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Description of key information

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:
2013-03-20 to 2013-03-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP and OECD guideline study without restrictions.
Reference:
Composition 0
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
06 July 2012
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymphnode assay (LLNA)
Test material information:
Composition 1
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: 11-12 weeks
- Weight at study initiation: 16.6 -20.9 g
- Housing: Grouped caging (5 animals/cage)
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice ad libitum
- Water: tap water ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 – 70 %
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

IN-LIFE DATES: From: 2013-03-13 To: 2013-03-25
Vehicle:
dimethylformamide
Concentration:
50 % (w/v)
25 % (w/v)
10 % (w/v)
5 % (w/v)
No. of animals per dose:
5 animals per dose
Details on study design:
RANGE FINDING TESTS:
- Compound solubility:
The test item was adequately miscible with all evaluated vehicles (Acetone: Olive oil 4:1 mixture (v/v) (AOO), Dimethylformamide (DMF), Methyl ethyl ketone (MEK), Dimethyl sulfoxide (DMSO)). Since N,N-Dimethylformamide (DMF) is one of the most preferred one by the relevant guidelines this vehicle was selected to be used for the test item formulations in the main test.
- Mortality:
No mortality was observed during the test. No significant, treatment related effect on the body weights was observed in any treatment groups. Signs of systemic toxicity were observed in all treatment groups after the treatment with different intensity. The observed symptoms were decreased activity, piloerection, narrow eyes and restlessness. Recovery of all animals was observed on the next day in the 100 % and 75 % dose groups, while in the 50% dose group animals recovered within few hours after the treatments. Although the body weights did not decrease significantly, the symptoms observed in the 100 % and 75 % dose groups were considered excessive. Although similar symptoms were observed also in the 50 % dose group based on the quick recovery of the animals these symptoms were considered acceptable. According to this 50 % (w/v) was used as maximum concentration in the main test in order to test highest concentration possible. Ensure validity of the test the test item was also tested at three additional, lower concentrations (25 %, 10 % and 5 % (w/v)) according to the relevant guidelines.
- Irritation: No significant sign of local irritation (indicated by an erythema score ≥ 3) was observed in any treatment group.
- Lymph node proliferation response: No significant sign of local irritation (indicated by an increase of ear thickness ≥ 25 %) were observed at the treatment site (ears) at the tested concentrations of the second preliminary test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Individual Approach of Local Lymph Node Assay
- Criteria used to consider a positive response:
Exposure to at least one concentration (non-irritating, non-toxic) of the test item which resulted in an incorporation of 3HTdR at least 3-fold or greater than recorded in control mice, as indicated by the stimulation index (SI ≥ 3). However, the strength of the dose-response, the statistical significance and the consistency of the solvent/vehicle and PC responses may also be used when determining whether a borderline result is declared positive.

TREATMENT PREPARATION AND ADMINISTRATION:
- Main test design
AOO was used as a vehicle control for the positive control substance. The negative control group for the test item was treated with DMF. The test item was administered at four different concentrations according to the results of the dose range finding test. The positive control substance (HCA) was tested at one concentration.
- In vivo Treatment
Each mouse was topically treated with 25 μL of the appropriate formulations of the test item, of the positive control substance (positive control group) or of the vehicles (AOO or DMF as negative control groups). The formulations were applied, with a pipette, on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Statistical analysis was performed by SPSS/PC+ (4.0.1) software package.
The measured DPM values corrected with the mean background value were used for statistical analysis of the proliferation data. The heterogeneity of variance between the groups treated with the test item and the relevant vehicle control (DMF) was checked by Bartlett's test. Since significant heterogeneity was detected, the normal distribution of data was examined by Kolmogorow-Smirnow test followed by the non-parametric method of Kruskal-Wallis One-Way analysis of variance. As a result of this analysis the inter-group comparisons was performed using Mann-Whitney U-test to asses the significance of inter-group differences. Significance of the positive control response was evaluated by T-test versus the relevant vehicle control (AOO).
Parameter:
SI
Remarks on result:
other: 50% (w/v): 31.43 25% (w/v): 21.06 10% (w/v): 8.24 5% (w/v): 2.24 The calculated EC3 value was 5.6 %.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
Mean DPM and SD 50% (w/v): 18157.7 +/- 6877.2 25% (w/v): 12167.7 +/- 6571.6 10% (w/v): 4762.1 +/- 1578.6 5% (w/v): 1293.1 +/- 728.1 Vehicle control (DMF): 577.7 +/- 140.6 Vehicle control (AOO): 684.9 +/- 356.8 Positive control (25% w/v HCA): 7775.3 +/- 2795.4

