaluated. Further test concentrations were achieved by formulation of the test item in Acetone: Olive oil 4:1 (v/v) mixture (AOO). Both the undiluted test item and the formulations were adequately applicable on the ears of animals. Concentration series of 100 %, 75 %, 50 %, 25 % and 10 % were tested in two preliminary irritation/toxicity tests.
- Irritation: Significant toxic and irritation effects were observed at 100 % and 75 % concentrations. The effects (toxicity an irritation) observed at 50 % were ambiguous. In order to test the highest concentration possible 50 % was selected as the maximum concentration used in the main test together with three additional, lower concentrations (25 %, 10 % and 5 %).
-Lymph node proliferation response: Visual appearance of the lymph nodes.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: Stimulation Index (SI) versus control.
- Other criteria examined: body weigth, clinical observations, including ear thickness, erythema and edema.

The test item is considered as a skin sensitizer, if the following criteria are fulfilled:
-Exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index.
- Data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

Positive control substance(s):

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

Statistics:

The stimulation index values were 22.6, 16.1, 7.1 and 1.8 at test item concentrations of 50 %, 25 %, 10 % and 5 %, respectively. Significance of the dose-response was evaluated by linear regression using SI values. Statistically significant dose-response was observed (p = 0.035; r value = 0.965). EC3 calculation was conducted by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve based on published method. The calculated EC3 value of Depanol I was 6.1 % in this LLNA.

Positive control results:

The positive control group animals were treated with 25 % (w/v) HCA solution (dissolved in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group.
Significant lymphoproliferative response (SI >3) was noted for HCA (SI = 3.6). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed validity of the assay.

Parameter:

other: Migrated information from in vivo LLNA study

Remarks on result:

other: Reading: other: Stimulation index (SI) = 1.8. Group: test group. Dose level: 5%. No with. + reactions: 0.0. Total no. in groups: 4.0.

Parameter:

other: Migrated information from in vivo LLNA study

Remarks on result:

other: Reading: other: Stimulation index (SI) = 7.1. Group: test group. Dose level: 10%. No with. + reactions: 0.0. Total no. in groups: 4.0.

Parameter:

other: Migrated information from in vivo LLNA study

Remarks on result:

other: Reading: other: Stimulation index (SI) = 16.1. Group: test group. Dose level: 25%. No with. + reactions: 0.0. Total no. in groups: 4.0.

Parameter:

other: Migrated information from in vivo LLNA study

Remarks on result:

other: Reading: other: Stimulation index (SI) = 22.6. Group: test group. Dose level: 50%. No with. + reactions: 4.0. Total no. in groups: 4.0. Clinical observations: increased ear thickness.

Parameter:

other: Migrated information from in vivo LLNA study

Remarks on result:

other: Reading: other: Stimulation index (SI) = 3.6. Group: positive control. Dose level: 25% HCA. No with. + reactions: 3.0. Total no. in groups: 4.0. Clinical observations: increased ear thickness.

Parameter:

other: Migrated information from in vivo LLNA study

Remarks on result:

other: Reading: other: Stimulation index (SI) = 1.0. Group: negative control. Dose level: AOO. Total no. in groups: 4.0.

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

The stimulation index (SI = the DPN of a treated (positive control or test item) group divided by the DPN of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result. Based on the results EC3 value = 6.1% (dose calculated to induce a stimulation index of 3) of the test item was calculated. Larger than the control lymph nodes was observed in the positive control group, in the 50 % and the 25 % dose group. Appearance of the lymph nodes was normal in the negative (vehicle) control group and in the 10 % or 5 % test item treated groups. In spite of normal visual appearance of the lymph nodes in the 10 % dose group significant (SI > 3) lymphoproliferative response was noted for Depanol I at concentrations of 50 %, 25 % and 10 %. The observed stimulation index values were 22.6, 16.1, 7.1 and 1.8 at test item concentrations of 50 %, 25 %, 10 % and 5 %, respectively. Significance of the dose-response was evaluated by linear regression using SI values. Statistically significant dose-response was observed (p = 0.035; r value = 0.965).

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

DPM (disintegration per minute) was measured for each treatment group in both Experiments. The measured DPM values were corrected with the background DPM value. The average of the two measured DPM values of 5 % (w/v) trichloracetic acid solutions was used as the background DPM value. The results were expressed as DPN (DPM divided by the number of pooled lymph nodes).

