6-methyl-3,4-dihydro-2H-1,4-benzoxazine

Endpoint summary

Description of key information

DPRA (OECD 442C): Solutions of the test substance were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. With moderate mean depletion of both peptides (22.7%) in the presence of the test item, the substance is therefore predicted by DPRA as positive and to be a potential skin sensitizer based on this assay.  

Luciferase (OECD 442D): It was concluded that the test substance gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the substance is a skin sensitizer.

h-CLAT (OECD 442E): The test substance with a log Pow of 2.14 activated THP-1 cells under the test conditions of this study. Therefore the substance is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

LLNA OECD 429: The test substance was found to be a skin sensitiser under the test conditions of this study.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 April 2019 - 14 May 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8 - 11 weeks
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%, artificial light 6.00 a.m. - 6.00 p.m.
Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Vehicle:

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

Concentration:

Low Dose: 2.5%
Mid Dose: 5%
High Dose: 10%

No. of animals per dose:

Four

Details on study design:

PRE-SCREEN TESTS:
A solubility experiment was performed according to the recommendations given by OECD 429. The highest test item concentration, which could be technically used was 100% (undiluted test item).
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 50 and 100% once daily each on three consecutive days. 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. 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. 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
Both animals showed an erythema of the ear skin (Score 1 to 2) and erythema of the scalp. The animals had to be sacrificed on day 2 and 3, respectively, due to clear symptoms of toxicity such as hunched posture, piloerection, partially closed eyes, sunken flanks, ataxia, apathy, laboured breathing, and weight loss. Additionally, staining of the urine was observed (orange).
Therefore, a second pre-test was performed using test item concentrations of 10 and 25%. Both animals showed an erythema of the ear skin (Score 1 to 2) and erythema of the scalp. Clear symptoms of toxicity were observed in the animal treated with 25% of the test item which included hunched posture, partially closed eyes, laboured respiration, and decreased activity. Furthermore, a loss in body weight of >10% was observed in this animal. Therefore, this concentration was considered to be inappropriate for use in the main study. In the animal treated with 10% of the test item, only mild and transient symptoms of toxicity were observed, namely partially closed eyes 1 hour after the third application, and hunched posture on days 4-6. Only a negligible loss in body weight (3%) was noted in this animal.

MAIN STUDY
The test item in the main study was assayed at 2.5, 5, and 10%. The highest concentration tested was the highest level that could be achieved whilst avoiding clear systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

ANIMAL ASSIGNMENT AND TREATMENT
The animals were distributed into the test groups at random.
- Criteria used to consider a positive response:
• 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 acetone/olive oil (4+1, v/v) was added (weight per weight). The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion.
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 2.5, 5, and 10% in acetone/olive oil (4+1, v/v). 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) was treated with an equivalent volume of the relevant vehicle alone (control animals).

Positive control substance(s):

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

Statistics:

All calculations were performed with a validated test script of “R”, a language and environment for statistical computing and graphics.

Positive control results:

Experiment performed in April 2019 (Envigo study number 1950400). Positive control substance: α-Hexylcinnamaldehyde
Vehicle: acetone:olive oil (4:1 v/v))
(See table 1)

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Control

Key result

Parameter:

SI

Value:

2.59

Test group / Remarks:

2.5% test item concentration

Key result

Parameter:

SI

Value:

4.48

Test group / Remarks:

5% test item concentration

Key result

Parameter:

SI

Value:

7.1

Test group / Remarks:

10% test item concentration

Key result

Parameter:

EC3

Value:

3.04

Test group / Remarks:

EC3 value calculated was 3.04 % (w/w)

Cellular proliferation data / Observations:

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.

EC3 CALCULATION :
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.

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.

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).

Table 1. Results of positive control

Test item concentration %	Group	Measurement DPM	Calculation			Result
			DPM-BG^a)	number of lymph nodes	DPM per lymph node^b)	S.I.
---	BG I	15	---	---	---	---
---	BG II	24	---	---	---	---
0	1	6866	6846.5	8	855.8	1.00
5	2	10186	10166.5	8	1270.8	1.48
10	3	15511	15491.5	8	1936.4	2.26
25	4	55457	55437.5	8	6929.7	8.10

1 = Control Group

2-4 = Test Group

^a)= The mean value was taken from the figures BG I and BG II

^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

Interpretation of results:

Category 1B (indication of skin sensitising potential) based on GHS criteria

Conclusions:

The test substance was found to be a skin sensitiser under the test conditions of this study.

Executive summary:

In the study the test item formulated in acetone/olive oil (4+1, v/v) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 2.5, 5, and 10%. The highest concentration tested was the highest concentration that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed by two pre-experiments.

No cases of mortality were observed. The animals treated with the test item showed an erythema of the ear skin (Score 1 to 2) and erythema of the scalp and scaly ears. Additionally, in animals treated with the high dose of the test item only, mild clinical symptoms such as partially closed eyes, hunched posture, and decreased activity as well as staining of the urine (orange) were observed transiently on test days 2, 3, and 4.

In this study Stimulation Indices (S.I.) of 2.59, 4.48, and 7.10 were determined with the test item at concentrations of 2.5, 5, and 10% in acetone/olive oil (4+1, v/v), respectively. A clear dose response was observed.

The test substance was found to be a skin sensitiser and an EC3 value of 3.04% (w/w) was derived.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

09 July 2018 - 26 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

activation of keratinocytes

Justification for non-LLNA method:

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The KeratinoSens™ in vitro skin sensitisation assay uses an immortalised adherent cell line derived from HaCaT human keratinocytes stably transfected with a selectable plasmid. The cell line contains the luciferase gene under the transcriptional control of a constitutive promoter fused with an ARE element from a gene that is known to be up-regulated by contact sensitisers. The luciferase signal reflects the activation by sensitisers of endogenous Nrf2 dependent genes, and the dependence of the luciferase signal in the recombinant cell line on Nrf2 has been demonstrated. This allows quantitative measurement (by luminescence detection) of luciferase gene induction, using well established light producing luciferase substrates, as an indicator of the activity of the Nrf2 transcription factor in cells following exposure to electrophilic test chemicals.

