4-Ethyl-2’-fluoro-4’-bromobiphenyl

Endpoint summary

Description of key information

OECD 442 C: no peptide depletion, negative

OECD 442 D: positive

OECD 442 E: no upregulation of expression of the cell surface markers, but inconclusive due to logP > 3.5

OECD 429: negative

The conflicting results observed in the OECD Guideline study 442 C and 442 D assay and the inconclucive results obtained in the OECD 442 E assay show that the in vitro battery was not be suitable for the assessment for skin sensitisation this compound. Due to these technical problems, for this endpoint no firm conclusion can be drawn, therefore, an in vivo LLNA was performed. In this GLP compliant OECD 429 assay, the test item was not a skin sensitiser.

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:

Jun 03 -Sep 17, 2019

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))

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:

Freie und Hansestadt Hamburg

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 65.69%.

Key result

Run / experiment:

other: cysteine run

Parameter:

other: mean peptide depletion [%]

Value:

0.82

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:

0.69

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Acceptance Criteria

The following criteria must be met for a run to be considered valid:
a) The standard calibration curve should have an r2 > 0.99.
b) The mean percent peptide depletion value of the three replicates for the positive control cinnamic aldehyde should be between 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide and the maximum standard deviation (SD) for the positive control replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
c) The mean peptide concentration of reference controls A should be 0.50 ± 0.05 mM and the coefficient of variation (CV) of peptide peak areas for the nine reference controls B and C in acetonitrile should be <15.0%.
If one or more of these criteria is not met, the run would have been repeated.

The following criteria must be met for a test item’s results to be considered valid:
a) The maximum standard deviation for the test item replicates should be < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion.
b) The mean peptide concentration of the three reference controls C in the appropriate solvent should be 0.50 ± 0.05 mM.
If these criteria were not met, the data would have been rejected and the run have been repeated for that specific test item.

Peptide Depletion in %

1) Cystein Depletion

Sample	Peak Area	Actual Peptide [mM]	Peak Are Ref Control C	Peptide Depletion [%]	Corr. peptide Depletion [%]
1	47.5989	0.475	47.8543	0.28	0.28
2	48.2216	0.482	48.4220	-1.02	0.00
3	46.6954	0.466	46.9235	2.17	2.17
Mean	47.5050	0.474	47.7333	0.48	0.82
Evaluation Reactivity Class. Negative

1) Lysine Depletion

Sample	Peak Area	Actual Peptide [mM]	Peak Are Ref Control C	Peptide Depletion [%]	Corr. peptide Depletion [%]
1	37.3825	0.477	37.3643	2.08	2.08
2	38.7725	0.496	38.4179	-1.56	0.00
3	40.5898	0.519	37.7442	-6.32	0.00
Mean	38.915	0.497	38.1755	-1.94	0.69
Evaluation Reactivity Class. Negative

Interpretation of results:

other: The data generated with this test should be considered in the context of integrated approached such as IATA.

Conclusions:

The test item revealed a mean cysteine and lysine peptide depletion of 0.76% and, hence, the test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Executive summary:

The purpose of this study was to determine the sensitising potential of the test item in a Direct Peptide Reactivity Assay (DPRA). The study was performed according to OECD guideline 442C. The DPRA is an in chemico method which quantifies the remaining concentration of cysteine- or lysine-containing peptide following 24 ± 2 hours incubation with the test item at 25 ± 2.5°C. Relative peptide concentration is measured by high-performance liquid chromatography (HPLC) with gradient elution and UV detection at 220 nm. Cysteine and lysine peptide percent depletion values are then calculated and used in a prediction model, which allows assigning the test item to one of four reactivity classes used to support the discrimination between sensitisers and non-sensitisers.

The test item was dissolved at a concentration of 100 mM in acetonitrile.

Two reference controls containing only 0.5 mM cysteine peptide solution or 0.5 mM lysine peptide solution and acetonitrile were also included in the HPLC run sequence and were used to verify the HPLC system suitability prior to analysis (reference controls A) and the stability of the reference controls over time (reference control B). To verify that the solvent used to dissolve the test item does not impact the percent peptide depletion the reference control C was prepared by adding acetonitrile (vehicle) to the peptide solution. The reference control C was used to calculate the percent peptide depletion for the test item. Each sample was tested in triplicate.