The symptoms (both local and systemic) observed in the 50% dose group were not significant hence it was considered that the proliferation values observed could not have been significantly affected by an adverse effect of the test item. Since no signs of irritation or systemic toxicity were observed in the other test item treated groups (25%, 10% and 5%) the proliferation values obtained, are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay.

No significant, treatment related effect on body weights was observed during the test. No mortality was observed during the test. Decreased activity was observed in the 50% dose group (4/4 animals) on Day 3 (after the third treatment), but the effect was slight and all animals were normal on the subsequent days (Day 4 to Day 6).

Increased ear thickness values compared to the vehicle control were observed in the 50% dose group on Day 3 (3/4 animals, range 9.1 % - 19.0%) and Day 6 (4/4 animals, range 13.6 % - 40.9 %). The increase was significant in case of one ear of one animal on Day 6 (animal No.: 768, 40.9 % increase), but the other measured values were not significant (did not exceed 25 %). No significant erythema (score ≥ 3) was observed during the study in any treatment group.

Interpretation of results:

sensitising

Remarks:

Migrated information moderate Criteria used for interpretation of results: EU

Conclusions:

Under the conditions of the present Local Lymph Node Assay Depanol I, tested at concentrations of 50 %, 25 %, 10 % and 5 % as homogenous formulations (solutions) in a suitable vehicle (AOO) was shown to have sensitization potential (sensitizer). Based on the EC3 value = 6.1% of calculated using dose-response analysis, Depanol I was considered a moderate sensitizer in this LLNA.

Executive summary:

Skin sensitization potential of Depanol I was tested in the Local Lymph Node Assay. The test item was a liquid, hence applicability of the 100 % concentration as well as formulations in Acetone: Olive oil 4:1 (v/v) mixture (AOO) at 75 %, 50 %, 25 % and 10 % were tested in two preliminary irritation/toxicity tests. Significant toxic and irritation effects were observed at 100 % and 75 % concentrations, whereas the effects (toxicity an irritation) observed at 50 % were ambiguous. 50 % was selected as the maximum concentration used in the main test together with three additional, lower concentrations (25 %, 10 % and 5 %).

In the main test 24 female CBA/Ca mice were allocated to 6 groups of four animals each, including 4 different concentrations of 50%, 25%, 10% and 5%, the negative control group (AOO), and positive control group (α-Hexylcinnamaldehyde HCA). Each substance was applied on the external surface of each ear (25 µL/ear) of the animals for three consecutive days (Day1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6 animals were intravenously injected via the tail vein with tritiated methyl thymidene (³HTdR), then sacrificed approximately 5 hours after the injection. Auricural lymph nodes were removed and processed. The cell proliferation in the local lymph nodes was measured by incorporation of ³HTdR and the obtained values were used to calculate stimulation indices (SI).

The positive control item ( 25% HCA in AOO) induced the appropriate stimulation over the control (SI = 3.6), thus confirming the validity of the assay.

No mortality was observed during the study. No significant effect on body weight was observed in any treatment group during the test. Decreased activity was observed in the 50% dose group (4/4 animals) on Day 3 (after the third treatment), but the effect was slight and all animal was normal on the subsequent days (Day 4 to 6). No significant erythema (score ≥3) was observed during the study in any treatment group. As a local effect increased ear thickness values (compared to the vehicle control) were observed in the 50% dose group on Day 3 (3/4 animals) and Day 6 (4/4 animals), but the increase did not exceed the 25% treshold (except in case of one ear of one animal on Day 6; animal No. 768, 40.9% increase) hence the effect was considered not significant.

Larger lymph nodes than the control was observed in the positive control group and in the 50% and 25% dose groups. Visual appearance of the lymph nodes was normal in the other groups (both vehicle and test item treated). The stimulation index values were 22.6, 16.1, 7.1 and 1.8 at test item concentrations of 50%, 25%, 10% and 5% respectively. Significance of the dose-response was evaluated by linear regression using SI values. Statistically significant dose-response was observed (p= 0.035; r valuea= 0.965).

The symptoms (both local and systemic) observed in the 50% dose group were not significant hence it was considered that the proliferation values observed could not been significantly affected by an adverse effect of the test item. Since no signs of irritation or systemic toxicity were observed in the other test item treated groups (25%, 10% and 5%) the proliferation values obtained are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay.

