3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane

Endpoint summary

Description of key information

Assessment of the skin sensitising potential of 3 methyl-3-[(2,2,3,3,3-pentafluoropropoxy)methyl]oxetane.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Type of information:

(Q)SAR

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

accepted calculation method

Justification for type of information:

According to Annex VII (Regulation (EC) No 1907/2006), the information needed for the classification or risk assessment of a substance should obtained through non-animal methods as a first step. Due to the complexity of the skin sensitisation endpoint, a combination of alternative test methods (e.g. in silico, in chemico and in vitro) in a weight of evidence approach needs to be considered to increase confidence in the final assessment of skin sensitisation (ECHA Guidance, Chapter R.7a, 2017). The in silico assessment below was performed with five computational tools: TOPKAT, CAESAR of Vega, Derek, Toxtree and OECD QSAR Toolbox.

Qualifier:

no guideline followed

Principles of method if other than guideline:

The prediction of the 3-methyl-3-[(2,2,3,3,3-pentafluoropropoxy)methyl]oxetane (CAS: 449177-94-0) skin sensitisation potential was performed with QSAR models TOPKAT 4.5 and CAESAR (VEGA 1.1.4), with expert system Derek 6.0.1 and Toxtree 2.6.13. The assessment with OECD QSAR Toolbox v4.1 was based on the profiler and read-across data.

GLP compliance:

not specified

Remarks:

Not required since using a calculation method to predict sensitisation.

Key result

Run / experiment:

other: QSAR

Parameter:

other:

Vehicle controls validity:

not applicable

Negative controls validity:

not applicable

Positive controls validity:

not applicable

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

TOPKAT predicted the query compound to be non-sensitizer; however, the prediction was low reliable due to the following uncertainty of the prediction: low structural similarity of the four structural analogues and moderate concordance of analogues with the predicted value. In addition, the Bayesian score was close to the best split value and probability of 0.60 indicated that likelihood of positive response in an experimental assay was within indeterminate range (0.30 - 0.70) according to the TOPKAT evaluation criteria.

CAESAR of Vega predicted the query compound to be not sensitizer with low reliability and indicated that the query structure is outside applicability domain of the model (global ADI: 0). In addition, one unknown fragment and two infrequent fragments of the query structure have been found in the compounds of the training set.

Derek predicted the query compound to not be a sensitizer in mammals. The query structure does not match any structural alerts or examples for skin sensitisation in Derek. Additionally, the query structure does not contain any unclassified or misclassified features and is consequently predicted to be a non-sensitizer.

Toxtree identified no alert in respect to skin sensitization reactivity domains for 3-methyl-3-[(2,2,3,3,3-pentafluoropropoxy)methyl]oxetane.

No alerts were triggered for 3-methyl-3-[(2,2,3,3,3-pentafluoropropoxy)methyl]oxetane in the protein binding alerts for skin sensitization by OASIS and according to GHS v1.4 in OECD QSAR Toolbox profiler.

No metabolites or transformation products were generated for the query compound with the autoxidation, skin metabolism and neutral hydrolysis simulators of OECD QSAR Toolbox. Therefore, the presence of pre- and pro-haptens may be excluded.

The read-across analysis from the nearest 5 neighbours was negative. The prediction was based on 5 values, 3 of them (60.0%) equal to predicted value. Prediction confidence was measured by the p-value: 0.5. The structural similarity of the nears neighbours ranged from 30 to 50%.

Table 1: Outcome of Model Predictions

Model	Prediction result	Reliability (model statistics)
TOPKAT	neg	Low (probability: 0.60)
CAESAR (VEGA)	neg	Low (global AD index: 0)
DEREK	no alert	Reliable
Toxtree	no alert	Restricted on 5 skin protein binding principles
OECD QSAR Toolbox
Protein binding alerts for skin sensitization by OASIS	no alert	Restricted on 11 mechanistic domains
Protein binding alerts for skin sensitization according to GHS	no alert	The absence of a protein binding alert should not be taken as an absence of toxicity
Autoxidation simulator:	no metabolites/transform. products
Skin metabolism simulator:	no metabolites/transform. products
Hydrolysis simulator (neutral):	no metabolites/transform. products
Read-across (Skin sensitisation II, ECETOC)	neg	Based on 5 values, 3 of them (60.0%) equal to predicted value; confidence by p-value 0.5

Interpretation of results:

GHS criteria not met

Conclusions:

Taking into account that all predictions showed negative skin sensitization results for the query compound and no pre- or pro-haptens were generated with the autoxidation, skin metabolism and neutral hydrolysis simulators in the OECD QSAR Toolbox, it can be concluded that 3 methyl-3-[(2,2,3,3,3-pentafluoropropoxy)methyl]oxetane is likely not a skin sensitizer.