Test item-treated samples revealed a cysteine peptide depletion of 0.82% and a lysine peptide depletion of 0.69% (mean peptide depletion of 0.76%) and, hence, were well below 6.38%. The test item is considered negative and predicted to be a non sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Cinnamic aldehyde was used as positive control at a concentration of 100 mM in acetonitrile. Treatment with the positive control item revealed a cysteine and lysine peptide depletion of 69.35% for cysteine and 65.40% for lysine peptide. These values are within the required range of 60.8% and 100% for the cysteine peptide and between 40.2% and 69.0% for the lysine peptide. The maximum standard deviation (SD) for the positive control replicates were < 14.9% for the percent cysteine depletion and < 11.6% for the percent lysine depletion. Therefore, the study can be regarded as valid.

The acceptance criteria of validity were fulfilled in this test.

The test item revealed a mean cysteine and lysine peptide depletion of 0.76% and, hence, the test item is considered negative and predicted to be a non-sensitiser (no or minimal reactivity) in the Direct Peptide Reactivity Assay (DPRA).

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

03 Jun - 26 Sep, 2019

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)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Remarks:

Freie und Hansestadt Hamburg, 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 (2.12 (experiment 1); 2.90 (experiment 2) .

Key result

Run / experiment:

other: 1

Parameter:

other: luciferase activity

Value:

5.47

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 1

Parameter:

other: cell viability [%]

Value:

50.91

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Luciferase determination run 1: EC 1.5

Parameter:

other: EC1.5 [µM]

Value:

2.77

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: 2

Parameter:

other: luciferase activity

Value:

4.97

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

positive indication of skin sensitisation

Key result

Run / experiment:

other: 2

Parameter:

other: cell viability [%]

Value:

134.17

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Key result

Run / experiment:

other: Luciferase determination run 2: EC 1.5

Parameter:

other: EC1.5 [µM]

Value:

0.98

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

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.

Numerical results for the test item

		Luciferase determinations		Cytotoxicity determinations
	Parameter	Imax	EC1.5[µM]	IC50[µM]	IC30[µM]
Test item	Repetition 1	5.47*	2.77	32.13	25.83
	Repetition 2	4.97*	<0.98	92.96	76.53
	Average	5.22 ± 0.36	-	54.65 ± 43.01	44.46 ± 35.85

*= statistically significantly different as compared to the negative control

Numerical results for the positive control (cinnamic aldehyde)

Positive control: Induction values Reference							Criteria#
Cinnamic aldehyde	4 µM	8 µM	16 µM	32 µM	64 µM	EC1.5	Induction 64 µM	EC1.5
Repetition 1	1.21	1.29	1.48	1.62*	2.12*	18.14	TRUE	TRUE
Repetition 2	1.15	1.30	1.35	1.98*	2.90*	19.84	TRUE	TRUE
Average	1.18	1.29	1.41	1.80	2.51	18.97	TRUE	TRUE

*= statistically significantly different compared to the negative control

# =the induction in the two replicates 64 µM should be between 2 and 8, the EC_1.5value should be between 7 µM and 30 µM.

Interpretation of results:

other: The data generated with this test should be considered in the context of integrated approached such as IATA.

Conclusions:

In this study under the given conditions the test item did 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 sensitiser.
The data generated with this test should be considered in the context of integrated approached such as IATA, combining the result with other complementary information, e.g. derived from in vitro assays addressing other key events of the skin sensitisation AOP.

Executive summary:

The test item was examined for sensitising properties in theARE-Nrf2 luciferase test method addressing the second molecular key event of the adverse outcome pathway (AOP), namely activation of keratinocytes by means of quantifying the luciferase activity in the transgenic cell line KeratinoSens™. Cytotoxicity was determined with the MTT assay. The GLP compliant study was performed according to OECD TG 442D.

