2-[4-(4-Alkoxyphenyl)-(pyridin-2-yl)-heteromonocyclyl]phenol

Endpoint summary

Description of key information

FAT 75637/B did not demonstrate dermal sensitization potential in the mouse LLNA.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Study initiation date - 30 April 2021; Experiment start date - 30 April 2021; Experiment end date - 17 June 2021; Study completion date - 02 July 2021.

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

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Specific details on test material used for the study:

Test Item: FAT 75637/B TE
Physical Appearance: Light yellow powder       
Purity: 99.4 % all organic constituents; 95.0 % main constituent
Batch No: AT-0063765400
Manufactured Date: 21st April 2020
Expiry Date: May 27th, 2025

Species:

mouse

Strain:

CBA/Ca

Sex:

female

Details on test animals and environmental conditions:

Mice were housed in an environment-controlled room at 20 to 23 °C and relative humidity of 65 to 67 percent. The photoperiod was 12 hours light and 12 hours darkness, light hours being 06:00 to 18:00 hours approximately. Adequate fresh air supply of 12.8 air changes/hour was maintained in the experimental room. The maximum and minimum temperature and relative humidity in the experimental room were recorded once daily. The relative humidity in the experimental room was calculated daily from dry and wet bulb temperature recordings. Animals were housed individually (to avoid licking of test item by cage mates) in solid floor standard polysulfone cages (Size: Approximately L 360 x B 205 x H 140 mm), with stainless steel top grill having facilities for providing pelleted food and drinking water in polycarbonate bottle with stainless steel sipper tubes. Steam sterilized corn cob was used as bedding and changed along with the cage once a week. Cages were placed on tiered racks. Mouse huts were provided in the cages as environment enrichment to minimize animal stress and promote overall well-being during the in-life phase of the study.

Vehicle:

other: 1 % Pluronic® L92 (1% L92)

Concentration:

1 % v/v

No. of animals per dose:

5 groups
Vehicle control (G1)
Positive control (G2)
Test item: Three concentrations (G3 to G5)
Six female mice per dose

Details on study design:

LLNA main study: Six female CBA/Ca mice per group received the vehicle (1 % L92) or 25 % α-hexylcinnamaldehyde (HCA: positive control in 1 % L92) or 10, 25 and 50 % w/v of test item in 1 % L92 on days 1 to 3. On day 6, the uptake of 3H-methyl thymidine into the auricular lymph nodes draining the site of test item application was measured five hours post administration. Proper conduct of the LLNA was confirmed via a positive response using 25 % α-hexylcinnamaldehyde, a contact sensitizer, which elicited proliferation with a Stimulation Index (SI) value of 6.39, in comparison to vehicle-treated mice.

Positive control substance(s):

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

Statistics:

A mean dpm value ± SD (standard deviation) was calculated for each group and the stimulation index (SI) was calculated using the absolute dpm value for each mouse as the numerator, and the mean dpm value from the vehicle-treated mice as the denominator.

1. The % increase in ear thickness was calculated for each ear using the following equation:
% Ear swelling = (B – A)/A x 100
Where, A = ear thickness measurement on Day 1 (µm); B = ear thickness measurement on Day 3 or 6 (µm)


2. The SI was calculated for each mouse using the following equation:
SI = Disintegrations per minute (dpm) of individual mouse/ Average dpm of the vehicle control mice.
Means and SD were generated for body weight data (absolute and gain) and LLNA response (dpm and SI values). The body weight and dpm data were analysed by one-way analysis of variance. When the differences were indicated by the ANOVA, a comparison of treated vs. control groups was done using a Dunnett’s t-test (p <0.05).

Positive control results:

25 % α-hexylcinnamaldehyde, a contact sensitizer, which elicited proliferation with a Stimulation Index (SI) value of 6.39, in comparison to vehicle-treated mice.

Parameter:

SI

Value:

1.08

Test group / Remarks:

10 %

Parameter:

SI

Value:

1.22

Test group / Remarks:

25 %

Parameter:

SI

Value:

1.5

Test group / Remarks:

50 %

Irritation Screening Test: Once daily topical applications of vehicle – 1 % Pluronic L92 (1 % L92), 5, 10, 20, 35 and 50 % w/v test item in 1 % L92 were performed to one animal at each dose level for 3 days. There were no clinical signs, no erythema at the site of application, no significant increase in the ear thickness and ear punch weights and no effect on body weight. Results from this screening study were used to determine the dosing concentrations of FAT 75637/B for the main LLNA study.

Main LLNA Study

The analyzed radioactivity of ³H-TdR working solution was 77 μCi/mL against the nominal concentration of 80 μCi/mL. There were no clinical signs, no erythema at the site of application and no significant effect on body weight gains. The sensitivity of this LLNA test was demonstrated via the response from the positive control (25 % HCA in 1 % L92), which elicited a stimulation index (SI) of 6.39, in comparison with the vehicle-treated mice. The Mean SI values for 10, 25, and 50 % w/v test item in 1 % L92 were 1.08, 1.22 and 1.50, respectively.