Executive summary:

According to Annex VII (Regulation (EC) No 1907/2006), the information needed for the classification or risk assessment of a substance should obtained through non-animal methods as a first step. Due to the complexity of the skin sensitisation endpoint, a combination of alternative test methods (e.g.in silico,in chemico andi n vitro) in a weight of evidence approach needs to be considered to increase confidence in the final assessment of skin sensitisation (ECHA Guidance, Chapter R.7a, 2017).

The present in silico assessment was performed with five computational tools: TOPKAT, CAESAR of Vega, Derek, Toxtree and OECD QSAR Toolbox.

Assessment of the data obtained by the various models by in silico assessment suggested that 3-methyl-3-[(2,2,3,3,3-pentafluoropropoxy)methyl] oxetane is likely not a skin sensitizer.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 November 2018 to 13 December 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

Details on the study design:

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.

Cell culture used: KeratinoSens™ with a stable insertion of the luciferase construct supplied by Givaudan (Dubendorf, Switzerland). The cells were 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).

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. 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.

Preparation of test solution: A stock solution of the test item, 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane, was prepared by weighing the test item into a tared glass container and diluting to 40 mg/mL in DMSO.

Preparation of solvent plates: To a 96 well plate was added 100 µL of DMSO except to column 12 and well H11 in test 1 and well B11 in test 2. 200 µL of the stock solution of the test item, 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane, 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.

To well H11 in test 1 and well B11 in test 2 was added 200 µL of the 6.4 mM stock solution of cinnamic aldehyde and serially diluted from column 11 to column 7.

The 100x solvent plate was replicated in a fresh 96 well plate by the addition of 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: 24 hours after the cell cultures were established. the medium was removed from the wells and replaced with 150 µL of assay medium. 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 in the first test. 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 PBS. In subsequent tests a 5 mg/mL MTT in PBS solution was used. After incubation, the transparent plate was removed from the incubator, the cell culture medium removed and 100 µL of 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 and 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 the second repeat of test 2. Frozen reconstituted reagent was used for test 1, test 2 and the repeat of test 2, and was thawed to room temperature before use.
After incubation the medium was removed from the wells of the triplicate white plates and 100 µL of fresh assay medium was added to each well before the addition of 100 µL of Steady-Glo® luciferase reagent to each well. 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.


Two independent tests each containing three replicates (i.e. n=6) were required to make a conclusion. Each independent test was performed on a different day with fresh stock solutions of the chemicals and independently harvested cells.

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 10.93 μM and 13.96 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 6.65 and 4.63 for test 1 and 2, respectively, which met the acceptance criterion of between 2 and 8.

The positive control results therefore meet the test acceptance criteria required.

Key result

Run / experiment:

other: Test 1

Parameter:

other: I max

Value:

6.65

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Run / experiment:

other: Test 2

Parameter:

other: I max

Value:

1.24

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Other effects / acceptance of results:

No apparent overall dose response for luciferase induction was noted in either test. The Imax for 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane was 1.38 in test 1 and 1.24 in test 2. The Imax for both tests was <1.5 fold compared to the DMSO control and therefore the EC1.5 could not be calculated. The cellular viability did not fall below 89.75% in test 1 and 90.97% in test 2 and therefore the IC30 and IC50 could not be calculated. The Keratinosen prediction was therefore assigned as negative.

- Acceptance criteria met for negative control: YES
The average coefficient of variation of the luminescence reading for the negative solvent control (DMSO) was 12.6% and 14.7% for test 1 and 2, respectively, which met the acceptance criterion of below 20%.

- Acceptance criteria met for positive control: YES
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 EC values of the positive control, cinnamic aldehyde, were 10.93 μM and 13.96 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 6.65 and 4.63 for test 1 and 2, respectively, which met the acceptance criterion of between 2 and 8.