Specific details on test material used for the study:

Test item: 6 -methyl-3,4 -dihydro-2H-1,4 -benzoxazine
CAS number: 71472-57-6
Batch number: 107562
Storage conditions: Refrigerated (2 - 8˚C), in the dark
Purity: 99% (area HPLC)
Appearance: Amber coloured viscous liquid
Molecular weight: 149.19 g/mol
Stability/expiry: 01 January 2019

Details on the study design:

Cell Culture
The cells used in this assay were the transgenic cell line KeratinoSens™ with a stable insertion of the luciferase construct supplied by Givaudan. The cells were routinely grown and subcultured in maintenance medium at 37°C ± 2°C in a humidified atmosphere containing 5% CO2 in air. Maintenance medium was 500 mL Dulbecco’s Modified Eagles Medium containing Glutamax (DMEM) (Gibco 21885), supplemented with 50 mL foetal bovine serum (FBS) (Gibco 10270) and 5.5 mL Geneticin (Gibco 10131).

Cell Culture from Frozen Stocks
Vials of KeratinoSens™ cells, stored frozen in cryotubes at -196C under liquid nitrogen, in DMEM containing 10% dimethyl sulphoxide and 20% FBS, were thawed rapidly at 37°C in a water-bath. The cells were then resuspended in a total of 10 mL of pre-warmed maintenance medium without geneticin and pelleted by centrifugation at 125 g for 5 minutes. The cell pellet was resuspended in maintenance medium without geneticin in tissue culture flasks. The flasks were incubated until 80-90% confluent cell monolayers had been obtained. Geneticin-containing medium was used in subsequent passages. Establishing cell cultures from frozen stocks and subsequent passage was conducted prior to the start of this study.

Cell Passage
Actively growing cell stocks were maintained and expanded by subculturing (passage). When the cells had reached 80 – 90% confluence, the medium from each flask was removed, the cells washed twice with Dulbecco’s phosphate buffered saline (DPBS) (Gibco 14190) and harvested using trypsin-EDTA solution. Cultures were incubated at 37 ± 2°C in a humidified atmosphere containing 5% CO2 in air until complete detachment and disaggregation of the cell monolayer had occurred. The cells were then resuspended in medium to neutralise the trypsin (cells from several flasks may have been pooled at this point). The cells were resuspended and distributed into flasks containing fresh maintenance medium. This passage procedure was repeated to provide a sufficient number of cells for a test, and were passaged at least twice before using the cells in a test. The passages of KeratinoSens™ cells were limited to 25 passages.

Preparation of Test Cell Cultures
The cells from flasks of actively growing cultures were detached and disaggregated as described above. The number of viable cells in the prepared cell suspension were determined by counting a trypan blue-stained cell preparation using an Improved Neubauer Haemocytometer. The cell suspension was diluted with maintenance medium without geneticin to give 1 x 105 viable cells/mL and 100 µL volumes pipetted into all wells except well H12 of sterile 96-well flat-bottomed microtitre plates. On each occasion four plates were prepared in parallel for each batch of up to seven proficiency chemicals: three white plates for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay. Well H12 of each plate received 100 µL maintenance medium without geneticin with no cells. The plates were incubated for 24 ± 2 hours at 37 ± 2C in a humidified atmosphere of 5% CO2 in air, to allow the cells to attach.

Preparation of the Positive Control
Cinnamic aldehyde (Sigma, 239968, lot: STBG0250V, expiry: July 2020) was prepared by weighing between 20 – 40 mg into a tared glass container and diluted to a final concentration of 200 mM in DMSO using the following formula:

V=5×((p÷100)×w)/MW-w/1000

Where
V = volume of DMSO in mL to be added
p = purity of the chemical in %
MW = molecular weight of the chemical in g/mol
w = exact weight of the chemical added to the vial in mg

The 200 mM cinnamic aldehyde solution was further diluted to a final concentration of 6.4 mM by adding 32 µL of the 200 mM solution to 968 µL of DMSO.

Results from the positive control were shared with other studies performed in the same assay.

Test Item Solubility
Prior to commencing testing, the solubility of the test item in DMSO was assessed. The test item was found to be soluble in DMSO at 200 mM, the highest concentration as recommended by the guideline this test follows.

Preparation of the Test Item
A stock solution of the test item was prepared by weighing between 20 – 40 mg into a tared glass container and diluting to 200 mM in DMSO using the formula above.

Test Procedure
Preparation of the 100x Solvent Plate
A 100x solvent plate was set up by adding 100 µL of DMSO to all wells of a 96 well plate except wells in column 12 and well H11 of the plate. 200 µL of the stock solution of the test item was added to one well in column 12. The test item was serially diluted across the plate by transferring 100 µL from column 12 to column 11 and then mixed by repeat pipetting (at least 3 times) and then 100 µL was transferred from column 11 to column 10 and so forth across the plate.

200 µL of the 6.4 mM stock solution of cinnamic aldehyde was added to well H11 and serially diluted from column 11 to column 7.

Preparation of the Dilution Plate
The 100x solvent plate was replicated into a fresh 96 well plate by adding 240 µL of assay medium to each well and then 10 µL solution per well from the 100x solvent plate was added to equivalent wells on the dilution plate. Assay medium was 495 mL DMEM (Gibco 21885), supplemented with 5.0 mL FBS.

Treatment of Cultured Plates
Approximately 24 hours after the test cell culture plates were established, the medium was removed from the wells by careful inversion of the plates and blotting onto sterile paper towel. 150 µL of assay medium was added to every well of the 96 well plates. 50 µL from each well of the dilution plate was transferred to equivalent wells in the 96 well plates. Three white plates were dosed for measuring luminescence and one transparent plate for measuring cell viability using the MTT assay.

The plates were then covered with a plate seal and placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air for 48 ± 2 hours.

