3-(benzothiazol-2-yl)-7-(diethylamino)-2-oxo-2H-1-benzopyran-4-carbonitrile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

Guideline adopted 22 July 2010; Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorised in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). Information on validation of IMDS and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).

Deviations:

yes

Remarks:

Measurement of cell counts instead of radioactive labelling. In addition, ear swelling and ear weights are determined to discriminate the irritating potential from the sensitizing potential of the test substance.

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July 2012

Principles of method if other than guideline:

The study was performed according to OECD 429. However, an alternative method was used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes. In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.
Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones.
Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round; Toxicology 212, 60-68 (2005a);
- Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005b).

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3-(benzothiazol-2-yl)-7-(diethylamino)-2-oxo-2H-1-benzopyran-4-carbonitrile

EC Number:

274-668-9

EC Name:

3-(benzothiazol-2-yl)-7-(diethylamino)-2-oxo-2H-1-benzopyran-4-carbonitrile

Cas Number:

70546-25-7

Molecular formula:

C21H17N3O2S

IUPAC Name:

3-(1,3-benzothiazol-2-yl)-7-(diethylamino)-2-oxo-2H-chromene-4-carbonitrile

impurity 1

Reference substance name:

unknown impurities

IUPAC Name:

unknown impurities

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: 27 - 34 g
- Housing: The animals were kept singly in MAKROLON cages (type II). Animals were not group-housed to prevent contact of the application sites.
- Diet (e.g. ad libitum): yes
- Water (e.g. ad libitum): yes
- Acclimation period: at least 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +/- 3°C
- Humidity (%): 55% +/- 10%
- Photoperiod (hrs dark / hrs light): 12hrs dark / 12 hrs light
- IN-LIFE DATES: From: 30 May 2018 To: 22 June 2018

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

Three concentrations of Macrolex Fluoreszenzrot G (5%, 10% and 25% (w/w)), suspended in acetone/olive oil (4:1, v/v) were tested in six female NMRI mice per group and compared to a vehicle control group. A 25% (w/w) concentration was the highest feasible concentration of the test item in acetone/olive oil (4:1, v/v), see Table 1.

No. of animals per dose:

6

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: suspension in acetone/olive oil (A/O, 4:1, v/v), see Table 1
- Irritation: no effects
- Systemic toxicity: no effects
- Ear thickness measurements: no effects
- Erythema scores: 0
A preliminary experiment was carried out in 3 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations out of the technically feasible concentrations (see Table below). Three concentrations of 5%, 10% and 25% (w/w) suspended in acetone / olive oil (4:1, v/v) were examined. Doses were selected based on the OECD guideline from the concentration series 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.5% (w/w) etc.
The preliminary experiment was conducted under conditions identical to the main LLNA study, except there was no assessment of lymph node proliferation and only 1 animal per concentration was used. Both ears of each mouse were observed for erythema. The test item suspension was administered to the dorsum of both animal's ears at an application volume of 25 µL/ear.
No irritating properties were observed in this preliminary experiment at concentrations of 5%, 10% and 25% (w/w), and no differences in ear weight and ear thickness were noted.


MAIN STUDY
The study was performed according to OECD 429, however not employing the use of radioactive labelling to measure cell proliferation, as the radioactive method proposed by the OECD guideline led to problems in various EU laboratories: such as (i) practical difficulties/complexity of the test, in particular the radiochemical steps, which sometimes resulted in loss of specimen/activity; this in turn led to variability in the results and to a poor reproducibility and (ii) radiation protection issues. However, the OECD guideline allows other endpoints for assessment of proliferation in form of lymph node cell counts and lymph node weights if justification and appropriate scientific support exist showing the validity of this method.
The alternative method used for the study employing the lymph node weight and lymph node cell count to assess proliferation has been established by an European inter-laboratory validation exercise, as described in the two publications by Ehling et al. 2005a and 2005b. This method has the advantage of (i) more simplistic experimental work, (ii) less variability, (iii) better reproducibility, (iv) faster results, (v) reduced costs.
In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item.
By additionally measuring simple inflammatory parameters such as ear thickness or ear weight, it is possible to reliably determine the degree of response that is attributable to irritation (Vohr and Ahr, 2005 ).
Hence, in addition, the acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control. The stimulation indices were calculated by dividing the average ear weight and average ear thickness on test day 4 per group to the test item treated animals by the vehicles treated ones. The cut-off threshold value for ear weight was set at 1.1.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: modified LLNA
- Criteria used to consider a positive response:
The so-called stimulation (or LLN-) indices to determine the sensitising potential (this value was fixed empirically during the interlaboratory validation of this method, for details see Ehling et al. 2005a and 2005b, page 12), were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones.
Thus, in case of no stimulating effect the index for the lymph node cell count is always below 1.4 (cut-off value). An index above 1.4 is considered positive.