Table 1            Results for 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane – Test 1

Test item conc. (µg/mL)	0.20	0.39	0.78	1.56	3.13	6.25	12.5	25	50	100	200	400
Mean fold induction	0.98	1.04	1.03	1.05	1.07	1.07	1.11	1.31	1.30	1.31	1.36	1.38
Statistically significant	No	No	No	No	No	No	No	No	No	No	No	No
Viability (%)	89.75	93.89	107.75	108.87	106.16	105.76	121.45	109.58	107.11	115.08	119.14	117.55
Imax	1.38
EC1.5(µg/mL)	N/A
IC30(µg/mL)	N/A
IC50(µg/mL)	N/A

 

 

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax>1.5 fold and statistically significant	No
Is the cellular viability >70% at the lowest concentration at the EC1.5 determining concentration	N/A
Is the EC1.5 value <1000µM	N/A
Is there an apparent overall dose-response for luciferase induction	No
KeratinoSens™ prediction	Negative

 

 Table 2            Results for Cinnamic Aldehyde – Test 1

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.41	1.38	1.71	2.54	6.65
Statistically significant	No	No	Yes	Yes	Yes
Viability (%)	119.38	121.85	118.98	114.28	102.89
Imax	6.65
EC1.5(µM)	10.93
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 (6.65)	Pass
EC1.5of positive control within two standard deviations of the historical mean (-6.97 to 43.47)	Yes (10.93)	Pass
CV% of blank values < 20%	Yes (12.6%)	Pass

 

 

Table 3            Results for 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane – Test 2

Test item conc. (µg/mL)	0.20	0.39	0.78	1.56	3.13	6.25	12.5	25	50	100	200	400
Mean fold induction	0.88	0.84	0.93	0.89	0.89	0.88	1.07	0.95	1.09	1.19	1.18	1.24
Statistically significant	No	No	No	No	No	No	No	No	No	No	No	No
Viability (%)	106.72	90.97	102.99	110.55	114.07	115.52	102.78	116.25	120.81	121.74	116.87	114.07
Imax	1.24
EC1.5(µg/mL)	N/A
IC30(µg/mL)	N/A
IC50(µg/mL)	N/A

 

Determination criteria for the skin sensitisation potential of the test item	Result
Is the Imax>1.5 fold and statistically significant	No
Is the cellular viability >70% at the lowest concentration at the EC1.5 determining concentration	N/A
Is the EC1.5 value <1000µM	N/A
Is there an apparent overall dose-response for luciferase induction	No
KeratinoSens™ prediction	Negative

 

 Table 4            Results for Cinnamic Aldehyde – Test 2

Positive control conc. (µM)	4	8	16	32	64
Mean fold induction	1.18	1.24	1.59	2.33	4.63
Statistically significant	No	No	Yes	Yes	Yes
Viability (%)	100.71	108.58	116.15	120.91	117.18
Imax	4.63
EC1.5(µM)	13.96
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 (4.63)	Pass
EC1.5 of positive control within two standard deviations of the historical mean (-6.97 to 43.47)	Yes (13.96)	Pass
CV% of blank values < 20%	Yes (14.7%)	Pass

 

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

It was concluded that 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane is not a skin sensitizer.

Executive summary:

The purpose of this study was to determine if the test item, 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane, is likely to be a skin sensitizer using the ARE-Nrf2 Luciferase Test (KeratinoSens™).

The I_max for 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane was 1.38 in test 1 and 1.24 in test 2. The I_max for both tests was <1.5 fold compared to the DMSO control and therefore the EC_1.5 could not be calculated. The cellular viability did not fall below 89.75% in test 1 and 90.97% in test 2 and therefore the IC₃₀ and IC₅₀ could not be calculated. Graphs for 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetaneshowed no overall dose-response for luciferase induction.

All acceptance criteria for the positive control and negative control were met.

It was concluded that 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane gave a negative response in the ARE-Nrf2 Luciferase Test (KeratinoSens™), supporting the prediction that 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane is not a skin sensitizer.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

06 November 2018 to 13 November 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)

Details on the study design:

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). 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.

The positive control chemical (Cinnamic Aldehyde) was prepared at a concentration of 100 mM in acetonitrile. A 100 mM stock solution of 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane was also prepared in acetonitrile.

Stability controls (Reference Control B), precision controls of both peptides were prepared at a concentration of 0.5 mM in acetonitrile/buffer.

Triplicate solutions each of the positive control and 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane stock solutions were diluted with the Cysteine peptide stock solution so as to prepare solutions containing 0.5 mM Cysteine and 5 mM of Cinnamic Aldehyde or 5 mM 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane. For the co-elution control, buffer solution was used in place of the Cysteine stock solution.