Cell viability measurement
A kit (Molecular Probes Vybrant MTT kit V13154) was used to determine cell viability. 1 vial from the kit was reconstituted by adding 1 mL of sterile PBS (Gibco 10010) and vortexed mixed until dissolved to give 5 mg/mL MTT in DPBS. After incubation, the transparent plate was removed from the incubator and the plate seal discarded. The cell culture medium was removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 100 µL fresh assay medium was added to each well. 10 µL of MTT solution was added to each well of the 96-well plate. The plate was incubated at 37 ± 2°C in a humidified atmosphere of 5% CO2 in air for 4 hours ± 10 minutes. The medium was then removed by careful inversion of the plate and blotted onto sterile paper towel to remove residual culture medium. 50 µL of DMSO was added to each well. The plate was then placed in the incubator at 37 ± 2°C, in a humidified atmosphere of 5% CO2 in air, protected from light, for at least 10 minutes. The absorbance value of each well was read using a plate reader with a 540 nm filter.

Luciferase measurement
Luciferase was measured using the Steady Glo® Luciferase Assay system kit supplied by Promega (E2550). Steady-Glo® luciferase reagent was prepared by transferring the contents of one bottle of Steady-Glo® buffer to one bottle of Steady-Glo® substrate. The reagent was mixed by inversion until the substrate had dissolved. The reconstituted reagent was used on the same day it was prepared for test 1. Frozen reconstituted reagent was used for test 2 and was thawed to room temperature before use.

After incubation the medium was removed from the wells of the triplicate white plates by careful inversion of the plates and blotting on sterile absorbent paper. 100 µL of fresh assay medium was added to each well before 100 µL of Steady-Glo® luciferase reagent was added to each well of the plate. The plates were shaken on a plate shaker for at least 5 minutes until the cells had lysed. Luminescence (emitted light) was measured using a SpectraMax L luminometer. Each plate was read for total photon count with an integration time of 1 second. The plates were dark adapted for 1 minute prior to measurement.

Positive control results:

The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5 in at least one of the tested concentrations (4 to 64 μM) in both tests

The EC1.5 values of the positive control, cinnamic aldehyde were 10.18 μM and 16.09 μM for test 1 and 2, respectively, which lay within the historical control range for this laboratory. The average induction in the three replicates for cinnamic aldehyde at 64 µM were 5.36 and 3.64 for test 1 and 2, respectively, which met the acceptance criterion of between 2 and 8.

Key result

Run / experiment:

other: Test 1

Parameter:

other: Imax

Value:

21.36

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: Imax

Value:

25.99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

The Imax for the tested substance was 21.36 in test 1 and 25.99 in test 2. The Imax for both tests was >1.5 fold and statistically significant as compared to the solvent vehicle control. The cellular viability did not fall below 93.63% in test 1 and therefore the IC30 and IC50 could not be calculated. In test 2 the cellular viability was 8.54% at 2000 µM, the IC30 was 1179.59 µM and the IC50 was 1446.55 µM. The EC1.5 for the substance was 12.75 µM and 35.29 µM for tests 1 and 2, respectively.

Results for test substance - Test 1

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	0.82	0.98	1.09	1.23	1.66	1.51	1.59	2.22	3.31	5.00	8.16	21.36
Statistically significant	No	No	No	No	Yes	Yes	Yes	Yes	Yes	Yes	Yes	Yes
Viability (%)	99.36	106.46	120.66	110.83	167.06	177.98	171.16	163.79	156.14	149.59	168.97	93.63
Imax	21.36
EC1.5(µM)	12.75
IC30(µM)	N/A
IC50(µM)	N/A

 

 

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax>1.5 fold and statistically significant	Yes
Is the cellular viability >70% at the lowest concentration at the EC1.5determining concentration	Yes
Is the EC1.5value <1000µM	Yes
Is there an apparent overall dose-response for luciferase induction	Yes
KeratinoSens™ prediction	Positive

 

 

 

 

Table 2: Results for Cinnamic Aldehyde – Test 1

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.23	1.35	1.90	3.05	5.36
Statistically significant	No	No	Yes	Yes	Yes
Viability (%)	117.65	120.11	124.20	126.39	111.65
Imax	5.36
EC1.5(µM)	10.18
IC30(µM)	N/A
IC50(µM)	N/A

 

Test Acceptance Criteria	Result
Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations	Yes	Pass
Average induction of positive control at 64 µM between 2 – 8	Yes (5.36)	Pass
EC1.5of positive control within two standard deviations of the historical mean (8.76 – 49.64)	Yes (10.18)	Pass
CV% of blank values < 20%	Yes (11.9%)	Pass

 

 

Table 3: Results for test substance – Test 2

Test item conc. (µM)	0.98	1.95	3.91	7.81	15.63	31.25	62.5	125	250	500	1000	2000
Mean fold induction	1.12	1.13	1.26	1.44	1.45	1.47	1.72	2.08	2.86	3.84	5.93	25.99
Statistically significant	No	No	No	No	No	No	Yes	Yes	Yes	Yes	Yes	Yes
Viability (%)	106.98	100.03	93.25	90.40	103.29	112.67	107.23	100.28	100.20	92.83	83.45	8.54
Imax	25.99
EC1.5(µM)	35.29
IC30(µM)	1179.59
IC50(µM)	1446.55

 

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax>1.5 fold and statistically significant	Yes
Is the cellular viability >70% at the lowest concentration at the EC1.5determining concentration	Yes
Is the EC1.5value <1000µM	Yes
Is there an apparent overall dose-response for luciferase induction	Yes
KeratinoSens™ prediction	Positive

 

 

 

Table 4: Results for Cinnamic Aldehyde – Test 2

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.17	1.25	1.50	2.12	3.64
Statistically significant	No	No	No	Yes	Yes
Viability (%)	104.55	111.83	110.91	111.58	102.37
Imax	3.64
EC1.5(µM)	16.09
IC30(µM)	N/A
IC50(µM)	N/A

 

Test Acceptance Criteria	Result
Luciferase activity induction obtained with the positive control statistically significant above the threshold of 1.5 in at least one of the test concentrations	Yes	Pass
Average induction of positive control at 64 µM between 2 – 8	Yes (3.64)	Pass
EC1.5of positive control within two standard deviations of the historical mean (8.76 – 49.64)	Yes (16.09)	Pass
CV% of blank values < 20%	Yes (14.8%)	Pass

 

 

Interpretation of results:

other: It was concluded that the test substance gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the substance is a skin sensitizer.