TREATMENT PREPARATION AND ADMINISTRATION:
The experimental schedule of the assay was as follows:
- Day 1:
The weight of each animal was individually identified. The weights and any clinical signs were recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer.
Open application of 25 µL of the appropriate dilution of the test item, the vehicle alone or the positive control (as appropriate) were administered to the dorsum of each ear.
- Days 2 and 3:
The application procedure carried out on day 1 was repeated.
- Day 4 (24 hours after the last application):
Ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer.
The animals were euthanized by carbon dioxide (CO2) inhalation and laparotomised.
Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance.
Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in PBS /0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.

Test formulation analysis was carried out non-GLP in 2 steps:
1) The analytical method was validated by LPT before in vivo testing. The following parameters were determined:
- Linearity, Accuracy, Precision, Sensitivity, Specificity and Stability (6 hours at room temperature)
2) 3 Samples of approximately 5 mL were taken during the in-life phase from the prepared formulations of 5%, 10% and 25% (w/w). The samples were stored at ≤ 20°C until analysis for concentration.

Observations of the animals daily for clinical signs, local irritation or systemic toxicity and body weight (day 1 and day 4) were included in the study.

Positive control substance(s):

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

Statistics:

The acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control.

Results and discussion

Positive control results:

The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid.(see Table 2)

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

< 1.4

Test group / Remarks:

the stimulation index (SI) for lymph node count is well below the threshold level of 1.4

Remarks on result:

other: no indication for skin sensitization

Results 2

Key result

Parameter:

SI

Value:

1.082

Test group / Remarks:

5% test item in A/O: the stimulation index (SI) for lymph node cell count is well below the threshold level of 1.4

Remarks on result:

other: no indication of skin sensitization

Results 3

Key result

Parameter:

SI

Value:

1.045

Test group / Remarks:

10% test item in A/O: the stimulation index (SI) for lymph node cell count is well below the threshold level of 1.4

Remarks on result:

other: no indication of skin sensitization

Results 4

Key result

Parameter:

SI

Value:

1.299

Test group / Remarks:

25% test item in A/O: the stimulation index (SI) for lymph node cell count is well below the threshold level of 1.4

Remarks on result:

other: no indication of skin sensitization

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
In the main study treatment with Macrolex Fluoreszenzrot G at concentrations of 5%, 10% and 25% (w/w) did not reveal any statistical significantly increased values for the lymph node cell count and lymph node weight. The stimulation index for the lymph node cell count did not exceed the threshold level of 1.4. (see Table 2)

The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted. (see Table 2)

The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid. (see Tabel 2)

DETAILS ON STIMULATION INDEX CALCULATION
The study was performed according to OECD 429. However, an alternative method was used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes. In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.
Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones.
Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).

EC 1.4 CALCULATION
The so-called stimulation (or LLN-) indices to determine the sensitising potential (this value was fixed empirically during the interlaboratory validation of this method, for details see Ehling et al. 2005a and 2005b, page 12), were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones. No values were above the threshold of 1.4 (lymph node cell count) or 1.1 (ear weight). Thus, under the present test conditions, Macrolex Fluoreszenzrot G at concentrations of 5%, 10% and 25% (w/w) in acetone/olive oil (4:1, v/v) did not reveal any skin sensitising properties in the local lymph node assay. A 25% (w/w) concentration of the test item in acetone/olive oil (4:1, v/v) was the maximum feasible concentration.

