[2-[[2-cyano-3-[4-(diethylamino)phenyl]-1-oxoallyl]oxy]ethyl][3-[[2-cyano-3-[4-(diethylamino)phenyl]-1-oxoallyl]oxy]propyl]dimethylammonium chloride

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

other: CBA/CaCrl

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River UK, Manston Road, Margate, Kent CT9 4LT, United Kingdom
- Age at study initiation: 8 – 9 weeks old
- Weight at study initiation: 17.1 ± 1.0 g (mean ± SD)
- Housing: group housing (5 animals per group) in Makrolon Type II / III cages, with wire mesh top (EHRET GmbH, 79302 Emmendingen, Germany)
- Diet: Pelleted standard diet, ad libitum (Harlan Laboratories B.V., 5960 AD Horst, Netherlands)
- Water: Tap water, ad libitum (Gemeindewerke, 64380 Rossdorf, Germany)
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Relative Humidity (%): 42 - 65
- Photoperiod (hrs dark / hrs light): 12 / 12 (6.00 p.m. - 6.00 a.m. / 6.00 a.m. - 6.00 p.m.)

Vehicle:

dimethylformamide

Concentration:

5% (w/w), 10% (w/w), 25% (w/w)

No. of animals per dose:

5 mice

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which can be technically used, was a 25% (w/w) dilution in dimethylformamide as vehicle. Vortexing and warming to about 37°C were necessary to formulate the test item. At higher concentrations, an applicable formulation of the test item was not achieved, neither by the use of other vehicles nor by using additional methods to formulate the test item (e.g. sonicating).
- Irritation: To determine the highest non-irritant test concentration that does not induce signs of systemic toxicity at the same time, a pre-test was performed in two animals. Two mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 10% and 25% (w/w) once daily each on three consecutive days. At the tested concentrations the animals did not show any signs of local skin irritation or systemic toxicity. Due to the intense colour of the test item local irritation reactions such as ear reddening could not be detected. Thus, the test item in the main study was assayed at 5%, 10%, and 25% (w/w).

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled: First, that exposure to at least one concentration of the test item resulted in an incorporation of ³HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index. Second, that the 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.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was placed into an appropriate container on a tared balance and dimethylformamide was added to achieve the required test item concentration. Vortexing and warming to about 37°C were necessary to formulate the test item. The different test item concentrations were prepared by serial dilution. The preparations were made freshly before each dosing occasion. Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% (w/w) in dimethylformamide. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (Ø ~ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) were treated with an equivalent volume of the relevant vehicle alone. On day 6, animals were intravenously injected wiht ³HTdR and sacrificed. Lymph nodes were removed and processed (individual approach).

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated for the body weights and for the DPM values (group mean DPM ± standard deviation) as well as for the ear weights, lymph node weights and lymph node cell counts.
The EC3 value was calculated according to the equation EC3 = (a-c) [(3-d)/(b-d)] + c, where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.
A statistical analysis was conducted on the DPM values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between test item groups and negative control group.
For all statistical calculations SigmaStat for Windows (Version 2.0) was used. A One-Way-Analysis-of-Variance was used as statistical method. In case of significant results of the One-Way-ANOVA, multiple comparisons were performed with the Dunnett test. Statistical significance was set at the five per cent level (p < 0.05). The Dean-Dixon-Test and Grubb’s test were used for identification of possible outliers (performed with Microsoft Excel 2003).
However, both biological and statistical significance were considered together.

Positive control results:

The periodic positive control experiment was performed with α-hexyl cinnamaldehyde dissolved in acetone:olive oil (4:1 v/v) (compound listed in OECD 429 Guideline) using CBA/CaCrl mice in May 2011. An EC3 value of 10.4% (w/v) was calculated.

Parameter:

SI

Remarks on result:

other: Stimulation Indices (S.I.) of 1.85, 1.92, and 1.72 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in dimethylformamide, respectively. The EC3 value could not be calculated, since all S.I.'s were below 3.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: The DPM values did not show a statistically significant or biologically relevant increase in any test item treated group in comparison to the vehicle control group. There were no conventional dose-response.

Calculation of Stimulation Indices per Dose Group

Test item concentration % (w/w)	Group Calculation
	Mean DPM    per animal^a)	SD^b)	S.I.
Vehicle (dimethylformamide)	748.8	179.7	1.00
5 % dye	1386.4	155.0	1.85
10 % dye	1440.6	464.3	1.92
25 % dye	1289.6	670.1	1.72

a)   Mean DPM/animal was determined by dividing the sum of the measured values from lymph
     nodes of all animals within a group by the number of animals in that group (5 animals)

b)   SD = Standard Deviation

 

 

Viability / Mortality: No deaths occurred during the study period.

Clinical Signs: No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period. Local irritation reactions such as ear reddening of the ear skin could not be detected due to the intense colouration caused by the test item.

Body Weights: The body weight of the animals, recorded prior to the first application and prior to treatment with ³HTdR, was within the range commonly recorded for animals of this strain and age.

Lymph Node Weights and Cell Counts: The measured lymph node weights and cell counts of all animals treated were recorded after sacrifice. No statistically significant and biologically relevant increase in lymph node weight was observed in all test groups in comparison to the vehicle control group (p<0.05). A statistically significant but not biologically relevant increase in lymph node cell count was observed in all test item treated groups in comparison to the vehicle control group. However, the cutoff-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was not exceeded at any dose group.

Ear Weights: The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant decrease in ear weights was determined at the high concentration of 25% (w/w) of test item. For BALB/c mice, a threshold for the ear weight index of 1.1 was reported for a positive response. However, the cutoff-value for a positive response regarding the ear weight index was not exceeded at any dose group and the mentioned cutoff-value has been determined using a different strain of mice and can thus not be implicitly adopted.

Interpretation of results:

not sensitising

Remarks:

Migrated information

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Skin sensitisation

In a dermal sensitisation study (BASF SE/Harlan CCR, 2011, according to OECD 429) 5 mice were dosed with the test item in concentrations of 5, 10 and 25 % (w/w, dissolved in dimethylformamide). The highest concentration tested was the highest concentration which could be technically achieved. Each concentration was applied on the external surface of each ear (25 µL/ear) of the animals for three consecutive days (days 1, 2 and 3). A further group of mice (control animals) was treated with an equivalent volume of the vehicle alone. 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), then sacrificed approximately 5 hours after the injection. Auricural lymph nodes were removed and processed (individual approach). 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). Ear punch weight determination method was also used for measurement of ear thickness. The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed. A statistically significant increase in ear weight was not observed in any group in comparison to the vehicle control group. In this study Stimulation Indices (S.I.) of 1.85, 1.92, and 1.72 were determined with the test item at concentrations of 5, 10 and 25% (w/w), respectively. No statistically significant or biologically relevant increase in DPM value and lymph node weights was observed in any dose group in comparison to the vehicle control group. A statistically significant but not biologically relevant increase in lymph node cell count was observed in all treated groups with the test item in comparison to the vehicle control group. The cutoff-value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was not exceeded in any dose group. Thus, the increase in the dose groups is not biologically relevant, especially as there was no dose response and the increased cell counts did not correlate with the marginal heightened S.I.s of incorporated ³HTdR. In conclusion, the test item was not a skin sensitiser under the test conditions of this study.

Migrated from Short description of key information:
The test item was not sensitising in the local lymph node assay.

Justification for selection of skin sensitisation endpoint:
only available reliable study

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the results obtained in the key skin sensitisation test, the test item is not subject to classification for sensitization according to Directive 67/548/EEC and Regulation (EC) No 1272/2008.