4-[4-(3,4-Difluorophenyl)-cyclohexyl]-cyclohexanone

Endpoint summary

Description of key information

According to an OECD TG 429 compliant study, the test item was found to be not a sensitiser (reference 7.4.1 -1).

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2003-02-26 to 2003-04-30

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)

Version / remarks:

06-2002

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd., Füllinsdorf, Switzerland
- Age at study initiation: 8 - 12 weeks
- Weight at study initiation: 15.6 - 20.6 g
- Housing: Groups of four mice in Makroion type III cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: day 1 To: day 5

Vehicle:

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

Concentration:

pre test for signs of irritation: 1, 2.5, 5, and 10 % (w/v)
main test: 2.5, 5 and 10 % (w/v)

No. of animals per dose:

pre test: 2 f
main test: 4 f per concentration

Details on study design:

RANGE FINDING TEST
In a non-GLP conform test in two mice, test item concentrations of 5, 10, 25 and 50 % (w/w) were tested on one ear each. No irritation effects were observed at the dosing sites applied at concentrations of 5 or 10 %. At the observation of 24 hours after the topical application, a moderate swelling and slight erythema were noted at the local sites dosed at >= 25 %. 10 % was the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation.

- Compound solubility: At 50 % solution in acetone:olive oil
- Irritation: Yes up to 50 % of test item
- Lymph node proliferation response: The proliferative response of the pooled lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph nodes (DPM) and as the ratio of 3HTdR incorporated into lymph node cells of lymph nodes of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/animal values were determined, mean scintillation background DPM was subtracted from test and control raw data.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION
The different test item concentrations were prepared individually. The preparations were made freshly before each dosing occasion. Application volume: 25 µL/ear/day.

OBSERVATIONS
In addition to the sensitising reactions the following observations and data were recorded during the test and observation period:
Mortality / Viability: Twice daily from acclimatization start to the termination
of in-life phase.
Body weights: At acclimatization start and prior to necropsy.
Lymph node weights: After excision, the lymph nodes were pooled for each experimental group and weighed immediately using an analytical balance.
Clinical signs (local / systemic): Clinical signs (systemic toxicity or local skin irritation) were recorded at least once daily. Especially the treatment sites were observed carefully.

Positive control substance(s):

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

Statistics:

The mean values and standard deviations were calculated in the body weight tables and for the DPM values (group mean DPM ± standard deviation).
A statistical analysis( One-Way-Analysis-of-Variance) was conducted on the DPM values.

Positive control results:

The sensitivity and reliability of the experimental technique employed was assessed by use of a substance which is known to have skin sensitization properties in CBA/CaOlaHsd mice. The validation- / positive control study was performed with ALPHA-HEXYLCINNAMALDEHYDE in acetone:olive oil, 4:1 (v/v) using CBA/CaOlaHsd mice (RCC Study Number 846871) between 08-JAN-2003 and 22-JAN-2003,

Stimulation Index: of 2.5, 3.7 and 9.7 were determined with the test item at concentrations of 5 %, 10 % and 25 % (w/v) in acetone:olive oil, 4:1 (v/v). The Estimated concentration for a S.I. of 3 (EC3) resulted 7.08 % (w/v).

Key result

Parameter:

SI

Value:

0.9

Test group / Remarks:

2.5%

Key result

Parameter:

SI

Value:

0.6

Test group / Remarks:

5%

Key result

Parameter:

SI

Value:

0.7

Test group / Remarks:

10%

Parameter:

other: disintegrations per minute (DPM)

Value:

1 032

Variability:

N/A: the lymph nodes of the animals of a dose group were pooled

Test group / Remarks:

2.5 %

Parameter:

other: disintegrations per minute (DPM)

Value:

687

Variability:

N/A: the lymph nodes of the animals of a dose group were pooled

Test group / Remarks:

5 %

Parameter:

other: disintegrations per minute (DPM)

Value:

808

Variability:

N/A: the lymph nodes of the animals of a dose group were pooled

Test group / Remarks:

10 %

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
Please refer to the respective table.