In a similar manner, triplicate solutions of each of the positive control and 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane stock solution were diluted with the Lysine peptide stock solution so as to prepare solutions containing 0.5 mM Lysine and 25 mM of Cinnamic Aldehyde or 25mM 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane. For the co-elution control, buffer solution was used in place of the Lysine stock solution.

The appearance of the 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane and positive control samples in the HPLC vials was documented following 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 initiation of the analysis run. Prior to initiation of the run the appearance of the samples in the vials was assessed and documented again.

The concentration of both the Cysteine and Lysine peptides in the presence of 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane and the associated positive controls was quantified by HPLC using UV detection.

The % peptide depletion was determined for each sample by the following equation;

% Peptide depletion = 100 - Peptide peak area in replicate depletion samples (x 100) / Mean Peptide peak area of reference control samples B

Run / experiment:

other: Cysteine

Parameter:

other: Mean peptide depletion (%)

Value:

9.98

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Lysine

Parameter:

other: Mean peptide depletion (%)

Value:

3.13

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

All analytical acceptance criteria were met and are tabulated below in Table 1. The depletion of peptide in the presence of 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane was 9.98% and 3.13% for cysteine and lysine respectively. Applying the following reactivity prediction depletion model (see Table 3 below), reactivity of 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane is classed as “Low” and the DPRA prediction is therefore positive. 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane is therefore predicted to be a potential skin sensitizer.

Table 1: List of acceptance criteria

		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, 0.38%, n=3)		B: 0.499 mM (CV 0.61%, n=6)		SD 0.46% (n=3)
		Lysine		r^2>0.999		59.4 (SD, 5.72%, n=3)		B: 0.510 mM (CV 0.99%, n=6)		SD 3.86% (n=3)

CV         Coefficient of Variation

SD         Standard deviation

 

Table 2: Table to show the depletion of peptide in the presence of 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane

		Mean peak area of peptide with test item(µV.sec)	Mean peptide depletion by 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane (%)
Cysteine	Control B: 791780 (n=6)	712790 (n=3)	9.98
Lysine	Control B: 779330 (n=6)	754950 (n=3)	3.13

Table 3         Individual Cysteine Peptide Depletion Values

Sample	Peak area (µV.sec)	Peptide concentration^1(µg/mL)	Peptide Depletion^2(%)	Mean Depletion (%)	SD  (%)
Positive control	212391	101	73.2	72.9	0.38
	218142	104	72.4
	213870	101	73.0
3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane	713744	338	9.86	9.98	0.46
	715821	339	9.59
	708792	336	10.5

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 791780 µV.sec(n=6)

Table 4          Individual Lysine Peptide Depletion Values

Sample	Peak area (µV.sec)	Peptide concentration^1(µg/mL)	Peptide Depletion^2(%)	Mean Depletion (%)	SD  (%)
Positive control	289958	149	62.8	59.4	5.72
	291131	149	62.6
	367714	188	52.8
3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane	781468	397	-0.274	3.13	3.86
	722306	367	7.32
	761073	386	2.34

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 779330 µV.sec(n=6)

Applying the following reactivity prediction depletion model, reactivity of 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane is classed as “Low” and the DPRA prediction is therefore positive. 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane is therefore predicted to be a potential skin sensitizer. 

Table 5: Reactivity Prediction Depletion Model Criteria

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

There was no co-elution peaks in either the Cysteine or Lysine assay.   

Interpretation of results:

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

Conclusions:

Solutions of 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane were successfully analyzed by the validated DPRA analytical method in both the Cysteine and Lysine containing synthetic peptides. With overall mean peptide depletion (reactivity) of 6.55% in the presence of the test item, 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane is therefore predicted by DPRA as positive and therefore 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 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane. 

Solutions of 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane were successfully analyzed by the validated DPRA analytical method in both the Cysteine and Lysine containing synthetic peptides. There was reactivity of both peptides in the presence of 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane. With an overall depletion of peptide of 6.55% the reactivity of 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane is classified as low and hence this test item is predicted by DPRA to be a potential skin sensitizer. 