Conclusions:

It was concluded that the test substance gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the substance is a skin sensitizer.

Executive summary:

The purpose of this study was to support a predictive, adverse-outcome-pathway evaluation of whether the test item, 6 -methyl-3,4 -dihydro-2H-1,4 -benzoxazine, is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The I_maxfor the test substance was 21.36 in test 1 and 25.99 in test 2. The I_maxfor both tests was >1.5 fold and statistically significant as compared to the solvent vehicle control. The cellular viability did not fall below 93.63% in test 1 and therefore the IC₃₀and IC₅₀could not be calculated. In test 2 the cellular viability was 8.54% at 2000 µM, the IC₃₀was 1179.59 µM and the IC₅₀was 1446.55 µM. The EC_1.5for the test substance was 12.75 µM and 35.29 µM for tests 1 and 2, respectively. Graphs for the test substance showed an overall dose-response for luciferase induction.

The luciferase activity induction obtained with the positive control, cinnamic aldehyde, was statistically significant above the threshold of 1.5 in at least one of the tested concentrations (4 to 64μM) in both tests.

The EC_1.5values of the positive control, cinnamic aldehyde were 10.18 μM and 16.09 μM for test 1 and 2, respectively, which lay within the historical control range for this laboratory. The average induction in the three replicates for cinnamic aldehyde at 64 µM were 5.36 and 3.64 for test 1 and 2, respectively, which met the acceptance criterion of between 2 and 8.

The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 11.9% and 14.8% for test 1 and 2, respectively, which met the acceptance criterion of below 20%. It was concluded that the tested substance gave a positive response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that the substance is a skin sensitizer.

Reference 3

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

27 June 2018 - 03 July 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442C (In Chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA))

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

direct peptide reactivity assay (DPRA)

Justification for non-LLNA method:

The DPRA test allows quantification of a chemical’s reactivity and is used to categorize a substance in one of four classes of reactivity to allow discriminating between skin sensitizing and non-sensitizing chemicals and thus assesses their sensitization potential.

Specific details on test material used for the study:

Chemical Name: 6-methyl-3,4-dihydro-2H-1,4-benzoxazine
Batch: 107562
CAS no.: 71472-57-6
Molecular formula: C9H11NO
Molecular weight: 149.19 g/mol
Retest date: 07 March 2023
Purity: 99%
Correction factor: Use as supplied
Storage conditions: Refrigerated condition (+2 to +8°C), parafilm sealed, avoid long exposure to air.

Details on the study design:

Analytical Procedure

Reagents
Acetonitrile (ACN): HPLC gradient grade
Trifluoroacetic acid (TFA): 99% Pure
Water: Deionised reverse osmosis
Ammonium Acetate: Analytical reagent
Sodium Phosphate, monobasic: Analytical reagent
Sodium Phosphate, dibasic: Analytical reagent
Ammonium Hydroxide: Analytical reagent
100 mM Phosphate buffer, pH 7.5: In house preparation
100 mM Ammonium Acetate buffer, pH 10.2: In house preparation
HPLC Mobile Phase A: 0.1% v/v TFA in Water, in house preparation
HPLC Mobile Phase B: 0.085% v/v TFA in ACN, in house preparation

Assessment of Test Item Solubility
The solubility of the test substance was assessed in a variety of solvents.

Preparation of Peptide Stock Solutions
Stock solutions of each peptide at concentrations of 0.667 mM were prepared by dissolution of pre-weighed aliquots of the appropriate peptide in ca 20 mL aliquots of the appropriate buffer solution (Cysteine in 100 mM phosphate buffer pH 7.5, Lysine in 100 mM Ammonium acetate buffer pH 10.2).

Preparation of Peptide Calibration Standards
Calibration standards of both peptides were prepared by diluting the requisite stock solution in the appropriate buffer and acetonitrile and contained each peptide at concentrations of 0.0167 mM, 0.0334 mM, 0.0667 mM, 0.133 mM, 0.267 mM and 0.534 mM. A buffer blank was also prepared.

Preparation of Reference Controls and Precision Controls
Stability controls (Reference Control B) and precision controls (Reference control A) of both peptides were prepared at a concentration of 0.5 mM in acetonitrile/buffer.

Preparation of Positive Control Solution and Test Item Stock Solution
The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of the test substance was prepared in acetonitrile.

Preparation of Positive Control and Cysteine Peptide Depletion Samples and Co-elution Controls
Accurate volume aliquots of the test substance and the positive control were diluted with the Cysteine peptide stock solution to prepare solutions containing 0.5 mM Cysteine and 5 mM of the test substance and 5 mM of the positive control. For the co-elution control, acetonitrile was used in place of the Cysteine stock solution.

Preparation of Positive Control and Lysine Peptide Depletion Samples and Co elution Controls
Accurate volume aliquots of the test substance and the positive control were diluted with the Lysine peptide stock solution to prepare solutions containing 0.5 mM Lysine and 25 mM of the test substance and 25 mM of the positive control. For the co-elution control, acetonitrile was used in place of the Lysine stock solution.

Incubation
The appearance of the test substance and positive control samples in the HPLC vials was documented after preparation and then the vials placed into the autosampler of the HPLC set at 25°C for a minimum of 22 hours incubation prior to injection of the samples as part of analytical run. Before initiation of the run the appearance of the samples in the vials was assessed and documented again.

Analysis
The concentration of both the Cysteine and Lysine peptides in the presence of the test substance and the associated positive controls was quantified by HPLC using UV detection as detailed in the chromatographic section.

