Reaction product of 9-[2-(ethoxycarbonyl)phenyl]-3,6-bis(ethylamino)-2,7-dimethylxanthylium chloride and Naphthalenesulfonic acids, reaction products with formaldehyde, ammonium salts

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:

2013-10-16 to 2013-10-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was conducted in accordance to GLP regulations and OECD/EU guideline.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

06 July 2012

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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: TOXI-COOP ZRT. H-1103, Budapest, Cserkesz u. 90., Hungary
- Age at study initiation: Young adult mice, 11 weeks
- Weight at study initiation: 18.1 -21.7 g
- Housing: Grouped caging (5 animals/cage)
- Diet (ad libitum): ssniff® SM R/M-Z+H complete diet
- Water (ad libitum): tap water from municipal supply
- Acclimation period: 14 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: 2013-10-16 To: 2013-10-22

Vehicle:

dimethylformamide

Concentration:

10 %, 5 %, 2.5 % and 1 % (w/v)

No. of animals per dose:

5 animals per treatment group

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Preliminary test item formulation evaluation was performed. Solubility of the test item was evaluated in the following vehicles:
1. N,N-Dimethylformamide (DMF)
2. Dimethyl sulfoxide (DMSO)
The test item was equally soluble in both vehicles with a maximum concentration of 10 % (w/v, based on concentration series recommended by the relevant guidelines). Since DMF is more preferred than DMSO by the relevant guidelines this vehicle was selected to be used for the test item formulations in the main test.
- Irritation and ear thickness response: No mortality or any signs of systemic toxicity were observed during the preliminary test. No significant, treatment related effect on body weights was observed. No signs of significant irritation (indicated by an increased ear thickness of ≥ 25 % on any day of measurement) were observed in the treatment groups.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymphnode assay (individual approach)
- Evaluation criteria: DPM was measured for each animal. The results were expressed as DPN (DPM divided by the number of lymph nodes). The mean DPM and DPN values and associated error terms were calculated for each treatment group. The stimulation index (SI = the mean DPN of a treated (positive control or test item) group divided by the DPN of the respective negative control group) was calculated for each treatment group. A stimulation index of 3 or greater is an indication of a positive result. All calculations were made by Microsoft Excel Software. Based on the results the EC3 value (dose calculated to induce a stimulation index of 3) of the test item was calculated.
- Criteria used to consider a positive response: That exposure to at least one concentration (non-irritating, non-toxic) 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 (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:
- 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.

PROLIFERATION ASSAY
- Injection of 3H -methyl-thymidine
On Day 6 each mouse was intravenously injected via the tail vein with 250 μL of sterile PBS (1 x PBS, diluted from 10x concentrate with purified water) containing 20 μCi of 3H-methyl-thymidine using a hypodermic needle with 1 mL sterile syringe. Once injected, the mice were left for 5 hours (± 30 minutes).
- Removal and Preparation of Draining Auricular Lymph Nodes
Five hours (± 30 minutes) after intravenous injection the mice were sacrificed by cervical dislocation. The draining auricular lymph nodes were excised by making a small incision in the skin between the jaw and the sternum, pulling the skin gently back towards the ears and exposing the lymph nodes. Then the lymph nodes were removed using forceps. Once removed, the lymph nodes of individual mice were collected separately in a Petri dish containing a small amount (1-2 mL) of PBS to keep the nodes wet before processing.
- Preparation of Single Cell Suspension of Lymph Node Cells
A single cell suspension (SCS) of lymph node cells (LNCs) of each individual animal was prepared and collected in disposable tubes by a gentle mechanical disaggregation of the lymph nodes through a cell strainer using the plunger of a disposable syringe. The cell strainer was washed with PBS (up to 10 mL). LNCs were pelleted with a relative centrifugal force (RCF) of approximately 190 x g for 10 minutes at 4°C. After centrifugation, the majority of the supernatant was aspirated off, leaving 1-2 mL supernatant above each pellet. The pellets were gently agitated and the LNCs were re-suspended in 10 mL of PBS. This washing procedure was repeated twice. This procedure was repeated for lymph nodes of each individual animal.
- Determination of Incorporated 3HTdR
After the final wash, each supernatant was removed leaving a small volume (< 0.5 mL) of supernatant above each pellet. The pellets were gently agitated before suspending the LNCs in 3 mL of 5 % (w/v) trichloroacetic acid (TCA, dissolved in purified water) for precipitation of the macromolecules. After incubation with 5 % (w/v) TCA at 2-8°C overnight (approx. 18 hrs), each precipitate was removed by centrifugation of the samples at approximately 190 x g for 10 minutes at 4°C and decanting the supernatants and the pellets were re-suspended in 1 mL of 5 % (w/v) TCA and dispersed using an ultrasonic water bath. Samples were transferred to suitable sized scintillation vials containing 10 mL of scintillation liquid, gently mixed and stored at room temperature protected from light until measurement.
For the measurement of incorporated radioactivity the vials were loaded into a β-scintillation counter and 3HTdR incorporation was measured for up to 10 minutes per sample. The β-counter expresses the 3HTdR incorporation as the amount of radioactive disintegration per minute (DPM). Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % (w/v) TCA.

