3-(diethylamino)-7-oxo-7H-[1]benzopyrano[3',2':3,4]pyrido[1,2-a]benzimidazole-6-carbonitrile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 February 2022 to 29 March 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

3-(diethylamino)-7-oxo-7H-[1]benzopyrano[3',2':3,4]pyrido[1,2-a]benzimidazole-6-carbonitrile

EC Number:

257-884-8

EC Name:

3-(diethylamino)-7-oxo-7H-[1]benzopyrano[3',2':3,4]pyrido[1,2-a]benzimidazole-6-carbonitrile

Cas Number:

52372-36-8

Molecular formula:

C23H18N4O2

IUPAC Name:

17-(diethylamino)-11-oxo-14-oxa-3,10-diazapentacyclo[11.8.0.0²,¹⁰.0⁴,⁹.0¹⁵,²⁰]henicosa-1(21),2,4,6,8,12,15(20),16,18-nonaene-12-carbonitrile

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment: temperature 22 ± 2°C
relative humidity approx. 45-65% (except for deviation)
artificial light 6.00 a.m. - 6.00 p.m.
ventilation: at least eight air changes per hour

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

5, 10, and 25%

No. of animals per dose:

4

Details on study design:

Vehicle and Dose Selection
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was a 25% suspension in propylene glycol. Vortexing was used to formulate the test item. At higher concentrations, an applicable formulation of the test item could not be achieved.
To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% once daily each on three consecutive days. One animal received a concentration of 10% and the other animal received a concentration of 25%. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. These ear-punch weights were compare to those of the historical controls. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. These ear-punch weights were compared to those of the historical controls. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6 (for detailed results see Appendix 1).
The animals showed mild, unspecific signs of discomfort such as intermittently piloerection and partially closed eyes. Possible redness of the ears could not be determined due to the inherent colour of the test item. During test days 1 and 2, additional observations were made in order to monitor the well-being of the animals closely, and an additional body weight check was performed on test day 2. These additional observations are described in the study raw data but are not reported in detail, since no clear signs of toxicity were observed, and all relevant clinical signs are reported within the scheduled observations. All clinical signs could be attributed to mild discomfort. Furthermore, both animals showed a normal gain of body weight over the study period, and no relevant increase in ear weights or ear thickness values was observed.
Thus, the test item in the main study was assayed at 5, 10, and 25%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

Test Item Preparation
The test item was placed into an appropriate container on a tared balance and propylene glycol was added (weight per weight).
The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer.
The preparations were made freshly before each dosing occasion.

Test Item Administration
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% in propylene glycol. The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (  8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

Administration of 3H-methyl-thymidine
Five days after the first topical application (day 6) 250 µL of phosphate-buffered saline containing 20.8 µCi of 3H-methyl thymidine (equivalent to 83.2 µCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

Terminal Procedure
Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.

Preparation of Single Cell Suspensions
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.

Determination of cellular proliferation (incorporation of 3HTdR)
The precipitates were then resuspended in 5 % trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a -scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % trichloroacetic acid. The -scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

Observations
Clinical Observations
All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.
Determination of Ear Thickness
In the pre-test, the ear thickness was determined prior to the first application of the test item (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer.
Determination of Ear Weights
In the pre-test, after the lymph nodes have been excised, both ears of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance. The values obtained were taken down manually. The results are described in the report.
Determination of Body Weights
The body weights were recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment).

Data Evaluation
Interpretation of raw data
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.
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.

General Calculations
The mean values and standard deviations were calculated in the body weight tables.
Where appropriate, the EC3 value were 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.
All calculations conducted on the DPM values were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.

Positive control substance(s):

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

Results and discussion

Positive control results:

valid

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.81

Test group / Remarks:

5%

Results 2

Parameter:

SI

Value:

1.09

Test group / Remarks:

10%

Results 3

Parameter:

SI

Value:

0.67

Test group / Remarks:

25% / highest concentration that could be formulated

Any other information on results incl. tables

Calculation and Results of Individual Data

Test item concentration %	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	21	---	---	---	---
---	BG II	13	---	---	---	---
0	1	8973	8956	8	1119.5	1.00
5	2	7286	7269	8	908.6	0.81
10	3	9737	9720	8	1215.0	1.09
25	4	5988	5971	8	746.4	0.67

BG = Background (1 ml 5% trichloroacetic acid) in duplicate

1 = Control Group

2-4 = Test Group

a) = The mean value was taken from the figures BG I and BG II and subtracted form measured DPM values for the pooled lymph nodes

b)=Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

Calculation of the EC3 value

The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Viability / Mortality

No deaths occurred during the study period.

 Clinical Signs

The animals did not show any signs of systemic toxicity during the course of the study. Possible redness of the ears could not be determined due to the inherent colour of the test item.

 Body Weights

The body weight of the animals, recordedprior to the first application and prior to treatment with³HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The test item Disperse Red 362 was not a skin sensitiser under the test conditions of this study.

Executive summary:

In order to study a possible skin sensitising potential of Disperse Red 362, three groups each of four female mice were treated once daily with the test item at concentrations of 5, 10, and 25% in propylene glycol by topical application to the dorsum of each ear for three consecutive days. The highest concentration tested was the highest concentration that could technically be achieved. A control group of four mice was treated with the vehicle (propylene glycol) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (³H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes, which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of³H-methyl thymidine measured in ab-scintillation counter.

All treated animals survived the scheduled study period and no signs of systemic toxicity were observed. Possible redness of the ears could not be determined due to the inherent colour of the test item.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in a 3-fold or greater increase in incorporation of³HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

In this study Stimulation Indices of 0.81, 1.09, and 0.67 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in propylene glycol, respectively. The EC3 value could not be calculated, since none of the tested concentrations induced a S.I. greater than the threshold value of 3.