 

Reference 4

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

07 November 2018 to 23 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD 442E; In Vitro Skin Sensitisation: In Vitro Skin Sensitisation Assays Annex I: In Vitro Skin Sensitisation: human Cell Line Activation Test (h-CLAT), June 2018.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

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

Details on the study design:

The following test concentrations were prepared by dilution of a 100 mg/mL solution of 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane in culture medium; 39.1, 78.1, 156, 313, 625, 1250, 2500 and 5000 µg/mL.

Cell Cultures: THP-1 cells (Human monocytic leukemia cell line) purchased from ATCC, #TIB-202 were used. Stocks of the THP-1 cell line were stored in liquid nitrogen in the cell bank of Envigo CRS GmbH (aliquots of cells in freezing medium at 1 x 10(+6) to 2 x 10(+6) cells/mL) allowing the repeated use of the same cell culture batch in experiments. Thawed stock cultures were propagated at 37 ± 1.5 °C in plastic flasks. The cell density should not exceed 1 x 10(+6) cells/mL. The passage numbers of the used THP-1 cells were 15 and 17 in the cytotoxicity tests and 20 and 21 in the h CLAT for runs 1 and 2, respectively.

Culture Medium: RPMI 1640 Medium, GlutaMAX™ Supplement including 25 mM HEPES, supplemented with 10 % FBS (v/v), 0.05 mM 2 mercaptoethanol, 4.5 g/L glucose, 1% (v/v) sodium pyruvate and appropriate antibiotics (100 U/mL of penicillin and 100 µg/mL of streptomycin) was used to culture the cells during the assay.

Preparation and Seeding of THP-1 Cells: For the cytotoxicity experiment, a volume of 500 µL with a cell density of 1.8 - 2 x 10(+6) THP-1 cells/mL was seeded in each well of a 24-well flat bottom plate.

For the main experiment (h-CLAT) 0.9 - 1 x 10(+6) cells/well in a volume of 500 µL were seeded in a 24-well plate before the treatment.

Cytotoxicity test (dose finding assay)
A volume (500 µL) of the dilutions of the test item and culture medium were added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. All dose groups were tested in one replicate for each cytotoxicity test. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.

Each concentration of the test item, culture medium and solvent control were prepared for the 7-AAD staining.

The cytotoxicity was analysed by flow cytometry using the software Cellquest Pro 6.0. The 7-AAD acquisition channel (FL-3) was set for the optimal detection of DNA-bound 7 AAD fluorescence signal.

The cell viability was measured by gating-out dead cells stained with 7-AAD. A total of 10,000 living cells were analysed. the cell viability (%) was determined as the number of living cells/number of acquired cells x 100.

The cytotoxicity test was considered to be acceptable if it met the following criteria:
-The cell viability of the medium and solvent control (if the test item is solved in DMSO) should be more than 90%.

Main Experiment (h-CLAT)
The mean of the two CV75 values was used to determine the dose-range for the main experiment (h-CLAT).

Eight final concentrations (µg/mL) were used for the test item in the main experiment (h-CLAT). The highest concentration used was 1.2 × mean CV75 and seven further concentrations of the test item were prepared by serial 1:1.2 dilution. The test item was tested in two independent runs. The tests were performed on different days. The test item was prepared separately for each run.

A volume (500 µL) of the dilutions of the test item, medium control, positive and DMSO control was added to the cells. The treated THP-1 cells were incubated for 24 ± 0.5 hours. At the end of the incubation period, the cell cultures were microscopically evaluated for morphological alterations.
Each concentration of the test item, medium control, positive and DMSO control were prepared in triplicates for the different staining (with FITC-labelled anti-CD86, CD54 antibody or mouse IgG1).

After staining with the antibodies, the cells were washed twice (2 -8 °C) with 1 mL FACS buffer and re-suspended in a final volume of 2 mL/tube FACS buffer. At least 10 minutes before the flow cytometry acquisition, 5 µL of a 7-AAD solution were added.

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.

Dead cells were gated-out by staining with 7-AAD. A total of 10,000 living cells were analysed. Mean fluorescence intensity (MFI) of viable cells and viability for each sample were recorded. 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).

The following acceptance criteria should be met when using the h-CLAT method:
• Cell viability of medium control and DMSO control should be more than 90%.
• 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.

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%. Except the CD54 RFI value of the positive control (2.0 µg/mL DNCB) in the first and second h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the CD54 RFI value of the positive control (3.0 µg/mL DNCB) in the first and second h-CLAT run exceeded the positive criteria. Full results are presented below.