According to evaluation criteria of the relevant guidelines, proliferation values above 3 at 50%, 25% and 10% and the significant dose-response observed, was considered evidence that Depanol I has sensitization potential. EC₃calculation was conducted by linear interpolation using data points lying immediately above and below the SI value of 3 on the LLNA dose-response curve based on published method. The calculated EC₃value of Depanol I was 6.1% in this LLNA. Using this EC₃value according to the published data for classification of contact allergens Depanol I can be ranked among moderate sensitizers ( 1 ≤EC₃< 10) in this LLNA.

In conclusion, under the conditions of the present Local Lymph Node Assay Depanol I, tested at concentrations of 50%, 25%, 10% and 5% as homogenous formulations (solutions) in a suitable vehicle (AOO) was shown to have sensitization potential (senitizer). Based on the EC₃values calculated using dose-response analysis, Depanol I was considered a moderate sensitizer in this LLNA.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Skin sensitization potential of Depanol I was tested in the Local Lymph Node Assay (TOXI-COOP ZRT, 2012). The test item was tested as a liquid at 100 % and formulation in Acetone: Olive oil 4:1 (v/v) mixture (AOO) at 75 %, 50 %, 25 % and 10 % in preliminary irritation/toxicity tests. Significant toxic and irritation effects were observed at 100 % and 75 % concentrations, therefore 50 % was selected as the maximum concentration used in the main test. In the main test 24 female CBA/Ca mice were allocated to 6 groups of four animals each, including 4 different concentrations of 50%, 25%, 10% and 5%, the negative control group (AOO), and positive control group (25% α-Hexylcinnamaldehyde HCA). Each substance was applied on the external surface of each ear (25µL/ear) of the animals for three consecutive days (Day1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6 animals were intravenously injected via the tail vein with tritiated methyl thymidene (3HTdR), then sacrificed approximately 5 hours after the injection and auricular lymph nodes were removed and processed. The cell proliferation in the local lymph nodes was measured by incorporation of 3HTdR and the obtained values were used to calculate stimulation indices (SI). The positive control item induced the appropriate stimulation over the control (SI = 3.6), thus confirming the validity of the assay.

No mortality nor significant effect on body weight was observed in any treatment group during the test. Decreased activity was observed in the 50% dose group (4/4 animals) on Day 3 (after the third treatment), but the effect was slight and all animals were normal on the subsequent days (Day 4 to 6). No significant erythema (score ≥3) was observed during the study in any treatment group. As a local effect, increased ear thickness values (compared to the vehicle control) were observed in the 50% dose group on Day 3 (3/4 animals) and Day 6 (4/4 animals), but the increase did not exceed the 25% threshold (except in case of one ear of one animal on Day 6; animal No. 768, 40.9% increase) hence the effect was considered not significant. Larger lymph nodes than the control was observed in the positive control group and in the 50% and 25% dose groups. Visual appearance of the lymph nodes was normal in the other groups (both vehicle and test item treated). The stimulation index values were 22.6, 16.1, 7.1 and 1.8 at test item concentrations of 50%, 25%, 10% and 5% respectively. Linear regression using SI values showed statistically significant dose-response (p= 0.035; r valuea= 0.965). The symptoms (both local and systemic) observed in the 50% dose group were not significant hence it was considered that the proliferation values observed could not been significantly affected by an adverse effect of the test item. Since no signs of irritation or systemic toxicity were observed in the other test item treated groups (25%, 10% and 5%) the proliferation values obtained, are considered to reflect the real potential of the test item to cause lymphoproliferation in the Local Lymph Node Assay. According to evaluation criteria of the relevant guidelines, Depanol I has sensitization potential. EC3 calculation by linear interpolation was 6.1% in this LLNA, which indicates that Depanol I can be ranked among moderate sensitizers (1 ≤EC3< 10) in this LLNA.

Migrated from Short description of key information:
A key Local Lymph Node Assay with Depanol I, tested at concentrations of 50%, 25%, 10% and 5% as homogeneous formulations (solutions) in a suitable vehicle (AOO) was shown to have sensitization potential. Based on the EC3 value calculated using dose-response analysis, Depanol I was considered a moderate sensitizer in this LLNA.

Justification for selection of skin sensitisation endpoint:
Key study with Depanol I

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Depanol I was classified as a sensitiser according to Annex I of Directive 67/548/EEC: according the EU labelling regulations Commission Directive 93/21/EEC symbol 'Xi' and risk phrase R 43 'MAY CAUSE SENSITISATION BY SKIN CONTACT'; according to CLP regulation (No. 1272/2008 of 16 December 2008), Category 1 classification is proposed with signal word 'WARNING' and hazard statement H317 'MAY CAUSE AN ALLERGIC SKIN REACTION'.