Irritation Screening Test

Body Weights (g) and clinical Signs

Mice Nos.	Dose Concentration	Day 1 (pre-dose)	Day 6	Body wt. gain (g)	Clinical Signs
Mh1911	Vehicle: 1 % L92	19.2	20.2	1.0	NAD
Mh1912	5 % w/v	18.9	19.9	1.0	NAD
Mh1913	10 % w/v	19.5	20.4	0.9	NAD
Mh1914	20 % w/v	20.1	21.0	0.9	NAD
Mh1915	35 % w/v	20.0	21.0	1.0	NAD
Mh1916	50 % w/v)	19.8	20.7	0.9	NAD

NAD: No Abnormality Detected

 

 

Erythema Scores (for both ears)

Mice Nos.	Dose Concentration	Day 1 (pre-dose)	Day 2	Day 3	Day 6
Mh1911	Vehicle: 1 % L92	0	0	0	0
Mh1912	5 % w/v	0	0	0	0
Mh1913	10 % w/v	0	0	0	0
Mh1914	20 % w/v	0	0	0	0
Mh1915	35 % w/v	0	0	0	0
Mh1916	50 % w/v)	0	0	0	0

Erythema scores – 0: No erythema

 

 

Ear thickness measurement and ear punch weight

Mice Nos.	Dose Concentration	Ear thickness (µm)						Ear punch weight (g)
		Day 1 (pre-dose)		Day 3		Day 6		Day 6
		L.E	R.E	L.E	R.E	L.E	R.E	L.E	R.E
Mh1911	Vehicle: 1 % L92	249	250	250	251	250	251	0.009	0.009
Mh1912	5 % w/v	263	262	264	264	264	264	0.010	0.010
Mh1913	10 % w/v	268	269	270	271	270	270	0.009	0.009
Mh1914	20 % w/v	257	256	259	258	259	258	0.009	0.009
Mh1915	35 % w/v	253	254	255	256	255	255	0.010	0.009
Mh1916	50 % w/v)	260	259	262	261	261	261	0.010	0.010

1 % L92: 1 % Pluronic L92

L.E: Left ear             

R.E: Right ear

 

TABLE 1 contd. Irritation Screening Test

 

D. Percent change in ear thickness

 

Mice Nos.	Dose concentration	% change in ear thickness
		Day 3		Day 6
		L.E	R.E	L.E	R.E
Mh1911	Vehicle: 1 % L92	0.40	0.40	0.40	0.40
Mh1912	5 % w/v	0.38	0.76	0.38	0.76
Mh1913	10 % w/v	0.75	0.74	0.75	0.37
Mh1914	20 % w/v	0.78	0.78	0.78	0.78
Mh1915	35 % w/v	0.79	0.79	0.79	0.39
Mh1916	50 % w/v)	0.77	0.77	0.38	0.77

1 % L92: 1 % Pluronic L92

L.E: Left ear             

R.E: Right ear   

 

 

TABLE 2. Summary of Body Weight, Body Weight Changesand Clinical signs

Group and Dose concentration	No. of mice		Body weight (g)
			Day 1 (Initial)	Day 6	Weight change (day 6 – Initial)	Clinical signs
G1 Vehicle: 1 % L92	6
		Mean	21.267	22.367	1.100	NAD
		SD	1.338	1.320	0.063
G2 25 % v/v HCA	6
		Mean	21.233	22.167	0.933	NAD
		SD	1.209	1.174	0.052
G3 10 % w/v test item	6
		Mean	21.133	22.117	0.983	NAD
		SD	1.029	1.026	0.041
G4 25 % w/v test item	6
		Mean	21.183	22.133	0.950	NAD
		SD	0.960	0.999	0.055
G5 50 % w/v test item	6
		Mean	21.150	22.083	0.933	NAD
		SD	0.855	0.854	0.082

1 % L92:1 % Pluronic L92

NAD: No Abnormality Detected

HCA: α – Hexylcinnamaldehyde

 

 

  

 

 TABLE 3.      Summary of Local Reaction Scores at the Site of Application

      

Group and Dose concentration	No. of Mice		Erythema Score of both ears (Mean ± SD)
			Pre-treatment	Day 2	Day 3	Day 6
G1 Vehicle: 1 % L92	6	Mean SD	0 0	0 0	0 0	0 0
G2 25 % v/v HCA	6	Mean SD	0 0	0.83 0.41	1.00 0.00	1.00 0.00
G3 10 % w/v test item	6	Mean SD	0 0	0 0	0 0	0 0
G4 25 % w/v test item	6	Mean SD	0 0	0 0	0 0	0 0
G5 50 % w/v test item	6	Mean SD	0 0	0 0	0 0	0 0

1 % L92: 1 % Pluronic L92

HCA: α – Hexylcinnamaldehyde   

Summary of Disintegrations Per Minute (DPM) for ³H-Methyl Thymidine Incorporation in Auricular Lymph Nodes and Stimulation Index (SI)