The test item was tested at 12 concentrations in the range from 0.98 to 2000 µM. The test item was completely dissolvedin treatment culture medium to a concentration of 125 µM. Test item precipitation was noted macroscopically starting at aconcentration of 250 µM.

Cinnamic aldehyde tested at five concentrations from 4 – 64 µMwas used as the positive control and the solvent (DMSO) was used as negative control.

Two independent repetitions with three parallel technical replicates were run with this same set-up, and one parallel plate was prepared for cytotoxicity determination.The maximal average fold induction of the luciferase activity (Imax) values were 5.47 or 4.97 fold and from the dose dependent increase, EC1.5 values of 2.77 or < 0.98 µM have been calculated for the first or second repetition, respectively. The corresponding cell viability was > 70% (101.32% and 102.05%) leading to IC50values of 32.13 or 92.96 µM in repetitions 1 and 2, respectively.

The solvent control and the positive control cinnamic aldehyde were run in all repetitions. All quality criteria for luciferase induction and variability of the solvent control and positive control required were fulfilled.

A dose response for luciferase activity induction was observed for each individual repetition as well as for an overall luciferase activity induction.

The test item revealed sensitising properties in the ARE-Nrf2 Luciferase test method.

Reference 3

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Aug 22 - Nov 28, 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guidelines for Testing of Chemicals, No. 442E: In Vitro Skin Sensitisation assays addressing the Key Event on activation of dendritic cells on the Adverse Outcome Pathway for Skin Sensitisation”, adopted 25 June 2018

Qualifier:

according to guideline

Guideline:

other: Human Cell Line Activation Test (h-CLAT) for Skin Sensitisation, DB-ALM Protocol n°158, July 1st, 2015

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany

Type of study:

activation of dendritic cells

Details on the study design:

The in vitro human cell line activation test (h-CLAT) enables detection of the sensitising potential of a test item by addressing the third molecular key event of the adverse outcome pathway (AOP), namely dendritic cell activation, by quantifying the expression of the cell surface markers CD54 and CD86 in the human monocytic cell line THP-1. The expression of the cell surface markers compared to the respective solvent controls is used to support discrimination between skin sensitisers and non-sensitisers.

Positive control results:

The positive control (DNCB) led to an upregulation of the expression of CD54 and CD86 in all
experiments.

The following relative fluorescense intensities and mean viabilities (values in %) have been detected:

Run Concentr. / [µg/mL] CD54 CD86 mean viability
1 4 349 527 74.2
2 4 425 367 80.1
3 4 289 311 90.0
4 4 215 244 78.6

The threshold of 150% for CD86 and 200% for CD54 were clearly exceeded.

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

292

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 14.2 µg/mL

Remarks:

Viability: 57.5%

Key result

Run / experiment:

other: 1

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

95

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 14.2 µg/mL

Remarks:

Viability: 57.5%

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

111

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 11.8 µg/mL

Remarks:

Viability: 68.8%

Key result

Run / experiment:

other: 2

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

140

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 11.8 µg/mL

Remarks:

Viability: 68.8%

Key result

Run / experiment:

other: 3

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

71

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 14.2 µg/mL

Remarks:

Viability: 90%

Key result

Run / experiment:

other: 3

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

71

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 14.2 µg/mL

Remarks:

Viability: 90%

Key result

Run / experiment:

other: 4

Parameter:

other: relative fluorescence intensity CD86 [%]

Value:

85

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 14.2 µg/mL

Remarks:

Viability: 83.2%

Key result

Run / experiment:

other: 4

Parameter:

other: relative fluorescence intensity CD54 [%]

Value:

75

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Concentration: 14.2 µg/mL

Remarks:

Viability: 83.2%

Other effects / acceptance of results:

Acceptance criteria:


The following acceptance criteria should be met when using the h-CLAT assay.
• The cell viabilities of medium and solvent/vehicle controls should be higher than 90%.
• In the solvent/vehicle control, RFI values of both CD86 and CD54 should not exceed the positive criteria (CD86 RFI ≥ 150% and CD54 RFI ≥ 200%). RFI values of the solvent/vehicle control are calculated by using the formula described in Section 6.1.
• For both medium and solvent/vehicle controls, the MFI ratio of both 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 RFI ≥ 150 and CD54 RFI ≥ 200) and cell viability should be more than 50%.
• For the test item, the cell viability should be more than 50% in at least four tested concentrations in each run.