Body Weight Measurement

No significant, treatment related effect on the body weights was observed in any treatment group.

Clinical Observations and Signs of Irritation

No mortality or symptoms of systemic toxicity were observed in any treatment group. No sign of significant irritation (indicated by an erythema score ≥ 3) or any other local effect was observed in any treatment groups.

Proliferation Assay

The lymph nodes in the positive control group were larger than the relevant vehicle control (AOO). Larger lymph nodes than the relevant vehicle control (DMF) was observed in the 50 %, 25 % and 10 % (w/v) test item treated groups. Visual appearance of the lymph nodes was normal in the negative (vehicle) control groups (both AOO and DMF) and in the 5 % (w/v) test item treated group.

Significantly increased lymphoproliferation (SI ≥ 3) was observed for the test item at concentrations of 50 %, 25 % and 10 % (w/v). No significantly increased lymphoproliferation was observed at concentration of 5 % (w/v). The corresponding stimulation index values were 31.43, 21.06, 8.24 and 2.24 at treatment concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively. Significance of the dose-response was evaluated by linear regression using the SI values. Statistically significant dose-related response was observed (p = 0.02, r = 0.98).

Statistical Analysis

Statistically significant increase of the proliferation value was observed in the positive control group (p < 0.01, T-test versus AOO control). Statistically significant increase of the proliferation values compared to the relevant vehicle control (DMF) was observed in the 50 %, 25 % and 10 % (w/v) test item treated groups (p < 0.01; Mann-Whitney U-test versus DMF control). No statistically significant increase of the proliferation values compared to the relevant vehicle control (DMF) was observed in the 5 % (w/v) test item treated group (Mann-Whitney U-test versus DMF control).

Reliability of the Assay

The positive control group animals were treated with 25 % (w/v) HCA solution (formulated in AOO) concurrent to the test item groups. No mortality, cutaneous reactions or signs of toxicity were observed in the positive control group.

The positive control substance induced the appropriate, statistically significant stimulation compared to the control (SI = 11.35; p < 0.01, T-test versus AOO control). The results of the positive control item demonstrated appropriate performance of the test in accordance with the relevant guidelines and confirmed sensitivity and validity of the assay.

 

Interpretation of Observations

Selection of test item concentrations based on the results of a formulation evaluation and also results of a preliminary irritation/toxicity test according to the relevant guidelines. The test item was a liquid hence miscible with an appropriate vehicle was evaluated. The test item was adequately miscible with all evaluated vehicles. Since N,N-Dimethylformamide (DMF) is one of the most preferred one by the relevant guidelines this vehicle was selected for the test item formulations in the main test.

Due to significant systemic toxicity observed in the preliminary irritation/toxicity test sensitization potential of the test item Aldehyde M was examined as 50 % (w/v, as the maximum applicable concentration) and as 25%, 10 % and 5 % (w/v) formulations in the selected vehicle (DMF) in the LLNA.

Since the test was valid and no sign of systemic toxicity or excessive irritation was observed, the proliferation values obtained are considered to reflect the real potential of the test item to cause/not cause lymphoproliferation in the Local Lymph Node Assay.

Visually larger lymph nodes than the relevant control (AOO or DMF) was observed in the positive control group and in the 50 %, 25 % and 10 % (w/v) test item treated groups.

