Table 1 ,
 Erythema Scores ,

No erythema,0,
Very slight erythema (barely perceptible),1,
Well-defined erythema,2,
Moderate to severe erythema,3,
Severe erythema (beet redness) to eschar formation preventing grading of erythema,4,
 Table 1 ,
 Recommended Reference chemicals for the LLNA PS. ,
    Presumed to be a non-sensitiser in humans based on the fact that no clinical patch test results were located, it is not included as a patch test kit allergen, and no case reports of human sensitisation were located.    ,
    GP data not available.    ,
    Chemicals should be prepared daily unless stability data demonstrate the acceptability of storage.    ,
    Because of the potential impact of different vehicles on the performance of the LLNA, the recommended vehicle for each reference chemical should be used (24) (32).    ,
    Mean value where more than one EC3 value was available. For negative substances ( i.e. with stimulation index < 3, the highest concentration tested is provided).    ,
    Number of LLNA studies from which data were obtained.    ,
    Commercially available as Kathon CG (CAS No 55965-84-9), which is a 3:1 mixture of CMI and MI. The relative concentrations of each component range from 1,1 % to 1,25 % (CMI) and 0,3 % to 0,45 % (MI). The inactive components are magnesium salts (21,5 % to 24 %) and copper nitrate (0,15 % to 0,17 %), with the remaining formulation 74 % to 77 % water. Kathon CG is readily available through Sigma-Aldrich and Rohm and Haas (now Dow Chemical Corporation).    ,
  Abbreviations: AOO = acetone: olive oil (4:1, v/v); CAS No = Chemical Abstracts Service Number; DMF = N,N -dimethylformamide; DMSO = dimethyl sulfoxide; DNCB = 2,4-dinitrochlorobenzene; EC3 = estimated concentration needed to produce a stimulation index of 3; GP = guinea pig test result ( i.e. B.6 or OECD Test Guideline 406) (13); HCA = hexyl cinnamic aldehyde; Liq = liquid; LLNA = murine local lymph node assay result ( i.e. B.42 or OECD Test Guideline 429) (1); MEK = methyl ethyl ketone; NA = not applicable since stimulation index < 3; NC = not calculated since data was obtained from a single study; Sol = solid; Veh = test vehicle.  ,

1,5-Chloro-2-methyl-4-isothiazolin-3-one (CMI)/2-methyl-4-isothiazolin-3-one (MI) ,26172-55-4/ 2682-20-4,Liq,DMF,0,009,1,0,0045-0,018,NC,+/+,+/+,
2,DNCB,97-00-7,Sol,AOO,0,049,15,0,025-0,099,0,02-0,094,+/+,+/+,
3,4-Phenylenediamine,106-50-3,Sol,AOO,0,11,6,0,055-0,22,0,07-0,16,+/+,+/+,
4,Cobalt chloride,7646-79-9,Sol,DMSO,0,6,2,0,3-1,2,0,4-0,8,+/+,+/+,
5,Isoeugenol,97-54-1,Liq,AOO,1,5,47,0,77-3,1,0,5-3,3,+/+,+/+,
6,2-Mercaptobenzothiazole,149-30-4,Sol,DMF,1,7,1,0,85-3,4,NC,+/+,+/+,
7,Citral,5392-40-5,Liq,AOO,9,2,6,4,6-18,3,5,1-13,+/+,+/+,
8,HCA,101-86-0,Liq,AOO,9,7,21,4,8-19,5,4,4-14,7,+/+,+/+,
9,Eugenol,97-53-0,Liq,AOO,10,1,11,5,05-20,2,4,9-15,+/+,+/+,
10,Phenyl benzoate,93-99-2,Sol,AOO,13,6,3,6,8-27,2,1,2-20,+/+,+/+,
11,Cinnamic alcohol,104-54-1,Sol,AOO,21,1,10,5-42,NC,+/+,+/+,
12,Imidazolidinyl urea,39236-46-9,Sol,DMF,24,1,12-48,NC,+/+,+/+,
13,Methyl methacrylate,80-62-6,Liq,AOO,90,1,45-100,NC,+/+,+/+,
14,Chlorobenzene,108-90-7,Liq,AOO,25,1,NA,NA,–/–,–/ ,
15,Isopropanol,67-63-0,Liq,AOO,50,1,NA,NA,–/–,–/+,
16,Lactic acid,50-21-5,Liq,DMSO,25,1,NA,NA,–/–,–/ ,
17,Methyl salicylate,119-36-8,Liq,AOO,20,9,NA,NA,–/–,–/–,
18,Salicylic acid,69-72-7,Sol,AOO,25,1,NA,NA,–/–,–/–,