The test mets the acceptance criteria.

Summarised Results of the hClat Assay - Relative Fluorescense Intensities (RFI) and cell viabilities

Run 1 and 2

Sample	Concentration / [µg/mL]	Experiment 1			Experiment 2
		RFI to vehicle control		Mean viability	RFI to vehicle control		Mean viability
		CD54	CD86	IgG/CD54/CD86	CD54	CD86	IgG/CD54/CD86
Medium Control	0	65	117	93.5	106	103	92.8
Vehicle Control (0.2% DMSO)	0	100	100	92.1	100	100	92.0
Positive Control (DNBC)	4.0	349	527	74.2	425	367	80.1
Test Item	8.2	183	210	80.8	154	100	77.9
	9.8	113	184	88.9	265	90	74.4
	11.8	98	264	71.1	140	111	68.8
	14.2	95	292	57.5	144	132	45.9
	17.0	176	452	12.9	108	114	49.8
	20.4	256	555	9.8	135	155	18.3
	24.4	257	863	6.2	158	181	16.3
	29.3	119	951	4.5	213	302	7.8

Run 3 and 4

Sample	Concentration / [µg/mL]	Experiment 3			Experiment 4
		RFI to vehicle control		Mean viability	RFI to vehicle control		Mean viability
		CD54	CD86	IgG/CD54/CD86	CD54	CD86	IgG/CD54/CD86
Medium Control	0	93	83	91.6	55	78	92.3
Vehicle Control (0.2% DMSO)	0	100	100	90.7	100	100	90.5
Positive Control (DNBC)	4.0	289	311	80.6	215	244	78.6
Test Item	4.0	65	78	90.5	66	67	88.9
	4.8	151	72	89.3	55	77	88.5
	5.7	124	90	89.6	45	84	85.6
	6.9	68	75	89.8	84	79	86.5
	8.2	65	69	89.5	56	80	85.1
	9.9	67	80	89.2	76	86	87.4
	11.9	58	73	87.7	45	81	87.8
	14.2	71	71	90.0	75	85	83.2

MFI - geometric mean fluorescense intensity
RFI -  relative fluorescense intensity

Interpretation of results:

other: The data generated with this test should be considered in the context of integrated approached such as IATA.

Conclusions:

In conclusion, the test material did not reveal any sensitising properties in the h-CLA T method.

Executive summary:

The test material was examined for sensitising properties in the h-CLAT assay, addressing the third molecular key event of the adverse outcome pathway (AOP), namely activation of dendritic cells by means of quantifying the expression of specific cell surface markers on a human monocytic leukaemia cell line (THP-1, (TIB-202™, ATCC)). The changes of surface marker expression (CD54 and CD86) were measured by flow cytometry following cell staining with fluorochrome-tagged antibodies. Cytotoxicity measurement was also conducted by analysing the propidium iodide (PI) uptake. The relative fluorescence intensity of surface markers compared to the solvent/vehicle control was calculated and used in the prediction model to support the discrimination between sensitisers and non-sensitisers.

In a preliminary experiment a <lose finding assay was performed to determine the CV75 (75% cell viability), and the test material was tested at eight concentrations in the

range of 7.8 - 1000 μg/mL culture medium. DMSO was used as solvent control at a concentration of 0.2%. The CV75 was calculated as 24.4 μg/mL and 29.3 μg/mL (1,2 x

CV75) was chosen as the maximum test concentration for the main experiment.

Hence, the test material was tested at 8 concentrations in the range from 8.2 to 29 .3 μg/mL. As the first two runs were not concordant for at least one of the markers (CD86 in experiment 1: RFI > 150% and CD54 in experiment 2: RFI >200%) and because the second run did not meet the acceptance criteria of more than 50% cell viability in at least four tested concentrations, a third and fourth run were needed. These third and fourth runs were tested at 8 concentrations in the range from 4.0 to 14.2 μg/mL.