Significantly increased lymphoprolipheration (≥ 3) was observed for the test item at concentrations of 50 %, 25 % and 10 % (w/v). The observed SI values were 31.43, 21.06, 8.24 and 2.24 at treatment concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively. The observed proliferation values at concentrations of 50 %, 25 % and 10 % (w/v) statistically significantly differed from the relevant vehicle (DMF) control (p < 0.01; Mann-Whitney U-test). Significant dose-related response was observed (linear regression, p = 0.02, r = 0.98).

According to evaluation criteria of the relevant guidelines, the significantly increased proliferation values observed at concentrations of 50 %, 25 % and 10 % (w/v) and the strong dose-response relationship are considered evidence that Aldehyde M is a skin sensitizer.

The EC3 (dose calculated to induce a stimulation index of 3) was calculated 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 Aldehyde M was 5.6 % in this LLNA. Using the calculated EC3 values Aldehyde M can be ranked among moderate skin sensitizers in this LLNA according to the published data for classification of contact allergens.

 

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the conditions of the present Local Lymph Node Assay, Aldehyde M tested at concentrations of 50 %, 25 %, 10% and 5% (w/v) as formulations in a suitable vehicle (DMF) was shown to have skin sensitization potential. Based on the EC3 value Aldehyde M was considered a moderate skin sensitizer in this LLNA.
Executive summary:

The aim of this study was to determine the skin sensitization potential of Aldehyde M in the Local Lymph Node Assay (LLNA). Individual approach was used in this test.

The maximum dose selection was performed according to the relevant guidelines and based on results of the formulation evaluation and a preliminary irritation / toxicity test. Due to significant systemic toxicity observed in the preliminary test at concentrations of 100% (the undiluted test item) and 75 % (w/v), the test item was examined in the main test at concentrations of 50 %, 25 %, 10 % and 5 % (w/v) as formulations in N,N-Dimethylformamide (DMF).

In the main test, 35 female CBA/Ca mice were allocated to seven groups of five animals each:

- four groups received Aldehyde M at 50 %, 25 %, 10 % or 5 % (w/v) in DMF,

- one control group (used as negative control for the test item treated groups) received the vehicle of the test item (DMF) only,

- one control group (used as negative control for the positive control substance treated group) received the vehicle of the positive control substance (AOO) only,

- the positive control group received 25 % (w/v) α-Hexylcinnamaldehyde (HCA) in AOO.

Each substance was applied on the external surface of each ear (25 μL/ear) of the animals for three consecutive days (Day 1, 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 thymidine (3HTdR), than sacrificed approximately 5 hours after the injection. 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 substance (25 % (w/v) HCA in AOO) induced the appropriate, statistically significant stimulation compared to the relevant control (SI = 11.35; p < 0.01, T-test versus the relevant vehicle (AOO) control), thus confirming the validity of the assay.

No mortality, significant, treatment related effect on the body weights or any other sign of systemic toxicity was observed in any treatment group. No sign of significant irritation (indicated by an erythema score ≥ 3) or any other local effect was observed in any treatment group.

Visually larger lymph nodes than the relevant vehicle control (AOO or DMF) was observed in the positive control, the 50 %, 25 % and the 10 % (w/v) test item treated groups.

Significantly increased lymphoproliferation (≥ 3) was observed for the test item at concentrations of 50 %, 25 % and 10 % (w/v). No significantly increased lymphoproliferation was observed at concentration of 5 % (w/v). The observed SI values were 31.43, 21.06, 8.24 and 2.24 at concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively. The observed proliferation values at concentrations of 50 %, 25 % and 10 % (w/v) statistically significantly differed from the relevant vehicle (DMF) control (p < 0.01; Mann-Whitney U-test). Significant dose-related response was observed (linear regression, p = 0.02, r = 0.98).

Since the test was valid and no sign of systemic toxicity or significant irritation was observed, the proliferation values obtained are considered to reflect the real potential of the test item to cause/not cause lymphoproliferation in the Local Lymph Node Assay.

According to evaluation criteria of the relevant guidelines, the significantly increased proliferation values observed at concentrations of 50 %, 25 % and 10 % (w/v) and the strong dose-response relationship are considered evidence that Aldehyde M is a skin sensitizer.