Run / experiment:

other: Test 1 - CD54 Antibody

Parameter:

other: RFI (%)

Value:

75.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Test 1- CD86 Antibody

Parameter:

other: RFI (%)

Value:

28.1

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Run / experiment:

other: Test 2 - CD54 Antibody

Parameter:

other: %RFI

Value:

56.4

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Run / experiment:

other: Test 2 - CD86 Antibody

Parameter:

other: %RFI

Value:

176.7

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Other effects / acceptance of results:

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 2500 µg/mL up to the highest tested concentration (5000 µg/mL) in both tests (threshold of cytotoxicity: < 75%). The mean CV75 value for both Cytotoxicity Tests was determined to be 1729.1 µg/mL. The mean CV75 value was used to determine the concentrations to be used in the main test (h-CLAT). These were determined as 579, 695, 834, 1001, 1201, 1441, 1729 and 2075 µg/mL.

In two independent runs, the relative fluorescence intensity (RFI) was calculated as an indicator of CD86 and CD54 expression. In addition, the cell viability was determined for the control cells, CD54 and CD86 cells. Full results are tabulated below.

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%).

In the first run the RFI of CD86 was determined to be ≥ 150% at a concentration of 1001, 1201 and 1441µg/mL (with cell viability ≥ 50%). In the second run, the RFI of CD86 was determined to be ≥ 150% at a concentration of 579, 695, 834, 1001 and 1201µg/mL (with cell viability ≥ 50%). The CD54 RFI value of the positive control (2.0 µg/mL DNCB) did not fulfil the positive criteria (CD54 ≥ 200%) in both tests.

Since the RFI of CD86 was equal or greater than 150% in at least one concentration of both independent runs, the h-CLAT prediction is considered positive for skin sensitisation for this test item.

The following acceptance criteria also was met when using the h-CLAT method and therefore the results are considered valid:
•The cell viability of the medium control was reported to be 96.21% and 96.69% in the two tests. The cell viability of the solvent control was determined to be 95.75% and 97.35% in the two tests. Since the cell viability of the medium control and DMSO control was more than 90% then the acceptance criteria has been met.
•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%. Except the CD54 RFI value of the positive control (2.0 µg/mL DNCB) in the first and second h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the CD54 RFI value of the positive control (3.0 µg/mL DNCB) in the first and second h-CLAT run exceeded the positive criteria.
•For the test chemical, the cell viability should be more than 50% in at least four tested concentrations in each run.

Table 1 Results of the first Cytotoxicity Test for the Test Item 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane

Test Group	Concentration [µg/mL]	Microscopic Evaluation / Cytotoxicity	Flow Cytometric Evaluation / Cell Viability [%]
Medium Control	-	no	97.71
Test Item	39.1	no	98.04
	78.1	no	98.07
	156	no	97.97
	313	no	98.11
	625	no	97.48
	1250	no	97.05
	2500^1	no	65.23
	5000^1	yes	2.80

¹ cytotoxic effects occurred by the Flow Cytometric Evaluation (< 75% cell viability)

The CV75 value of the first Cytotoxicity Test: 2020.75 µg/mL.

Table 2 Results of the second Cytotoxicity Test for the Test Item 3-methyl-3- (2,2,3,3,3-pentafluoropropoxy)methyl oxetane

Test Group	Concentration [µg/mL]	Microscopic Evaluation / Cytotoxicity	Flow Cytometric Evaluation / Cell Viability [%]
Medium Control	-	no	97.30
Test Item	39.1	no	97.98
	78.1	no	97.70
	156	no	97.01
	313	no	97.11
	625	no	95.36
	1250	no	93.49
	2500^1	no	1.72
	5000^1	yes	0.54

¹ cytotoxic effects occurred by the Flow Cytometric Evaluation (< 75% cell viability)

The CV75 value of the second Cytotoxicity Test: 1437.35 µg/mL. The mean CV75 value of both Cytotoxicity Tests:1729.1µg/mL.

