Fluoren-9-one

Endpoint summary

Description of key information

Two in vitro assays have ben performed to adress key elements of the AOP for skin sensitisation:

OECD 442 C: negative

OECD 442 D: negative

Both assays are negative and provide no evidence for skin sensitisation within the AOP framework.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in chemico

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-10-19 to 2017-10-27

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: Direct Peptide Reactivity Assay (DPRA) for Skin Sensitization Testing, DB-ALM Protocol n°154, January 12, 2013

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

other: (in chemico) reactivity against synthetic peptides with a thiol or amino group

Details on the study design:

The in chemico direct peptide reactivity assay (DPRA) enables detection of the sensitising potential of a test item by quantifying the reactivity of test chemicals towards synthetic peptides containing either lysine or cysteine.

Positive control results:

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.16%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

0.29

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: lysine run

Parameter:

other: mean peptide depletion [%]

Value:

0

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptance Criteria

The run meets the acceptance criteria if:
- the standard calibration curve has a r² > 0.99,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 60.8% and 100% for the cysteine peptide and the maximum standard deviation (SD) for the
positive control replicates is < 14.9%,
- the mean percent peptide depletion (PPD) value of the three replicates for the positive control is
between 40.2% and 69.0% for the lysine peptide and the maximum SD for the positive control
replicates is < 11.6%,
- the mean peptide concentration of the three reference controls A replicates is 0.50 ± 0.05 mM,
- the coefficient of variation (CV) of peptide peak areas for the six reference control B replicates and three reference control C replicates in acetonitrile is < 15.0%.

The results of the test item meet the acceptance criteria if:
- the maximum standard deviation (SD) for the test chemical replicates is < 14.9% for the cysteine
percent depletion (PPD),
- the maximum standard deviation (SD) for the test chemical replicates is < 11.6% for the lysine
percent depletion (PPD),
- the mean peptide concentration of the three reference controls C replicates in the appropriate solvent is 0.50 ± 0.05 mM.

Both peptide runs and the test item results met the acceptance criteria of the test.

Cysteine and Lysine Values of the Calibration Curve

Sample	Cysteine Peptide		Lysine Peptide
	Peak Area at 220 nm	Peptide Concentration [mM]	Peak Area at 220 nm	Peptide Concentration [mM]
STD1	3309.9390	0.5340	4455.4287	0.5340
STD2	1616.4630	0.2670	2248.6604	0.2670
STD3	764.3784	0.1335	1103.8511	0.1335
STD4	380.1269	0.0667	555.1791	0.0667
STD5	195.6536	0.0334	278.7628	0.0334
STD6	99.5359	0.0167	141.0930	0.0167
STD7	0.0000	0.0000	0.0000	0.0000

Depletion of the Cysteine Peptide

Cysteine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	809.6354	0.1341	74.93	75.35	0.40	0.53
	794.7175	0.1317	75.39
	783.8397	0.1299	75.72
Test Item	3335.1116	0.5412	0.00	0.29	0.50	173.21
	3201.0125	0.5196	0.87
	3236.5881	0.5253	0.00

Depletion of the Lysine Peptide

Lysine Peptide
Sample	Peak Area at 220 nm	Peptide Conc. [mM]	Peptide Depletion [%]	Mean Peptide Depletion [%]	SD of Peptide Depletion [%]	CV of Peptide Depletion [%]
Positive Control	1768.9691	0.2117	57.97	56.98	1.07	1.87
	1804.7944	0.2160	57.12
	1858.1759	0.2224	55.85
Test Item	4236.3945	0.5071	0.00	0.00	0.00	--
	4228.3511	0.5061	0.00
	4216.3423	0.5047	0.00

Prediction Model 1

Cysteine 1:10/ Lysine 1:50 Prediction Model ¹

Mean Cysteine andLysine PPD	Reactivity Class	DPRA Prediction²
0.00% ≤ PPD ≤ 6.38%	No or Minimal Reactivity	Negative
6.38% < PPD ≤ 22.62%	Low Reactivity	Positive
22.62% < PPD ≤ 42.47%	Moderate Reactivity
42.47% < PPD ≤ 100%	High Reactivity