Instrumentation Parameters
High performance liquid chromatograph (HPLC): Waters Alliance 2695 separation module and 2487 dual wavelength detector
Column: Agilent Zorbax SB C18, 3.5m, 100 x 2.1 mm
Guard column: Phenomenex AJO4286
Column temperature: 30°C
Sample temperature: 25°C
Mobile Phase (MP) A: 0.1% TFA in Water
Mobile Phase (MP) B: 0.085% TFA in ACN
Gradient: Time (minutes) MP A
(%) MP B
(%)
0 90 10
20 75 25
21 10 90
23 10 90
23.5 90 10
30 90 10
Flow rate: 0.35 mL/minute
Stroke volume 25 µL
Detector wavelength: UV, 220 nm
Injection volume: 2 µL (slow draw rate)
Run time: 30 minutes
Approximate retention time (Cysteine) 11 minutes
Approximate retention time (Lysine) 7 minutes
2.4.12 Calculations
The peak area response for the peptide in each calibration chromatogram was measured. Calibration curves were constructed by linear regression of standard response versus standard concentration. The area responses of the peptide peak observed at the characteristic retention time of each peptide in each sample chromatogram was measured. Peptide depletion was determined using the following equation:
% Peptide depletion = 100 - Peptide peak area in replicate depletion samples (x 100)
Mean Peptide peak area of reference control samples B

2.5 Major Computerized Systems
Chromatography data handling: Waters Empower
Test item management: Pristima, Xybion Medical Systems Corporation
Version numbers of the systems are maintained by Envigo.

Positive control results:

Mean Cysteine peptide depletion: 72.9%
Mean Lysine peptide depletion: 54.3%

Run / experiment:

other: Cysteine peptide deplation

Parameter:

other: Mean percentage depletion

Value:

21.8

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

Run / experiment:

other: Lysine peptide depletion

Parameter:

other: Mean percentage depletion

Value:

23.6

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

valid

 Solubility Assessment

The solubility of the test substance in acetonitrile at a nominal concentration of 100 mM was achieved.

Reactivity Assessment

All analytical acceptance criteria for each peptide run were met:

 

		Peptide		Standard Linearity		Positive control depletion (%)		Reference controls		Test item
Acceptance criteria		Cysteine		r^2>0.99		60.8-100 (SD <14.9%)		0.45-0.55 mM (CV <15%)		SD <14.9%
		Lysine		r^2>0.99		40.2-69.0 (SD <11.6%)		0.45-0.55 mM (CV <15%)		SD <11.6%
Achieved results		Cysteine		r^2>0.999		72.9 (SD, 0.17%, n=3)		B: 0.502 mM (CV 4.32%, n=6)		SD 1.64% (n=3)
		Lysine		r^2>0.999		54.3 (SD, 4.50%, n=3)		B: 0.505 mM (CV 1.56%, n=6)		SD 2.60% (n=3)

CV         Coefficient of Variation

SD         Standard deviation

The depletion of peptide in the presence of the test substance was:

	Mean peak area of reference control(µV.sec)	Mean peak area of peptide with test item(µV.sec)	Mean peptide depletion by BN-02 (%)
Cysteine	Control B: 771020 (n=6)	602950 (n=3)	21.8
Lysine	Control B: 768530 (n=6)	587250 (n=3)	23.6

Applying the following depletion model (below), reactivity is classed as “moderate” hence the DPRA prediction is positive and the test substance is therefore predicted to be a sensitizer. 

Mean of Cysteine and Lysine% depletion	Reactivity Class	DPRA Prediction
0%≤ mean% depletion ≤6.38%	No or minimal reactivity	Negative
6.38%< mean% depletion ≤22.62%	Low reactivity	Positive
22.62%< mean% depletion ≤42.47%	Moderate reactivity
42.47%< mean% depletion ≤100%	High reactivity

In both the Cysteine and Lysine assays, in the presence of the test substance the concentration of the protein decreases and the responses of protein adducts increases over time. This is a clear indication of a reaction occurring between the test substance and the proteins.

There were no co-elution peaks in the Cysteine assay. There was a small response in the Lysine assay (2.34% of the mean peak area of Lysine in the presence of the test substance). This is considered to have minimal significant impact on the results. 

Overall Achieved Depletion Values

Test item		Cysteine peptide depletion (%)		Lysine peptide depletion (%)		Overall mean depletion (%)		Reactivity class		DPRA prediction
6 -methyl-3,4 -dihydro-2H-1,4 -benzoxazine		21.8		23.6		22.7		Moderate		Positive

 Cysteine Peptide Depletion

Sample	Peak area (µV.sec)	Peptide concentration^1(µg/mL)	Peptide Depletion^2(%)	Mean Depletion (%)	SD  (%)
Positive control	207864	100	73.0	72.9	0.17
	208868	101	72.9
	210480	101	72.7
6 -methyl-3,4 -dihydro-2H-1,4 -benzoxazine	617127	301	20.0	21.8	1.64
	599234	293	22.3
	592494	289	23.2

SD      Standard Deviation

¹                Samples prepared at a nominal concentration of 376 µg/mL (0.5 mM)

²                Calculated against a mean Reference Control B area of 771020 µV.sec(n=6)

   Lysine Peptide Depletion

Sample	Peak area (µV.sec)	Peptide concentration^1(µg/mL)	Peptide Depletion^2(%)	Mean Depletion (%)	SD  (%)
Positive control	355704	181	53.7	54.3	4.50
	382908	195	50.2
	314158	160	59.1
6 -methyl-3,4 -dihydro-2H-1,4 -benzoxazine	609888	311	20.6	23.6	2.60
	579784	295	24.6
	572083	291	25.6

SD      Standard Deviation

¹                Samples prepared at a nominal concentration of 388 µg/mL (0.5 mM)

²          Calculated against a mean Reference Control B area of 768530 µV.sec(n=6)

Interpretation of results:

other: positive and to be a potential skin sensitizer based on this assay.

Conclusions:

Solutions of the test substance were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both Cysteine and Lysine containing synthetic peptides. With moderate mean depletion of both peptides (22.7%) in the presence of the test item, the substance is therefore predicted by DPRA as positive and to be a potential skin sensitizer based on this assay.

Executive summary:

The purpose of this study (based on the OECD guideline for the testing of chemicals, In chemico Skin Sensitisation: Direct Peptide Reactivity Assay (DPRA), OECD/OCDE document TG 442C) was to assess the reactivity and sensitizing potential of 6 -methyl-3,4 -dihydro-2H-1,4 -benzoxazine. 

