Evaluation of non-radioactive endpoints of ex vivo local lymph node assay-BrdU to investigate select contact sensitizers

Figures & data

Table 1.  Lymph node weight, and cell count after treatment of mice with different concentrations of formaldehyde, PPD, cinnamal, and eugenol in AOO. Each value shown is the mean ± SD (n = 5/group).

Chemical	Vehicle	Concentration (% w/v)	Lymph node weight (mg)	Cell count (107/ml)
Formaldehyde	AOO	—	15 ± 1.0	2.00 ± 0.50
		0.10	18 ± 1.20	3.60 ± 0.21
		0.25	38 ± 2.0	4.00 ± 0.55
		0.50	49 ± 5.2*	6.00 ± 0.26*
		1.00	60 ± 1.3*	8.00 ± 2.21*
PPD	AOO	—	18 ± 2.0	4.00 ± 1.00
		0.025	22 ± 5.0	5.30 ± 0.65
		0.050	25 ± 10.0	9.30 ± 1.33
		0.100	37 ± 6.0*	10.66 ± 2.06†
		0.250	57 ± 0.4*	13.30 ± 2.66*
Cinnamal	AOO	—	16 ± 1.0	8.00 ± 0.20
		0.5	20 ± 2.0	11.20 ± 3.36
		1.0	28 ± 6.0†	13.60 ± 2.46†
		5.0	30 ± 3.0*	17.60 ± 2.37*
		10.0	35 ± 0.7*	25.60 ± 5.23*
Eugenol	AOO	—	15 ± 3.0	8.00 ± 0.20
		2.5	23 ± 7.0	10.50 ± 3.36
		10.0	33 ± 2.0*	13.00 ± 2.26*
		20.0	35 ± 3.0*	13.00 ± 1.27*
		50.0	47 ± 0.6*	18.00 ± 1.36*

^†p < 0.05, *p < 0.01; Significant differences from vehicle control (Dunnett’s test).

Table 2.  OD₄₅₀. SI. and EC₃ values from ex vivo BrdU labeling studies.

Chemical	Vehicle	Concentration (w/v%)	OD450*	SI	EC3 (%)
Formaldehyde	AOO	—	0.232 ± 0.016	1.00	0.29
		0.10	0.397 ± 0.023	1.71
		0.25	0.548 ± 0.027	2.36
		0.50	1.398 ± 0.071	6.03
		1.00	1.669 ± 0.065	7.19
PPD	AOO	—	0.426 ± 0.012	1.00	0.09
		0.025	0.517 ± 0.026	1.21
		0.050	0.812 ± 0.017	1.91
		0.100	1.452 ± 0.056	3.41
		0.250	1.523 ± 0.061	3.57
Cinnamal	AOO	—	0.117 ± 0.011	1.00	1.91
		0.5	0.217 ± 0.018	1.85
		1.0	0.305 ± 0.026	2.60
		5.0	0.511 ± 0.023	4.36
		10.0	1.076 ± 0.058	9.19
Eugenol	AOO	—	0.117 ± 0.011	1.00	16.60
		2.5	0.173 ± 0.021	1.48
		10.0	0.223 ± 0.023	1.90
		20.0	0.371 ± 0.042	3.17
		50.0	0.532 ± 0.059	4.54

* OD₄₅₀ values shown are in terms of mean ± SD (n = 4/group).

Table 3.  EC₃ values for the four test chemicals using standard LLNA and ex-vivo LLNA-BrdU methods.*

Chemicals	EC3 (%) (Standard LLNA)	Category	Reference	EC3 (%) (ex vivo LLNA-BrdU*)	Category
Formaldehyde	0.40	Strong	Kimber et al. (2003)	0.29	Strong
PPD	0.06–0.18	Extreme or strong	Basketter et al. (2007)	0.09	Extreme
Cinnamal	1.70–3.10	Moderate	Basketter et al. (2007)	1.91	Moderate
Eugenol	4.90–15.00	Moderate or weak	Basketter et al. (2007)	16.60	Weak

*The data from this study.

Figure 1.  Cytokine levels in cultures of auricular lymph node cells from mice treated daily (3 days) with formaldehyde. ^†p < 0.05, *p < 0.01; Significant differences from vehicle control (Dunnett’s test).

Figure 2.  Cytokine levels in cultures of auricular lymph node cells from mice treated daily (3 days) with PPD. ^†p < 0.05, *p < 0.01; Significant differences from vehicle control (Dunnett’s test).

Figure 3.  Cytokine levels in cultures of auricular lymph node cells from mice treated daily (3 days) with cinnamal. ^†p < 0.05, *p < 0.01; Significant differences from vehicle control (Dunnett’s test).

Figure 4.  Cytokine levels in cultures of auricular lymph node cells from mice treated daily (3 days) with eugenol. ^†p < 0.05, *p < 0.01; Significant differences from vehicle control (Dunnett’s test).

Table 4.  Cytokine SI values for auricular lymph node cells from mice treated daily (3 days) with formaldehyde, PPD, cinnamal, or eugenol.

Chemical	Concentration (w/v%)	SI IL-2	SI IFNγ	SI IL-4	SI IL-5
Formaldehyde in AOO	AOO	1.00	1.00	1.00	1.00
	0.10	2.64	1.52	1.39	1.38
	0.25	7.02	3.74	2.72	1.50
	0.50	8.29	8.91	3.89	2.05
	1.00	13.31	13.99	5.08	3.23
PPD in AOO	AOO	1.00	1.00	1.00	1.00
	0.025	2.07	3.84	5.55	1.55
	0.050	4.15	4.91	11.96	4.63
	0.100	7.75	8.10	19.12	10.04
	0.250	13.83	11.82	44.41	19.97
Cinnamal in AOO	AOO	1.00	1.00	1.00	1.00
	0.50	2.39	2.28	1.52	1.71
	1.00	3.06	3.39	3.26	2.30
	5.00	5.88	4.98	4.84	3.92
	10.00	6.88	8.05	7.36	6.08
Eugenol in AOO	AOO	1.00	1.00	1.00	1.00
	2.5	1.25	1.33	1.77	1.34
	10.0	2.56	2.99	2.03	1.63
	20.0	3.16	8.19	3.94	5.70
	50.0	4.90	13.05	6.13	7.93

Kimber, I., Basketter, D. A., Butler, M., Gamer, A., Garrigue, J. L., Gerberick, G. F., Newsome, C., Steiling, W. Vohr, H. W. 2003. Classification of contact allergens according to potency: Proposals. Food Chem. Toxicol. 41:1799–1809.

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Basketter, D. A., Gerberick, F. Kimber, I. 2007. The local lymph node assay and the assessment of relative potency: Status of validation. Contact Derm. 57:70–75.

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