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 results 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). Significance of the dose-response was evaluated by linear regression made with Microsoft Excel Software.

Positive control results:

The lymph nodes in the positive control group were larger than the relevant control (AOO). The positive control substance induced the appropriate, statistically significant stimulation compared to the control (SI = 9.1; 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.

Parameter:

SI

Remarks on result:

other: Test item: 10 % (w/v): 5.4 5 % (w/v): 6.0 2.5 % (w/v): 5.6 1 % (w/v): 2.7 Positive control: 25 % (w/v) HCA: 9.1

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

Mean DPM and SD Vehicle control for positive control: 3109.5 +/- 2011.3 Positive control (25 % (w/v) HCA): 28390.5 +/- 4073.9 Test item (10 % (w/v)): 4340.7 +/- 2053.6 Test item (5 % (w/v)): 4781.5 +/- 3640.0 Test item (2.5 % (w/v)): 4513.8 +/- 1618.7 Test item (1 % (w/v)): 2142.1 +/- 635.6 Vehicle control for the test item: 800.7 +/- 344.7

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 or any other local effect was observed in any treatment group. Although erythema could not be reliably observed due to intensive red colour of the test item an erythema score < 3 was considered in the test item treated groups. No erythema was observed in the control groups.

 

Proliferation Assay

The lymph nodes in the positive control group were larger than the relevant control (AOO). Visual appearance of the lymph nodes was normal in the negative (vehicle) control groups (both AOO and DMF) and in the test item treated groups (compared to the relevant control of DMF). In spite of normal visual appearance of the lymph nodes significant lymphoproliferation (indicated by an SI ≥ 3) was observed for the test item at concentrations of 10 %, 5 % and 2.5 % (w/v). The corresponding stimulation index values were 5.4, 6.0, 5.6 and 2.7 at treatment concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively. Unexpectedly low DPM values were measured in the 2.5 % (w/v) dose group in case of two samples. Therefore this concentration was not considered in the statistical analysis.

 

Statistical Analysis

Individual DPM values (corrected with the mean background value) were statistically evaluated in the positive control and in the 10 %, 5 % and 1 % (w/v) dose groups.

Statistically significant (p < 0.01) increase of the proliferation value was observed in the positive control group by T-test versus AOO control. Statistically significant (p < 0.01) increase of the proliferation values were observed in the evaluated test item treated groups: Mann-Whitney U-test versus the relevant vehicle control (DMF) was performed. Significance of the dose-response was evaluated by linear regression using the SI values of the statistically evaluated groups. Although dose-related response was observed its linearity was not significant (p = 0.48, r = 0.73).

 

Interpretation of Observations

Selection of test item concentrations based on the results of a formulation evaluation and also resulted from a preliminary irritation/toxicity test according to the relevant guidelines. Based on the preliminary test results the test item was examined in the LLNA at 10 % (as the maximum attainable concentration, based on solubility) and at 5 %, 2.5 % and 1 % (w/v) as formulations in N,N-Dimethylformamide DMF.

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.

Unexpectedly low DPM values were measured in the 2.5 % (w/v) dose group in case of two samples. It was considered that these outlier values were observed due to possible error in sample processing as the other values in this dose group were in accordance with the observed trend. Even so, although an informatory SI value was calculated, this dose group was excluded from the further statistical analysis since evaluation of less than 4 values per dose groups did not meet with assay acceptance criteria.

Based on the calculated SI values significant lymphoproliferation (SI ≥ 3) was observed for the test item at concentrations of 10 %, 5 % and 2.5 % (w/v). The corresponding stimulation index values were 5.4, 6.0, 5.6 and 2.7 at treatment concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively.

The measured DPM values corrected with the mean background value were statistically evaluated in the 10 %, 5 % and 1 % (w/v) dose groups. Statistically significant differences compared to the relevant vehicle control (DMF) were observed at concentrations of 10 % and 5 % (w/v) (p < 0.01). Although the observed SI value of 2.7 did not exceed the threshold value of 3, the increase compared to the relevant control was statistically significant (p < 0.01) in the 1 % (w/v) dose group also. The response was considered dose-related however its linearity was not statistically significant.