DETAILS ON STIMULATION INDEX CALCULATION
The proliferative response of lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3HTdR incorporated into lymph node cells of test lymph nodes relative to that recorded for control lymph nodes (STIMULATION INDEX). Before DPM/NODE values were determined, mean scintillation-background DPM was subtracted from test and control raw data.


EC3 CALCULATION
An exact EC3 value could not be calculated because this calculation requires data lying immediately above and below S.I. value of 3. The results showed a negative dose response, demonstrated by decreasing S.I. values. As no test item related findings such as, significant body weight loss or local/systemic findings were observed, it might be considered that the effect obtained is based on local cytotoxicity to the Langerhans cells as the immuno targets.

CLINICAL OBSERVATIONS:
No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

BODY WEIGHTS
The body weight of the animals, recorded at the start of acclimatization period and prior to necropsy, was within the range commonly recorded for animals of this strain and age.

Viability / Mortality
No deaths occurred during the study period

The following results were obtained:

Test item concentration % (w/w)		Measurement dpm	Calculation			Result
			dpm - BGa)	number of lymph nodes	dpm per lymph nodeb)	S.I.
--	BG I	0	--	--	--	--
--	BG II	1	--	--	--	--
--	CG 1	9297	9296	8	1162	--
2.5	TG 2	7225	7224	7	1032	0.9
5	TG 3	5498	5497	8	687	0.6
10	TG 4	6463	6462	8	808	0.7

 

Interpretation of results:

GHS criteria not met

Conclusions:

An EC3 value could not be calculated because this calculation requires data lying immediately above and below S.I. value of 3. The test item was found to be a non-sensitizer when tested at concentrations of up 10 % (w/w).

Executive summary:

The LLNA test was conducted according to the OECD guideline 429 adopted 22 July 2012. In order to study a possible contact allergenic potential of the test item, three groups each of four female mice were treated daily with the test item at concentrations of 2.5%, 5% and 10% (w/w) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (acetone:olive oil, 4:1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a (3-scintillation counter. In this study Stimulation Indices (S.I.) of 0.9, 0.6, and 0.7 were determined with the test item at concentrations of 2.5, 5, and 10% (w/w) in acetone:olive oil, 4:1 (v/v), respectively. The results obtained, showed a negative dose response, demonstrated by decreasing S.I. values. As no test item related findings such as, significant body weight loss or local/systemic findings were observed, it might be considered that the effect obtained is based on local cytotoxicity to the Langerhans cells as the immuno targets. Thus, an extrapolated EC3 could not be calculated, since a clear dose response could not be observed. In conclusion, the test item was found to be a non-sensitizer when tested at concentrations of up to 10 % (w/w).

Reference 2

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The LLNA test was conducted according to the OECD guideline 429 adopted 22 July 2012.

In order to study a possible contact allergenic potential of the test item, three groups each of four female mice were treated daily with the test item at concentrations of 2.5%, 5% and 10% (w/w) in acetone:olive oil, 4:1 (v/v) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (acetone:olive oil, 4:1 (v/v)) only. Five days after the first topical application the mice were injected intravenously into a tail vein with radio-labelled thymidine (3H-methyl thymidine). Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the incorporation of 3H-methyl thymidine measured in a (3-scintillation counter. In this study Stimulation Indices (S.I.) of 0.9, 0.6, and 0.7 were determined with the test item at concentrations of 2.5, 5, and 10% (w/w) in acetone:olive oil, 4:1 (v/v), respectively. The results obtained, showed a negative dose response, demonstrated by decreasing S.I. values. As no test item related findings such as, significant body weight loss or local/systemic findings were observed, it might be considered that the effect obtained is based on local cytotoxicity to the Langerhans cells as the immuno targets. Thus, an extrapolated EC3 could not be calculated, since a clear dose response could not be observed. In conclusion, the test item was found to be a non-sensitizer when tested at concentrations of up to 10 % (w/w).

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on this information, the substance is not considered to be classified for skin sensitisation under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) 2019/521.