Table 3 Results of the first h-CLAT run for the Test Item 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability ISO (%)
Medium Control	-	100.0	100.0	96.21
DMSO Control	-	100.0	100.0	95.76
Positive Control (DNCB)	2.0	139.8^#	187.7*	91.62
	3.0	310.7*	308.0*	85.44
Test Item	579	75.6	112.1	95.21
	695	94.5	110.2	95.27
	834	51.2	28.1	95.92
	1001	85.0	190.5*	92.01
	1201	11.8	150.4*	79.89
	1441	82.7	189.6*	62.83
	1729^1	-387.4	2521.6*	7.01
	2075^1	-1020.5	3845.5*	2.79

¹ cell viability below 50%, are excluded from the evaluation

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

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

Table 4 Results of the second h-CLAT run for the Test Item 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane

	Concentration (µg/mL)	RFI (%) CD54 Antibody	RFI (%) CD86 Antibody	Cell Viability ISO (%)
Medium Control	-	100.0	100.0	96.69
DMSO Control	-	100.0	100.0	97.35
Positive Control (DNCB)	2.0	89.2^#	255.2*	93.50
	3.0	305.4*	332.4*	90.26
Test Item	579	56.4	176.7*	94.34
	695	95.7	222.4*	89.93
	834	88.0	470.9*	67.79
	1001	177.8	439.1*	70.64
	1201	144.4	305.0*	63.05
	1441^1	653.0*	-140.4	18.72
	1729^1	735.9*	-224.1	2.65
	2075^1	553.8*	-262.9	2.80

¹ cell viability below 50%, are excluded from the evaluation

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

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

Interpretation of results:

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

Conclusions:

The test item 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane 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:

An 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 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyloxetane which formed a stable suspension/dispersion 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 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetanewas previously determined by two cytotoxicity tests.

 

Cytotoxic effects were observed following incubation with the test item starting with the concentration of 2500 µg/mL up to the highest tested concentration (5000 µg/mL) in both cytotoxicity tests (threshold of cytotoxicity: < 75%). The mean CV75 value of both cytotoxicity tests was calculated as 1729.1µg/mL.

 

The following concentrations of the test item were tested in the main experiments (h-CLAT): 579, 695, 834, 1001, 1201, 1441, 1729 and 2075 µg/mL

In 2 independent runs, the RFI of CD86 was equal or greater than 150% in at least one concentration. 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%. Except the CD54 RFI value of the positive control (2.0 µg/mL DNCB) in the first and second h-CLAT run did not exceed the positive criterion (CD54 ≥ 200%). However, this is considered to be acceptable since the CD54 RFI value of the positive control (3.0 µg/mL DNCB) in the first and second h-CLAT run exceeded the positive criteria.

 

This human cell line activation test is used in a weight of evidence approach for the assessment of the skin sensitisation potential of 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane. With the result of the h-CLAT providing a positive result for skin sensitisation then further tests are required.

Reference 5

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Phase: 20 February 2019 to 20 March 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)

Deviations:

yes

Remarks:

The relative humidity in the animal room was for a few hours between approximately 13 - 45 % instead of 45 - 65% as stated in the study plan. This deviation to the study plan is not considered to affect the validity of the study.

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: Female CBA/Ca (CBA/CaOlaHsd) strain mice were supplied by Envigo RMS B.V, Inc., Postbus 6174, 5960 AD Horst, The Netherlands.
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Pre-test: 11 to 12 weeks; Main Test: 9 to 10 weeks
- Weight at study initiation: Not Stated
- Housing: Caged by group in cages with granulated soft wood bedding.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: At least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 2°C
- Humidity (%): 45 to 65%
- Photoperiod (hrs dark / hrs light): 12 hours in light, 12 hours in darkness

Vehicle:

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

Concentration:

Control = vehicle only
Low dose = 25% v/v
Mid dose = 50% v/v
High dose = 100% v/v

No. of animals per dose:

Four per dose (Plus four for the control)

Details on study design:

PRELIMINARY TEST

The systemic toxicity and irritancy potential of the test item was assessed in a preliminary screening test performed using two mice at 50 or 100% v/v test substance respectively. No signs of systemic toxicity were observed; however, very slight erythema of the ear skin was observed in both animals. Additionally, the animal treated with 100% test item concentration showed scaly ears on day 6. No signs of excessive local skin erythema were present in both animals.

Based on this information the undiluted test item and the test item at concentrations of 50% and 25% v/v in acetone/olive oil 4:1 were selected for the main test.

MAIN TEST

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 25% v/v and 50% v/v, in acetone/olive oil (4+1 v/v), and 100% (undiluted). The application volume, 25 µL/ear/day, was spread over the entire dorsal surface (approximately 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 19.98 µCi of 3H-methyl thymidine (equivalent to 79.9 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 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 β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL aliquots of 5 % trichloroacetic acid. The β-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 micrometre.