1 The numbers refer to statistically generated threshold values and are not related to the precision of the measurement.

2 DPRA predictions should be considered in the framework of an IATA.

Prediction Model 2

Cysteine 1:10 Prediction Model

Cysteine PPD	ReactivityClass	DPRA Prediction²
0.00% ≤ PPD ≤ 13.89%	No or Minimal Reactivity	Negative
13.89% < PPD ≤ 23.09%	Low Reactivity	Positive
23.09% < PPD ≤ 98.24%	Moderate Reactivity
98.24% < PPD ≤ 100%	High Reactivity

Categorization of the Test Item

Prediction Model	Prediction Model 1 (Cysteine Peptide and Lysine Peptide / Ratio: 1:10 and 1:50)			Prediction Model 2 (Cysteine Peptide / Test Item Ratio: 1:10)
Test Substance	Mean Peptide Depletion [%]	Reactivity Category	Prediction	Mean Peptide Depletion [%]	Reactivity Category	Prediction
Test Item	0.14	Minimal Reactivity	no sensitiser	0.29	Minimal Reactivity	no sensitizer
Positive Control	66.16	High Reactivity	sensitizer	75.35	Moderate Reactivity	sensitizer

Interpretation of results:

other: Study cannot be used as stand-alone for conclusion on classification

Conclusions:

In this study under the given conditions the test item showed minimal reactivity towards both peptides. The test item can be considered as “non-sensitiser”.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

In the present study the test material, 9 -Fluorenone, was dissolved in acetonitrile, based on the results of the pre-experiments. Based on a molecular weight of 180 g/mol a 100 mM stock solution was prepared. The test item solutions were tested by incubating the samples with the peptides containing either cysteine or lysine for 24 ± 2 h at 25 ± 2.5 °C. Subsequently samples were analysed by HPLC.

For the 100 mM solutionof the test item no turbidity or precipitation was observed when diluted with the cysteine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Slight precipitation was observed for the samples of the positive control (including co-elution control). Samples were not centrifuged prior to the HPLC analysis.

For the 100 mM solution of the test item no turbidity or precipitation was observed when diluted with the lysine peptide solution. After the 24 h ± 2 h incubation period but prior to the HPLC analysis samples were inspected for precipitation, turbidity or phase separation. Phase separation (small droplets on the bottom of the vial) was observed for the samples of the positive control (excluding the co-elution control). Samples were not centrifuged prior to the HPLC analysis.

Since the acceptance criteria the depletion range of the positive control was fulfilled, the observed precipitations and phase separation were regarded as insignificant.

No co-elution of test item with the peptide peaks was observed. Sensitising potential of the test item was predicted from the mean peptide depletion of both analysed peptides (cysteine and lysine) by comparing the peptide concentration of the test item treated samples to the corresponding reference control C (RC C_acetonitrile).

The 100 mM stock solution of the test item showed minimal reactivity towards the synthetic peptides. The mean depletion of both peptides was ≤ 6.38% (0.14%). Based on the prediction model 1 the test item can be considered as non-sensitiser.

The 100 mM stock solution of the positive control (cinnamic aldehyde) showed high reactivity towards the synthetic peptides. The mean depletion of both peptides was 66.16%.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2017-12-15 to 2018-02-19

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

other: KeratinoSens™, EURL ECVAM DB-ALM Protocol No. 155, July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of keratinocytes

Details on the study design:

The in vitro KeratinoSens™ assay enables detection of the sensitising potential of a test item by
addressing the second molecular key event of the adverse outcome pathway (AOP), namely
activation of keratinocytes, by quantifying the luciferase activity in the transgenic cell line
KeratinoSens™. The luciferase activity, assessed by luminescence measurement, compared
to the respective solvent controls is used to support discrimination between skin sensitisers
and non-sensitisers.

Positive control results:

The luciferase activity induced by the positive control at a concentration of 64 µM was between 2 and 8 (4.26 (experiment 1); 3.17 (experiment 2).