DNCB (2,4-dinitrochlorobenzene) was used as the positive control for CD86/CD54 expression measurement at a final single concentration of 4.0 μg/mL. Each experiment

consisted of two independent runs for cytotoxicity and CD86/CD54 expression measurement. All quality criteria required were fulfilled. The h-CLAT prediction was considered negative as: The RFI of CD54 was below 200% and the RFI of CD86 was below 150% in the third and fourth experiment at all concentrations with a cell viability of more than

50%.

In conclusion, the test material did not reveal any sensitising properties in the h-CLA T method.

Reference 4

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Jan 08, 2020 - Apr 06, 2020

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:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc Postbus 6174 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: Pre-test and Main test: 18.5 (16.9-20.1) g
- Housing: grouped per dose
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 + 2°C
- Humidity (%): 45-65%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: day 1 To: day 6

Vehicle:

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

Concentration:

1, 2.5, 5, and 10% (The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
ASSAY I:
- Compound concentration: 25 and 50 % in AOO
- Irritation: moderate to severe erythema of the ear skin (up to Score 3)
- Systemic effects: progressive loss in body weight and due to systemic signs of toxicity
- Lymph node proliferation response: -

ASSAY I:I
- Compound concentration: 5 and 10 % in AOO
- Irritation: very slight to well defined erythema of the ear skin (Score 1 to 2)
- Systemic effects: no systemic toxicity
- Lymph node proliferation response: -

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: OECD 429
- Criteria used to consider a positive response: Stimulation index > 3

Positive control substance(s):

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

Statistics:

Standard statistical methods have been applied for data processing.

Positive control results:

Conc. SI
0%: 1.0
5% 1.58
10% 1.78
25% 8.19

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

Test Group: 1% in AOO

Key result

Parameter:

SI

Value:

1.1

Test group / Remarks:

Test Group: 2.5% in AOO

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

Test Group:5% in AOO

Key result

Parameter:

SI

Value:

2.2

Test group / Remarks:

Test Group: 10% in AOO

Cellular proliferation data / Observations:

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

CLINICAL OBSERVATIONS: No signs of systemic toxicity were observed during the study period. The animals treated with test item concentrations of 5 and 10% showed a very slight to well defined erythema of the ear skin (Score 1 to 2). Animals treated with 1 and 2.5% test item concentration did not show any signs of local skin irritation.

BODY WEIGHTS: The body weight of the 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.

Calulation of  Stimulation Indices per Dose Group

Test Item concentration	Group Calculation
	Mean DPM per anminal (2 lymph nodes)	SD	S.I.
Vehicle Control Group (acetone/olive oil = 4/1, v,v)	1435.0	483.2	1.0
1%	17.39.2	825.7	1.2
2.5 %	1616.6	441.2	1.1
5%	1411.8	283.5	1.0
10%	3088.2 *	1045.8	2.2

a) Mean DPM/animal was determined by dividing the sum of the measured values from lymph nodes of all animals within a group by the number of animals in that group (5 animals)
*statistically significant (p<0.05) vs. vehicle control

Interpretation of results:

GHS criteria not met

Conclusions:

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

Executive summary:

In the study the test item formulated in methyl 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 1, 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.

The animals did not show any signs of systemic toxicity during the course of the study and no cases of mortality were observed. The animals treated with test item concentrations of 5 and 10% showed a very slight to well defined erythema of the ear skin (Score 1 to 2). Animals treated with 1 and 2.5% test item concentration did not show any signs of local skin irritation.

In this study Stimulation Indices (S.I.) of 1.2, 1.1, 1.0, and 2.2 were determined with the test item at concentrations of 1, 2.5, 5, and 10% in acetone/olive oil (4+1, v/v), respectively.

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

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

The available experimental test data are reliable and suitable for justification for classification purposes under Regulation (EC) No 1272/2008. Based on available data, the test item does not require classification according to Regulation (EC) No 1272/2008 (CLP).