19,Sodium lauryl sulphate,151-21-3,Sol,DMF,8,1,5,4,05-16,2,1,5-17,1,+/–,+/–,
20,Ethylene glycol dimethacrylate,97-90-5,Liq,MEK,28,1,14-56,NC,+/–,+/+,
21,Xylene,1330-20-7,Liq,AOO,95,8,1,47,9-100,NC,+/ ,+/–,
22,Nickel chloride,7718-54-9,Sol,DMSO,5,2,NA,NA,–/+,–/+,
 Table 1 ,
 Reference Chemicals  ,
    These Reference Chemicals are a subset of the Reference Chemicals used in the validation study.    ,
     In vivo score in accordance with B.4 and OECD Test Guideline 404 (4).    ,
    Under this Test Method, the UN GHS optional Category 3 (mild irritants) (1) is considered as No Category.    ,
    The UN GHS optional Category3 is not applicable under the EU CLP.    ,

naphthalene acetic acid,86-87-3,0,Solid,No Cat.,
isopropanol,67-63-0,0,3,Liquid,No Cat.,
methyl stearate,112-61-8,1,Solid,No Cat.,
heptyl butyrate,5870-93-9,1,7,Liquid,  No Cat.    ( Optional Cat. 3 ) ,   ,
hexyl salicylate,6259-76-3,2,Liquid,  No Cat.    ( Optional Cat. 3 ) ,   ,
cyclamen aldehyde,103-95-7,2,3,Liquid,Cat. 2,
1-bromohexane,111-25-1,2,7,Liquid,Cat. 2,
potassium hydroxide (5 % aq.),1310-58-3,3,Liquid,Cat. 2,
1-methyl-3-phenyl-1-piperazine,5271-27-2,3,3,Solid,Cat. 2,
Heptanal,111-71-7,3,4,Liquid,Cat. 2,
 Table 2 ,
 Acceptability ranges for negative control OD values ,

≥ 0,6,≤ 1,5,
≥ 1,0,≤ 2,5,
≥ 1,2,≤ 2,5,
 Table 3 ,
 Examples of QC batch release criteria ,

IC 50 = 1,0 mg/ml,IC 50 = 3,0 mg/ml,
ET 50 = 4,8 hr,ET 50 = 8,7 hr,
ET 50 = 4,0 hr,ET 50 = 9,0 hr,
 Table 1 ,
 Minimum List of Reference Chemicals for Determination of Accuracy and Reliability Values for Similar or Modified RhE Skin Irritation Methods  ,
    The chemical selection is based on the following criteria: (i) the chemicals are commercially available; (ii) they are representative of the full range of Draize irritancy scores (from non-irritant to strong irritant); (iii) they have a well-defined chemical structure; (iv) they are representative of the chemical functionality used in the validation process; and (v) they are not associated with an extremely toxic profile ( e.g. carcinogenic or toxic to the reproductive system) and they are not associated with prohibitive disposal costs.    ,
    Chemicals that are irritant in the rabbit but for which there is reliable evidence that they are non-irritant in humans (31) (32) (33).    ,
    Under the UN GHS, not in the EU CLP    ,