CLINICAL OBSERVATIONS and BODY WEIGHTS
No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment.

Any other information on results incl. tables

In a preliminary experiment, concentrations of 5%, 10% and 25% (w/w) of Macrolex Fluoreszenzrot G, employing 1 animal per concentration, were examined. No irritating properties were observed in this preliminary experiment at concentrations of 5%, 10% and 25% (w/w), and no differences in ear weight and ear thickness were noted. A 25% (w/w) concentration was the highest feasible concentration of Macrolex Fluoreszenzrot G in acetone/olive oil (4:1, v/v).

Table 2: Stimulation indices (SI) in the main experiment (mean of 6 animals per group):

Parameter	negative control (A/O)	5% (w/w) test item in A/O	10% (w/w) test item in A/O	25% (w/w) test item in A/O	positive control (20% alpha-hexyl cinnamic aldehyde (v/v) in A/O)
Lymph node cell count	1.000	1.082	1.045	1.299	1.610*
Lymph node weight	1.000	1.114	1.023	1.136	1.477*
Ear weight	1.000	0.988	0.982	1.006	1.161*
Ear thickness	1.000	1.004	1.016	1.029	1.053

* significantly different from control at p ≤ 0.01

The analysis of the test item vehicle suspensions of the 5%, 10% and 25% (w/w) concentrations for the actual test item levels was carried out (non-GLP) under conditions employing a validated analytical method. The analysis resulted in actual levels of 111.4% (5% (w/w) concentration), 112.3% (10% (w/w) concentration) and 104.4% (25% (w/w) concentration) of the nominal concentration.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Executive summary:

The purpose of this study was to determine the skin sensitising potential of Macrolex Fluoreszenzrot G in the modified local lymph node assay in mice. The study was performed according to OECD 429. However, an alternative method was used employing the lymph node weight and lymph node cell count to assess proliferation of lymphocytes. In addition, the acute inflammatory skin reaction is measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.

Stimulation indices were calculated for the lymph node cell count, lymph node weight, ear weight and ear thickness by dividing the average values per group of the test item treated animals by the respective vehicle treated ones.

Values above 1.4 (lymph node cell count to identify sensitisation) or 1.1 (ear weight to identify irritation) are considered positive (these values were fixed empirically during the interlaboratory validation of this method (Ehling et al. 2005a and 2005b)).

Three concentrations of Macrolex Fluoreszenzrot G (5%, 10% and 25% (w/w)), suspended in acetone/olive oil (4:1, v/v) were tested in six female NMRI mice per group and compared to a vehicle control group. Acetone/olive oil (A/O, 4:1, v/v) was selected as it provided a suitable suspension of the test item both for administration and adherence to the mouse ear. A 25% (w/w) concentration was the highest feasible concentration in A/O. Methyl ethyl ketone, N,N-dimethylformamide, propylene glycol and dimethyl sulfoxide, other recommended vehicles, did not provide higher concentrated suitable suspensions.

In the main study treatment with Macrolex Fluoreszenzrot G at concentrations of 5%, 10% and 25% (w/w) did not reveal any statistical significantly increased values for the lymph node cell count and lymph node weight. The stimulation index for the lymph node cell count did not exceed the threshold level of 1.4. The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted. The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). Therefore, the study can be regarded as valid. No signs of local or systemic intolerance were recorded. The animal body weight was not affected by the treatment.

In conclusion, under the present test conditions, Macrolex Fluoreszenzrot G at concentrations of 5%, 10% and 25% (w/w) in acetone/olive oil (4:1, v/v) did not reveal any skin sensitising properties in the local lymph node assay. A 25% (w/w) concentration of the test item in acetone/olive oil (4:1, v/v) was the maximum feasible concentration.