Solutions of the test substance were successfully analysed by the validated DPRA analytical method (Envigo analytical method FIA/M101/15) in both the Cysteine and Lysine containing synthetic peptides. With moderate depletion of both peptides in the presence of the test substance, the substance is therefore classified by DPRA as positive and hence to be a potential skin sensitizer.

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 July 2018 - 16 August 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for the Testing of Chemicals: OECD 442E; In Vitro Skin Sensitisation: In Vitro Skin Sensitisation Assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation.

Version / remarks:

Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), October 2017.

The technical proficiency of the h-CLAT with the OECD 442E guideline recommended proficiency substances was demonstrated.

Deviations:

yes

Remarks:

The cytotoxicity measurement and estimation of the CV75 value of the dose finding assay is performed by XTT test instead of flow cytometry.

Principles of method if other than guideline:

This study was performed based on the procedures indicated by the following recommendations:
1. Nukada Y, Ashikaga T, Miyazawa M, Hirota M, Sakaguchi H, Sasa H, Nishiyama N. (2012). Prediction of skin sensitization potency of chemicals by human Cell Line Activation Test (h-CLAT) and an attempt at classifying skin sensitization potency. Toxicol In Vitro. 2012 Oct; 26(7):1150-60.
2. Ashikaga T, Sakaguchi H, Sono S, Kosaka N, Ishikawa M, Nukada Y, Miyazawa M, Ito Y, Nishiyama N, Itagaki H. (2010). A comparative evaluation of in vitro skin sensitisation tests: the human cell-line activation test (h-CLAT) versus the local lymph node assay (LLNA). Altern Lab Anim. 2010 Aug; 38(4):275-84.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Human Cell Line Activation Test (h-CLAT)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Chemical Name: 6-methyl-3,4-dihydro-2H-1,4-benzoxazine
- Batch: 107562
- CAS No.: 71472-57-6
- Purity: 98.8% (NMR), dose calculation was not adjusted to purity
- Partition coefficient (n-octanol/water): log Pow: 2.14
- Water solubility: Insoluble
- Appearance: Amber coloured viscous liquid
- Expiry Date: 01 January 2019
- Storage Conditions: In the refrigerator, under N2 atomosphere
- Stability in Solvent: Stable in water (not quantified)

Details on the study design:

Skin sensitisation (In vitro test system) - Details on study design:

FLOW CYTOMETRY ACQUISITION
Before using the flow cytometer (FACSCalibur, Becton Dickinson GmbH), the device was calibrated with appropriate beads in accordance with the manufacturer’s instructions.
The expression of cell surface antigens (CD54, CD86) was analysed by flow cytometry using the software Cellquest Pro 6.0. The FITC acquisition channel (FL-1) was set for the optimal detection of the FITC fluorescence signal, and the 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7-AAD fluorescence signal.

PREPARATION OF THE ACQUISITION
The following acquisition plots were prepared:
• 2D plot consisting of FSC (Forward Scatter) versus SSC (Side Scatter)
• Histogram plot of each channel (FL-1 and FL-3, respectively)
The voltage of FSC and SSC was set with untreated cells to appropriate levels. FSC and SSC are not needed for the analysis, but the FSC/SSC plot was checked to make sure that a single population appears without contamination or excessive debris. The FL-1 and FL-3 voltage were set and compensate to appropriate position. The FL-1 voltage was set using the FITC labelled-mouse IgG1 medium-treated cells tube, as such that the MFI of control cells was set in the range between 1.0 and 4.0 (Geo Mean) and in the range between 3.0 and 4.0 (Geo Mean) with the FITC labelled CD54 medium-treated cells (FACSCalibur, Becton Dickinson GmbH).
The cell viability was additionally detected by setting an R1-gate (dead cells are gated-out by staining with 7-AAD). Therefore, the R1 gate was set approximately at the middle position between the peak of the negative fraction and the positive fraction in the FL-3 histogram using DNCB-treated cells. The negative fraction corresponds to the living cells and was kept for the subsequent analyses. For each control and all test item concentrations, the cell viability was recorded from the isotype control cell tube (stained with FITC labelled-mouse IgG1), and the CD54 and CD86 cell tubes, where only the isotype control cells were used for the cell viability evaluation.
The maintenance of the flow cytometer was in accordance with the manufacturer’s instructions. The process of washing was conducted very carefully since insoluble chemicals could flow into the flow line.

ACQUISITION
Dead cells were determined by staining with 7-AAD. Gating by FSC (forward scatter) and SSC (side scatter) was not done. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were used for analysis. The other tubes were acquired without changing the settings of the cytometer. The MFI was recorded for each condition. The relative fluorescence intensity (RFI) was calculated, but excluded from the evaluation, if the cell viability was less than 50% (due to diffuse labelling of cytoplasmic structures that could be generated due to cell membrane destruction).

FLOW CYTOMETRY ANALYSIS
The RFI is used as an indicator of CD86 and CD54 expression, and is calculated as follows for each concentration of every chemical:
RFI [%] = (((MFI _of test item treated cells ) - (MFI _of test item treated isotype control cells )) / ((MFI_of solvent control cells ) - (MFI_of solvent isotype control cells))) ×100
MFI = Geometric Mean Fluorescence Intensity (GeoMean)

The cell viability of the h-CLAT experiment is calculated as follows for each concentration of every chemical:
Cell Viability [%] = ((Mean cytotox_of solvent control cells) / (Mean cytotox _of test item treated cells) ) ×100
where the Mean cytotox is the mean of GeoMean(7-AAD) isotype control, GeoMean(7-AAD) CD54 and GeoMean(7-AAD) CD86.

In addition, the cell viability from the isotype control cells, CD54 and CD 86 cells is calculated according to the following equation:
Cell Viability [%]= (Number of living cells) / (Number of acquired cells) × 100
Where only the isotype control cells (which are stained with mouse IgG1 (isotype) antibodies) are used for the cell viability evaluation.