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 DOCU Red 116 was 1.36 % in this LLNA.

 

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, DOCU Red 116 tested at up to the maximum attainable concentration of 10 % (w/v) was shown to have skin sensitization potential. Based on the EC3 value of 1.36 % DOCU Red 116 was considered a moderate skin sensitizer in this LLNA.

Executive summary:

The aim of the study was to determine the skin sensitization potential of DOCU Red 116 in the Local Lymph Node Assay (LLNA). Individual approach was used in this test.

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 maximum concentration in a suitable vehicle was 10 % (w/v). Based on the preliminary test results the test item was examined in the LLNA at 10 % (as the maximum attainable concentration) and at 5 %, 2.5 % and 1 % (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 DOCU Red 116 at 10 %, 5 %, 2.5 % and 1 % (w/v) respectively 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 induced the appropriate, statistically significant stimulation compared to the relevant control (SI = 9.1; p < 0.01, T-test versus 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. Although erythema could not be reliably observed due to intensive red colour of the test item an erythema score < 3 was considered in the test item treated groups. No erythema was observed in the control groups. No any other local effect was observed in any treatment group.

Visually larger lymph nodes than the relevant vehicle control (AOO) was observed in the positive control group. Visual appearance of the lymph nodes was normal in the vehicle (both AOO and DMF) control groups and in the test item treated groups.

In spite of normal visual appearance of the lymph nodes SI values above 3 were observed for the test item at concentrations of 10 %, 5% and 2.5 % (w/v). The observed SI values were 5.4, 6.0, 5.6 and 2.7 at treatment concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively.

The measured DPM values corrected with the mean background value were statistically evaluated in the 10 %, 5 % and 1 % (w/v) dose groups. Due to outlier values observed, the 5 % (w/v) dose group was excluded from the statistical evaluation although an informatory SI value was calculated. Statistically significant differences compared to the relevant vehicle control (DMF) were observed at all evaluated concentrations (p < 0.01, Mann Whitney U-test). Dose-related response was observed, although its linearity was not statistically significant (linear regression, p = 0.48, r = 0.73).

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 and the dose-related response are considered evidence that DOCU Red 116 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 DOCU Red 116 was 1.36 % in this LLNA. Using the calculated EC3 values DOCU Red 116 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:

The aim of the study was to determine the skin sensitization potential of DOCU Red 116 in the Local Lymph Node Assay (LLNA). Individual approach was used in this test.

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 maximum concentration in a suitable vehicle was 10 % (w/v). Based on the preliminary test results the test item was examined in the LLNA at 10 % (as the maximum attainable concentration) and at 5 %, 2.5 % and 1 % (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 DOCU Red 116 at 10 %, 5 %, 2.5 % and 1 % (w/v) respectively 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 induced the appropriate, statistically significant stimulation compared to the relevant control (SI = 9.1; p < 0.01, T-test versus 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. Although erythema could not be reliably observed due to intensive red colour of the test item an erythema score < 3 was considered in the test item treated groups. No erythema was observed in the control groups. No any other local effect was observed in any treatment group.

Visually larger lymph nodes than the relevant vehicle control (AOO) was observed in the positive control group. Visual appearance of the lymph nodes was normal in the vehicle (both AOO and DMF) control groups and in the test item treated groups.

In spite of normal visual appearance of the lymph nodes SI values above 3 were observed for the test item at concentrations of 10 %, 5% and 2.5 % (w/v). The observed SI values were 5.4, 6.0, 5.6 and 2.7 at treatment concentrations of 10 %, 5 %, 2.5 % and 1 % (w/v), respectively.

The measured DPM values corrected with the mean background value were statistically evaluated in the 10 %, 5 % and 1 % (w/v) dose groups. Due to outlier values observed, the 5 % (w/v) dose group was excluded from the statistical evaluation although an informatory SI value was calculated. Statistically significant differences compared to the relevant vehicle control (DMF) were observed at all evaluated concentrations (p < 0.01, Mann Whitney U-test). Dose-related response was observed, although its linearity was not statistically significant (linear regression, p = 0.48, r = 0.73).

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 and the dose-related response are considered evidence that DOCU Red 116 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 DOCU Red 116 was 1.36 % in this LLNA. Using the calculated EC3 values DOCU Red 116 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:
In a Local Lymph Node assay according to OECD TG 429 DOCU Red 116 in DMF was shown to have skin sensitising properties. The EC3 value was determined to be 1.36 %.

Justification for selection of skin sensitisation endpoint:
Only one study available

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

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