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

1.44

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 31.25 µM

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

82.8

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 1

Parameter:

other: EC1.5 [µM]

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

1.75

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 250 µM

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

30.6

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: EC1.5 [µM]

Value:

1.75

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

not determinable

Other effects / acceptance of results:

Acceptance Criteria

The test meets acceptance criteria if:
- the luciferase activity induction of the positive control is statistically significant above the threshold of 1.5 (using a t-test) in at least one of the tested concentrations
- the average induction in the three technical replicates for the positive control at a concentration of
64 µM is between 2 and 8
- the EC1.5 value of the positive control is within two standard deviations of the historical mean
- the average coefficient of variation (CV; consisting of 6 wells) of the luminescence reading for the
negative (solvent) control DMSO is <20% in each repetition.

The controls fullfilled the validity criteria of the test.

Results of the Cytotoxicity Measurement

	Concentration [µM]	Cell Viability [%]
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	100	100	100	0.0
Positive Control	4.00	102.8	102.9	102.9	0.0
	8.00	113.1	103.3	108.2	7.0
	16.00	113.5	105.8	109.7	5.4
	32.00	125.3	108.1	116.7	12.1
	64.00	131.2	107.3	119.2	16.8
Test Item	0.98	113.7	95.0	104.3	13.2
	1.95	104.3	94.6	99.5	6.8
	3.91	95.0	94.0	94.5	0.7
	7.81	98.7	98.9	98.8	0.1
	15.63	88.0	93.1	90.6	3.7
	31.25	82.8	90.9	86.9	5.7
	62.50	73.2	94.4	83.8	15.0
	125.00	58.2	75.2	66.7	12.0
	250.00	0.0	30.6	15.3	21.6
	500.00	0.2	0.0	0.1	0.1
	1000.00	0.4	0.5	0.5	0.1
	2000.00	0.5	1.3	0.9	0.6

Induction of Luciferase Activity Experiment 1

Experiment 1	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.13	1.14	1.12	1.13	0.01
	8.00	1.20	1.12	1.07	1.13	0.07
	16.00	1.33	1.61	1.23	1.39	0.19
	32.00	2.10	1.76	1.72	1.86	0.21	*
	64.00	4.82	4.21	3.76	4.26	0.53	*
Test Item	0.98	1.10	1.33	1.66	1.36	0.28
	1.95	1.21	1.63	1.44	1.43	0.21
	3.91	1.21	1.53	1.29	1.34	0.16
	7.81	1.26	1.21	1.18	1.22	0.04
	15.63	1.37	1.39	1.28	1.35	0.06
	31.25	1.57	1.22	1.55	1.44	0.20
	62.50	1.14	1.29	1.23	1.22	0.07
	125.00	1.54	1.46	1.33	1.44	0.11
	250.00	0.00	0.85	0.00	0.29	0.49
	500.00	0.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.00	0.00	0.00	0.00

 * = significant induction according to Student’s t-test, p < 0.05

Induction of Luciferase Activity Experiment 2

Experiment 2	Concentration [µM]	Fold Induction					Significance
		Rep. 1	Rep. 2	Rep. 3	Mean	SD
Solvent Control	-	1.00	1.00	1.00	1.00	0.00
Positive Control	4.00	1.16	1.21	1.35	1.24	0.10
	8.00	1.09	1.24	1.16	1.17	0.08
	16.00	1.57	1.62	1.59	1.59	0.02	*
	32.00	1.91	2.23	2.04	2.06	0.16	*
	64.00	2.89	3.14	3.48	3.17	0.30	*
Test Item	0.98	1.21	1.22	1.40	1.28	0.11
	1.95	0.94	1.21	1.10	1.08	0.14
	3.91	1.12	1.07	1.14	1.11	0.04
	7.81	1.02	1.11	1.16	1.10	0.07
	15.63	1.84	1.11	1.02	1.33	0.45
	31.25	1.19	1.17	1.20	1.19	0.02
	62.50	1.24	1.26	1.19	1.23	0.04
	125.00	1.27	1.51	1.26	1.35	0.14
	250.00	2.12	1.66	1.47	1.75	0.33	*
	500.00	0.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00	0.00
	2000.00	0.02	0.00	0.00	0.01	0.01