The EC3 (dose calculated to induce a stimulation index of 3) was calculated 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 Aldehyde M was 5.6 % in this LLNA. Using the calculated EC3 values Aldehyde M can be ranked among moderate skin sensitizers in this LLNA according to the published data for classification of contact allergens.

 

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

LLNA

The aim of this study was to determine the skin sensitization potential of Aldehyde M in the Local Lymph Node Assay (LLNA). Individual approach was used in this test.

The maximum dose selection was performed according to the relevant guidelines and based on results of the formulation evaluation and a preliminary irritation / toxicity test. Due to significant systemic toxicity observed in the preliminary test at concentrations of 100% (the undiluted test item) and 75 % (w/v), the test item was examined in the main test at concentrations of 50 %, 25 %, 10 % and 5 % (w/v) as formulations in N,N-Dimethylformamide (DMF).

In the main test, 35 female CBA/Ca mice were allocated to seven groups of five animals each:

- four groups received Aldehyde M at 50 %, 25 %, 10 % or 5 % (w/v) in DMF,

- one control group (used as negative control for the test item treated groups) received the vehicle of the test item (DMF) only,

- one control group (used as negative control for the positive control substance treated group) received the vehicle of the positive control substance (AOO) only,

- the positive control group received 25 % (w/v) α-Hexylcinnamaldehyde (HCA) in AOO.

Each substance was applied on the external surface of each ear (25 μL/ear) of the animals for three consecutive days (Day 1, 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 thymidine (3HTdR), than sacrificed approximately 5 hours after the injection. 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 substance (25 % (w/v) HCA in AOO) induced the appropriate, statistically significant stimulation compared to the relevant control (SI = 11.35; p < 0.01, T-test versus the relevant vehicle (AOO) control), thus confirming the validity of the assay.

No mortality, significant, treatment related effect on the body weights or any other sign of systemic toxicity was observed in any treatment group. No sign of significant irritation (indicated by an erythema score ≥ 3) or any other local effect was observed in any treatment group.

Visually larger lymph nodes than the relevant vehicle control (AOO or DMF) was observed in the positive control, the 50 %, 25 % and the 10 % (w/v) test item treated groups.

Significantly increased lymphoproliferation (≥ 3) was observed for the test item at concentrations of 50 %, 25 % and 10 % (w/v). No significantly increased lymphoproliferation was observed at concentration of 5 % (w/v). The observed SI values were 31.43, 21.06, 8.24 and 2.24 at concentrations of 50 %, 25 %, 10 % and 5 % (w/v), respectively. The observed proliferation values at concentrations of 50 %, 25 % and 10 % (w/v) statistically significantly differed from the relevant vehicle (DMF) control (p < 0.01; Mann-Whitney U-test). Significant dose-related response was observed (linear regression, p = 0.02, r = 0.98).

Since the test was valid and no sign of systemic toxicity or significant irritation was observed, the proliferation values obtained are considered to reflect the real potential of the test item to cause/not cause lymphoproliferation in the Local Lymph Node Assay.

According to evaluation criteria of the relevant guidelines, the significantly increased proliferation values observed at concentrations of 50 %, 25 % and 10 % (w/v) and the strong dose-response relationship are considered evidence that Aldehyde M is a skin sensitizer.

The EC3 (dose calculated to induce a stimulation index of 3) was calculated 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 Aldehyde M was 5.6 % in this LLNA. Using the calculated EC3 values Aldehyde M can be ranked among moderate skin sensitizers in this LLNA according to the published data for classification of contact allergens.

 


Migrated from Short description of key information:
Under the conditions of the present Local Lymph Node Assay, Aldehyde M tested at concentrations of 50 %, 25 %, 10 % and 5 % (w/v) as formulations in a suitable vehicle (DMF) was shown to have skin sensitization potential.

Justification for selection of skin sensitisation endpoint:
Only one GLP and guideline study available.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available experimental data the test item is classified as skin sensitizer H317, cat. 1B according to Regulation (EC) No 1272/2008 (CLP) and as Xi, R43 according to Directive 67/548/EEC.