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.

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).

Positive control substance(s):

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

Positive control results:

The postive control (run as a separate study) responded as expected and confirmed the responsiveness of the test system in the laboratory.

Key result

Parameter:

SI

Value:

0.93

Test group / Remarks:

25% v/v test substance

Key result

Parameter:

SI

Value:

0.76

Test group / Remarks:

50% v/v test substance

Key result

Parameter:

SI

Value:

0.66

Test group / Remarks:

100% v/v test substance

There were no deaths on the study.  No signs of systemic toxicity were observed during the study period. On day 3, animals treated at all test item concentrations showed a very slight erythema of the ear skin.

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

Interpretation of results:

GHS criteria not met

Conclusions:

The test item 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane was not a skin sensitiser under the test conditions of this study.

Executive summary:

Introduction

The study was performed to assess the skin sensitisation potential of the test substance in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was designed to meet the requirements of the following guidelines:

• OECD Guideline for the Testing of Chemicals No. 429 "Skin Sensitisation: Local Lymph Node Assay" (adopted 22 July 2010)
  
  
• Method B42 Skin Sensitisation (Local Lymph Node Assay) of Commission Regulation (EC) No. 440/2008 (updated 06 July 2012)

 

Method

Following preliminary screening test animals showed a very slight erythema of the ear skin. No clinical signs of toxicity were noted at a concentration of 100% and this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay.

Three groups, each of four animals, were treated with 50 μL (25 μL per ear) of the test item at 25% and 50%, in acetone/olive oil (4 +1 v/v), and 100% (undiluted). A further group of four animals was treated with acetone/olive oil 4:1 alone.

Results

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. On day 3, animals treated at all test item concentrations showed a very slight erythema of the ear skin.

The Stimulation Indices expressed as the mean radioactive incoporation for each treatment group divided by the mean radioactive incorporation of the vehicle control group are as follows:

Concentration (%v/v)	Stimulation Index	Result
25	0.93	Negative
50	0.76	Negative
100	0.66	Negative

 

 

Conclusion

The test item 3-methyl-3-(2,2,3,3,3-pentafluoropropoxy)methyl oxetane was not a skin sensitiser under the test conditions of this study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The following battery of QSAR and in vitro investigations were undertaken to assess the skin sensitising potential of 3 methyl-3-[(2,2,3,3,3-pentafluoropropoxy)methyl]oxetane:

Assessment Method	Result
QSAR assessment was performed with five computational tools: TOPKAT, CAESAR of Vega, Derek, Toxtree and OECD QSAR Toolbox	The assessed substance is unlikely to be a skin sensitiser (NEGATIVE)
h-CLAT	The test item is considered positive for skin sensitisation (POSITIVE)
ARE-Nrf2 Luciferase Test (Keratinosens)	The test item is considered negative for skin sensitisation (NEGATIVE)
Direct Peptide Reactivity Assay	Thetest item is considered positie for skin sensitsation (POSITIVE)

 

The results collected in the QSAR assessment and in vitro tests were not conclusive with regards to the skin sensitising potential of 3 methyl-3-[(2,2,3,3,3-pentafluoropropoxy)methyl]oxetane and for this reason a confirmatory in vivo skin sensitisation study (Local Lymph Node Assay, EU B.42 / OECD TG 429) was performed to confirm skin sensitisation status. This LLNA test gave a negative response with no indication of any dose response (SI values of 0.93, 0.76 and 0.66 for test concentrations of 25, 50 and 100% v/v respectively).

The results from the h-CLAT and Direct Peptide Assay in vitro tests may indicate that the substance can have effects in certain parts of the Adverse Outcome Pathway for skin sensitisation, but the lack of response in the  ARE-Nrf2 Luciferase and LLNA tests indicate that it cannot intiate the full sequence of events intiating skin sensitisation and on this basis 3 methyl-3-[(2,2,3,3,3-pentafluoropropoxy)methyl]oxetane is considered to not be a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The results from the h-CLAT and Direct Peptide Assay in vitro tests may indicate that the substance can have effects in certain parts of the Adverse Outcome Pathway for skin sensitisation, but the lack of response in the  ARE-Nrf2 Luciferase and LLNA tests indicate that it cannot intiate the full sequence of events intiating skin sensitisation and on this basis 3 methyl-3-[(2,2,3,3,3-pentafluoropropoxy)methyl]oxetane is considered to not be a skin sensitiser.