* = significant induction according to Student’s t-test, p < 0.05

Induction of Luciferase Activity – Overall Induction

Overall Induction	Concentration [µM]	Fold Induction				Significance
		Experiment 1	Experiment 2	Mean	SD
Solvent Control	-	1.00	1.00	1.00	0.00
Positive Control	4.00	1.13	1.24	1.19	0.08
	8.00	1.13	1.17	1.15	0.03
	16.00	1.39	1.59	1.49	0.14
	32.00	1.86	2.06	1.96	0.14	*
	64.00	4.26	3.17	3.71	0.77	*
Test Item	0.98	1.36	1.28	1.32	0.06
	1.95	1.43	1.08	1.26	0.24
	3.91	1.34	1.11	1.23	0.16
	7.81	1.22	1.10	1.16	0.08
	15.63	1.35	1.33	1.34	0.02
	31.25	1.44	1.19	1.31	0.18
	62.50	1.22	1.23	1.22	0.01
	125.00	1.44	1.35	1.39	0.07
	250.00	0.29	1.75	1.02	1.04
	500.00	0.00	0.00	0.00	0.00
	1000.00	0.00	0.00	0.00	0.00
	2000.00	0.00	0.01	0.00	0.01

 * = significant induction according to Student’s t-test, p < 0.05

Additional Parameters

Parameter	Experiment 1	Experiment 2	Mean	SD
EC1.5[µM]	n/a	172.70	172.70	-
Imax	1.44	1.75	1.60	0.22
IC30[µM]	75.70	139.56	107.63	45.15
IC50[µM]	142.57	195.67	169.12	37.54

Acceptance Criteria

Criterion	Range	Experiment 1	pass/fail	Experiment 2	pass/fail
CV Solvent Control	< 20%	7.8	pass	9.0	pass
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.0	pass	3.0	pass
EC1.5 PC	7 < x < 34 µM	19.77	pass	14.27	pass
Induction PC at 64 µM	2.00 < x < 8.00	4.26	pass	3.17	pass

Historical Data

Acceptance Criterion	Range	Mean	SD	N
CV Solvent Control	< 20%	11.3	3.3	41
No. of positive control concentration steps with significant luciferase activity induction >1.5	≥ 1	2.3	0.6	41
EC1.5 PC	7 < x < 34 µM	20.4	6.7	41
Induction PC at 64 µM	2.00 < x < 8.00	3.3	1.1	41

 

Interpretation of results:

other: Study cannot be used as stand-alone for conclusion on classification

Conclusions:

In this study under the given conditions the test item did not induce the luciferase activity in the transgenic KeratinoSens™ cell line in at least two independent experiment runs. Therefore, the test item can be considered as non sensitiser.
The data generated with this method may be not sufficient to conclude on the absence of skin sensitisation potential of chemicals and should be considered in the context of integrated approach such as IATA.

Executive summary:

In the present study 9 -Fluorenone was dissolved in DMSO.

Based on a molecular weight of 180 g/mol a stock solution of 200 mM was prepared.

Based on the stock solution a set of twelve master solutions in 100% solvent was prepared by serial dilution using aconstant dilution factor of 1:2. These master solutions were diluted 1:100 in cell culture medium and a stable suspension was formed. The following concentration range was tested in the assay:

2000, 1000, 500, 250, 125, 62.5, 31.25, 15.63, 7.81, 3.91, 1.95, 0.98 µM

Cells were incubated with the test item for 48 h at 37°C. After exposure cells were lysed and luciferase activity was assessed by luminescence measurement.

In the first experiment, no significant luciferase induction >1.5 was found in the tested concentration range.Therefore, no EC_1.5 value could be calculated.

In the secondexperiment, a max luciferase activity (I_max) induction of 1.75 was determined at a test item concentration of 250 µM, but the corresponding cell viability was 30.6%. No further significant luciferase induction >1.5 was found in the tested concentration range. The calculated EC_1.5 was < 1000 µM (172.70).

No dose response for luciferase activity induction was observed for each individual run as well as for an overall luciferase activity induction.

Under the condition of this study the test item is therefore considered as non sensitiser.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the provided information there is no need for classification according to the EU Regulation (EC) No 1272/2008 on Classification,Lab elling and Packaging of Substances and Mixtures, as amended for the 10th time in Regulation (EU) No 2017/776.