Group and Dose concentration	No. of mice		DPM / Mouse	SI
G1 Vehicle: 1 % L92	6
		Mean	716.500	0.998
		SD	53.795	0.076
G2 25 % v/v HCA	6		+
		Mean	4575.500	6.387
		SD	841.411	1.177
G3 10 % w/v test item	6		+
		Mean	776.667	1.085
		SD	53.541	0.073
G4 25 % w/v test item	6		+
		Mean	876.833	1.225
		SD	84.353	0.118
G5 50 % w/v test item	6		+
		Mean	1075.667	1.502
		SD	217.467	0.306

+: Significantly higher than the vehicle control group

1 % L92 : 1 % Pluronic L92

HCA: α – Hexylcinnamaldehyde

Interpretation of results:

GHS criteria not met

Conclusions:

A topical application with 10, 25 and 50 % w/v of test item in 1 % Pluronic L92 (1 % L92) elicited a stimulation index (SI) of 1.08, 1.22 and 1.50, respectively. The test item FAT 75637/B did not demonstrate dermal sensitization potential in the mouse LLNA, as the lymph nodes draining the area of topical application did not elicit a proliferative response greater than the 3X threshold.

Executive summary:

Local Lymph Node Assay (LLNA) was conducted to evaluate the potential of the test item FAT 75637/B to cause contact sensitization by measuring the lymphocyte proliferative response from auricular lymph nodes following topical application to the female CBA/Ca mouse ear. This study was conducted according to OECD test guideline 429 in a GLP-certified laboratory. 

Screening Study: Once daily topical application of the vehicle 1 % Pluronic L92 (1 % L92) and 5, 10, 20, 35 and 50 % w/v test item in 1 % L92 were performed on one animal at each dose level. There were no clinical signs, no erythema at the site of application, no significant increase in the ear thickness and ear punch weights and no effect on body weight. The results of this screening test were used to determine the dosing concentration for the main study.

LLNA main study: Six female CBA/Ca mice per group received the vehicle (1 % L92) or 25 % α-hexylcinnamaldehyde (HCA: positive control in 1 % L92) or 10, 25 and 50 % w/v of test item in 1 % L92 on days 1 to 3. On day 6, the uptake of ³H-methyl thymidine into the auricular lymph nodes draining the site of test item application was measured five hours post administration. Proper conduct of the LLNA was confirmed via a positive response using 25 % α-hexylcinnamaldehyde, a contact sensitizer, which elicited proliferation with a Stimulation Index (SI) value of 6.39, in comparison to vehicle-treated mice. There were no clinical signs, no local skin reactions at the tested concentrations and treatment had no significant effect on body weight gain. The test item FAT 75637/B at dose concentrations of 10, 25 and 50 % w/vof test item in 1 % L92 elicited proliferative response with SI of 1.08, 1.22 and 1.50, respectively, in comparison with the vehicle-treated mice. The test item FAT 75637/B did not demonstrate dermal sensitization potential in the mouse LLNA, as the lymph nodes draining the area of topical application did not elicit a proliferative response greater than the 3X threshold.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Local Lymph Node Assay (LLNA) was conducted to evaluate the potential of the test item “FAT 75637/B” to cause contact sensitization by measuring the lymphocyte proliferative response from auricular lymph nodes following topical application to the female CBA/Ca mouse ear. This study was conducted according to OECD test guideline 429 in a GLP-certified laboratory. 

Screening Study: Once daily topical application of the vehicle 1 % Pluronic L92 (1 % L92) and 5, 10, 20, 35 and 50 % w/v test item in 1 % L92 were performed on one animal at each dose level. There were no clinical signs, no erythema at the site of application, no significant increase in the ear thickness and ear punch weights and no effect on body weight. The results of this screening test were used to determine the dosing concentration for the main study.

LLNA main study: Six female CBA/Ca mice per group received the vehicle (1 % L92) or 25 % α-hexylcinnamaldehyde (HCA: positive control in 1 % L92) or 10, 25 and 50 % w/v of test item in 1 % L92 on days 1 to 3. On day 6, the uptake of ³H-methyl thymidine into the auricular lymph nodes draining the site of test item application was measured five hours post administration. Proper conduct of the LLNA was confirmed via a positive response using 25 % α-hexylcinnamaldehyde, a contact sensitizer, which elicited proliferation with a Stimulation Index (SI) value of 6.39, in comparison to vehicle-treated mice. There were no clinical signs, no local skin reactions at the tested concentrations and treatment had no significant effect on body weight gain. The test item FAT 75637/B at concentrations of 10, 25 and 50 % w/vof test item in 1 % L92 elicited proliferative response with SI of 1.08, 1.22 and 1.50, respectively, in comparison with the vehicle-treated mice.

The test item FAT 75637/B did not demonstrate dermal sensitization potential in the mouse LLNA, as the lymph nodes draining the area of topical application did not elicit a proliferative response greater than the 3x threshold.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

FAT 75637 was found to considered to have no skin sensitisation potential from the results of a LLNA study, hence, it does not warrant classification for sensitisation according to Regulation EC No. 1272/2008 (CLP).