ACCEPTANCE CRITERIA
The following acceptance criteria should be met when using the h-CLAT method:
• Cell viability of medium control is adjusted to 100% and the cell viability of the DMSO control should be more than 90% in comparison to the medium control.
• In the solvent/vehicle control (i.e. DMSO), RFI values compared to the medium control of both CD86 and CD54 should not exceed the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%).
• For both medium and solvent/vehicle controls (i.e. DMSO), the MFI ratio of CD86 and CD54 to isotype control should be > 105%.
• In the positive control (DNCB), RFI values of both CD86 and CD54 should meet the positive criteria (CD86 ≥ 150% and CD54 ≥ 200%) and the cell viability should be > 50% in at least one concentration of the two tested positive control concentrations.
• For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.
Negative results are acceptable only for test items exhibiting a cell viability of < 90% at the highest concentration tested (i.e. 1.2 × CV75). If the cell viability at 1.2 × CV75 is ≥ 90% the negative result should be discarded. In such case it is recommended to try to refine the dose selection by repeating the CV75 determination. It should be noted that when 5000 μg/mL in saline (or medium or other solvents/vehicles), 1000 μg/mL in DMSO or the highest soluble concentration is used as the maximal test concentration of a test chemical, a negative result is acceptable even if the cell viability > 90% (OECD 442E guideline).

ACCEPTABILITY OF THE ASSAY
The XTT test is considered to be acceptable if it meets the following criteria:
• mean absorbance of the medium control is ≥ 0.5
• mean viability of the solvent control is ≥ 90% in comparison to the medium control

PREDICTION MODEL
For CD86/CD54 expression measurement, each test item is tested in at least two independent runs to derive a single prediction (POSITIVE or NEGATIVE). An h-CLAT prediction is considered POSITIVE if at least one of the following conditions is met in 2 of 2 or in at least 2 of 3 independent runs (OECD 442E guideline):
− The RFI of CD86 is ≥ 150% at any tested concentration (with cell viability ≥ 50%);
− The RFI of CD54 is ≥ 200% at any tested concentration (with cell viability ≥ 50%).
Otherwise, the h-CLAT prediction is considered NEGATIVE (see attached)).
Based on the above, if the first two runs are both positive for CD86 and/or are both positive for CD54, the h-CLAT prediction is considered POSITIVE and a third run does not need to be conducted. Similarly, if the first two runs are negative for both markers, the h-CLAT prediction is considered NEGATIVE without the need for a third run. If, however, the first two runs are not concordant for at least one of the markers (CD54 or CD86), a third run is needed and the final prediction will be based on the majority result of the three individual runs (i.e. 2 out of 3). In this respect, it should be noted that if two independent runs are conducted and one is only positive for CD86 (hereinafter referred to as P1) and the other is only positive for CD54 (hereinafter referred to as P2), a third run is required. If this third run is negative for both markers (hereinafter referred to as N), the h-CLAT prediction is considered NEGATIVE. On the other hand, if the third run is positive for either marker (P1 or P2) or for both markers (hereinafter referred to as P12), the h-CLAT prediction is considered POSITIVE. An h-CLAT prediction should be considered in the framework of an IATA (OECD 442E guideline).

Test chemicals with a Log Pow > 3.5 tend to produce false negative results. Therefore negative results with test chemicals with a Log Pow > 3.5 should not be considered (according to the OECD guideline). However, positive results obtained with test chemicals with a Log Pow > 3.5 could still be used to support the identification of the test chemical as a skin sensitiser (OECD 442E guideline).

Positive control results:

The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%) and the cell viability was >50%, with one exception. The CD54 RFI value of the positive control (2.0 µg/mL DNCB) in the first h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this one exception is considered to be acceptable since the CD54 RFI value of the positive control (3.0 µg/mL DNCB) in the first h-CLAT run exceeded the positive criteria.

Key result

Run / experiment:

other: Mean of CD86 (%)

Parameter:

other: RFI

Value:

150 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: Mean of CD54 (%)

Parameter:

other: RFI

Value:

200 %

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Other effects / acceptance of results:

The test item with a log Pow of 2.14 was tested in 2 independent runs. The RFI of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of the first run and the RFI of CD86 was equal or greater than 150% in at least one concentration of the second run. Therefore the h-CLAT prediction is considered positive for the tested test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).


ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: not examined
- Acceptance criteria met for positive control: yes

1. Resultsof the Dose Finding Assay (XTT Test)

Results of the first XTT test for Test Item

 

		Microscopic Evaluation	Photometric Evaluation
Test Group	Concen-tration [µg/mL]	Cytotoxicity	Mean Ab-sorbance*^§	Standard-Deviation	Chem. Blank^§	Mean Ab-sorbance – Chemical Blank	Absorbance in % of Solvent Control**
Medium Control	-	no	0.604	0.022	0.213	0.391	97.66
Solvent Control	-	no	0.618	0.056	0.218	0.400	100.00
Test Item	9.8	no	0.649	0.028	0.228	0.421	105.24
	19.5	no	0.629	0.047	0.246	0.383	95.65
	39.1	no	0.748	0.084	0.273	0.475	118.75
	78.1	no	0.675	0.059	0.319	0.356	89.08
	156.3^P	no	0.693	0.069	0.408	0.286	71.37
	312.5^P	yes	0.689	0.036	0.560	0.130	32.42
	625^P	yes	0.853	0.027	0.816	0.038	9.44
	1250^P	yes	1.330	0.031	1.259	0.071	17.67

Shaded test groups:            cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

P             precipitation

§             Given absorbances are rounded values.

*             mean absorbance (absolute) of 6 wells

**          relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 102.4%.

The CV75 value of the first XTT test: 140.2 µg/mL

Results of the second XTT test for Test Item

 

		Microscopic Evaluation	Photometric Evaluation
Test Group	Concen-tration [µg/mL]	Cytotoxicity	Mean Ab-sorbance*^§	Standard-Deviation	Chem. Blank^§	Mean Ab-sorbance – Chemical Blank	Absorbance in % of Solvent Control**
Medium Control	-	no	0.736	0.078	0.200	0.536	85.03
Solvent Control	-	no	0.827	0.102	0.197	0.630	100.00
Test Item	9.8	no	0.910	0.184	0.213	0.697	110.58
	19.5	no	0.787	0.144	0.227	0.560	88.86
	39.1	no	0.913	0.166	0.240	0.673	106.87
	78.1	no	0.875	0.155	0.278	0.597	94.71
	156.3	yes	0.907	0.092	0.373	0.534	84.76
	312.5	yes	0.753	0.083	0.507	0.246	39.03
	625	yes	0.721	0.037	0.698	0.024	3.78
	1250	yes	0.943	0.019	0.888	0.055	8.81

Shaded test groups: cytotoxic effects occurred in the photometric evaluation (< 75% cell viability)

§             Given absorbances are rounded values.