1-bromo-4-chlorobutane,6940-78-9,Liquid,0,Cat. 2,No Cat.,
diethyl phthalate,84-66-2,Liquid,0,No Cat.,No Cat.,
naphthalene acetic acid,86-87-3,Solid,0,No Cat.,No Cat.,
allyl phenoxy-acetate,7493-74-5,Liquid,0,3,No Cat.,No Cat.,
isopropanol,67-63-0,Liquid,0,3,No Cat.,No Cat.,
4-methyl-thio-benzaldehyde,3446-89-7,Liquid,1,Cat. 2,No Cat.,
methyl stearate,112-61-8,Solid,1,No Cat.,No Cat.,
heptyl butyrate,5870-93-9,Liquid,1,7,No Cat.,No Cat.,
hexyl salicylate,6259-76-3,Liquid,2,No Cat.,No Cat.,
Cinnamaldehyde,104-55-2,Liquid,2,Cat. 2,  No Cat.    ( Optional Cat. 3 )   ,
 1-decanol  , 112-30-1 , Liquid , 2,3 , Cat. 2 , Cat. 2 ,
cyclamen aldehyde,103-95-7,Liquid,2,3,Cat. 2,Cat. 2,
1-bromohexane,111-25-1,Liquid,2,7,Cat. 2,Cat. 2,
2-chloromethyl-3,5-dimethyl-4-methoxypyridine HCl,86604-75-3,Solid,2,7,Cat. 2,Cat. 2,
 di-n-propyl disulphide  , 629-19-6 , Liquid , 3 , No Cat. , Cat. 2 ,
potassium hydroxide (5 % aq.),1310-58-3,Liquid,3,Cat. 2,Cat. 2,
benzenethiol, 5-(1,1-dimethylethyl)-2-methyl,7340-90-1,Liquid,3,3,Cat. 2,Cat. 2,
1-methyl-3-phenyl-1-piperazine,5271-27-2,Solid,3,3,Cat. 2,Cat. 2,
Heptanal,111-71-7,Liquid,3,4,Cat. 2,Cat. 2,
Tetrachloroethylene,127-18-4,Liquid,4,Cat. 2,Cat. 2,
 Table 2 ,
 Required predictive values for sensitivity, specificity and overall accuracy for any similar or modified method to be considered valid ,

≥ 80 %,≥ 70 %,≥ 75 %,
 Table 1 ,
 Cell treatment and harvest times for the MNvit assay ,
Lymphocytes, primary cells and cell lines treated with cytoB,+ S9,  Treat for 3-6 hrs in the presence of S9;    remove the S9 and treatment medium;    add fresh medium and cytoB;    harvest 1,5-2,0 normal cell cycles later.  ,
  – S9    Short exposure  ,  Treat for 3-6 hrs;    remove the treatment medium;    add fresh medium and cytoB;    harvest 1,5-2,0 normal cell cycles later.  ,
  – S9    Extended exposure  ,   Option A: Treat for 1,5-2 normal cell cycles in the presence of cytoB;    harvest at the end of the exposure period.     Option B: Treat for 1,5-2,0 normal cell cycles;    remove the test substance;    add fresh medium and cytoB;    harvest 1,5-2,0 normal cell cycles later.  ,
  Cell lines treated without cytoB    (Identical to the treatment schedules outlined above with the exception that no cytoB is added)  ,
    The reference chemicals are the recommended chemicals for use. Substitution or adding of chemicals to the list of reference chemicals can be done if their activity is known and if they induce micronuclei by the same mechanisms of action, and if they are shown to be relevant to the chemicals that will be tested using the MNvit procedure. Depending on the purpose, justification could also include a validation study employing a broad variety of substances or focused on a narrower spectrum based on the chemical class of the test substance or the mechanism of damage being studied.    ,


,Cytosine arabinoside,147-94-4,205-705-9,
,Mitomycin C,50-07-7,200-008-6,

,Benzo(a)pyrene,50-32-8,200-028-5,
,Cyclophosphamide,50-18-0,200-015-4,

,Colchicine,64-86-8,200-598-5,
,Vinblastine,143-67-9,205-606-0,

,Di(2-ethylhexyl)phthalate,117-81-7,204-211-0,
,Nalidixic acid,389-08-2,206-864-7,
,Pyrene,129-00-0,204-927-3,
,Sodium chloride,7647-14-5,231-598-3,
 Table 1 ,
 Erythema Scores ,

No erythema,0,
Very slight erythema (barely perceptible),1,
Well-defined erythema,2,
Moderate to severe erythema,3,
Severe erythema (beet redness) to eschar formation preventing grading of erythema,4,
 Table 1 ,
 Erythema Scores ,

No erythema,0,
Very slight erythema (barely perceptible),1,
Well-defined erythema,2,
Moderate to severe erythema,3,
Severe erythema (beet redness) to eschar formation preventing grading of erythema,4,