*             mean absorbance (absolute) of 7 wells

**          relative absorbance [rounded values]

The mean viability of the solvent control in comparison to the medium control was 117.6%.

The CV75 value of the second XTT test: 189.6 µg/mL

The mean CV75 value of both XTT tests: 164.9 µg/mL

2. Results of the h-CLAT Test

Results of the first h-CLAT run for the Test Item

	Concentration (µg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)	R1-gate Cell Viability (%)**
Medium Control	-	100.0	100.0	100.0	94.79
DMSO Control	-	100.0	100.0	100.0	95.14
Positive Control (DNCB)	2.0	189.5^#	178.9*	87.1	88.58
	3.0	303.5*	275.7*	81.3	86.38
Test Item	55	178.9	153.8*	86.4	93.01
	66	188.3	98.5	82.6	92.84
	80	223.4*	149.4	86.1	91.54
	95	231.3*	121.1	87.3	91.91
	115	185.9	144.6	83.3	91.02
	138	204.7*	163.2*	80.7	88.57
	165	196.1	162.6*	76.3	86.89
	198	221.1*	210.5*	67.4	82.46

^#    CD54 RFI value of the positive control (2.0 µg/mL DNCB) did not fulfil the positive criteria (CD54 ≥ 200%).

*   RFI value of CD86 or CD54 fulfilled the positive criteria (CD86≥150% and CD54≥200%).

** cell viability of the isotype control cells (which are stained with mouse IgG1 (isotype) antibodies)

Results of the second h-CLAT run for the Test Item

	Concentration (µg/mL)	RFI (%) CD 54 Antibody	RFI (%) CD 86 Antibody	Cell Viability (%)	R1-gate Cell Viability (%)**
Medium Control	-	100.0	100.0	100.0	96.05
DMSO Control	-	100.0	100.0	100.0	96.23
Positive Control (DNCB)	2.0	201.6*	205.8*	85.2	91.26
	3.0	330.6*	419.6*	75.0	85.15
Test Item	55	123.5	175.3*	79.0	90.23
	66	160.9	132.7	78.7	90.96
	80	186.1	160.3*	82.2	90.55
	95	174.8	231.7*	77.9	86.65
	115	151.3	165.7*	75.0	88.10
	138	135.7	255.6*	77.0	86.38
	165	185.2	247.8*	71.1	83.67
	198	168.7	340.0*	59.5	75.97

*   RFI value of CD86 or CD54 exceeded the positive criteria (CD86≥150% and CD54≥200%).

** cell viability of the isotype control cells (which are stained with mouse IgG1 (isotype) antibodies)

Interpretation of results:

other: study on its own cannot be used for classification

Conclusions:

The test substance with a log Pow of 2.14 activated THP-1 cells under the test conditions of this study. Therefore the test item is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Executive summary:

This in vitro Human Cell Line Activation Test (h-CLAT) was performed to assess the dendritic cell activation potential (third key event of a skin sensitization AOP) of the test substance dissolved in culture medium when administered to THP-1 cells for 24 ± 0.5 hours. The highest test item concentration for the main experiment (h-CLAT) of the test substance was previously determined by two XTT tests.

Cytotoxic effects by photometric evaluation were observed following incubation with the test item starting with the concentration of 156.25 µg/mL up to the highest tested concentration (1250 µg/mL) in the first XTT test and starting with the concentration of 312.5 µg/mL up to the highest tested concentration (1250 µg/mL) in the second XTT test (threshold of cytotoxicity: < 75%). The mean CV75 value of both XTT tests was calculated as 164.9 µg/mL.

The following concentrations of the test item were tested in the main experiments (h-CLAT):

55, 66, 80, 95, 115, 138, 165 and 198 µg/mL

The test item with a log Pow of 2.14 was tested in 2 independent runs. The RFI of CD86 and CD54 was equal or greater than 150% and 200%, respectively, in at least one concentration of the first run, and the RFI of CD86 was equal or greater than 150% in at least one concentration of the second run. Therefore the h-CLAT prediction is considered positive for the test item in this h-CLAT.

In the DMSO control, RFI values compared to the medium control of both CD54 and CD86 did not exceed the positive criteria (CD54 ≥ 200% and CD86 ≥ 150%).The RFI values of the positive controls (DNCB) for CD54 and CD86 exceeded the positive criteria(CD54 ≥ 200% and CD86 ≥ 150%)and the cell viability was >50%, with one exception. The CD54 RFI value of the positive control (2.0 µg/mL DNCB) in the first h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this one exception is considered to be acceptable since the CD54 RFI value of the positive control (3.0 µg/mL DNCB) in the first h-CLAT run exceeded the positive criteria.

In conclusion, the test substance with a log Pow of 2.14 activated THP-1 cells under the test conditions of this study. Therefore the substance is considered positive for the third key event of the skin sensitisation Adverse Outcome Pathway (AOP).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

Several skin sensitisation tests to OECD test guidlines and acceptable quality standards show that the test substance is a skin sensitiser under the conditions of the tests.

Justification for selection of skin sensitisation endpoint:
GLP compliant guideline studies with a klimisch score of 1

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

In several GLP compliant, guideline skin sensitisation tests based on in-chemico/in-vitro and in-vivo procedures 6 -methyl-3,4 -dihydro-2H-1,4 -benzoxazine was considered to be a skin sensitiser under the conditions of the tests. Based on the EC3 value of 3.04% (w/w) derived in the LLNA the substance is classified Category 1B (indication of skin sensitisation potential) based on GHS criteria.