SEND Example.In vitro Neutral Red Cytotoxicity Assay

This example shows trial design and results data for in vitro Neutral Red Cytotoxicity Assay, Relative Absorbance.

Note that Variable Names and Domain abbreviations may change (have not been checked against existing and are not vetted)

red font - indicates potential for CT code lists
green font - links to other domains
purple font - remaining questions to resolve

The following shows the tabulated data as it is represented in a report for this study. This report includes summary statistics (e.g. Average, Standard Deviation, Coeff. Var.); however, statistics are not included in the SEND standard and will not be represented in the SEND domains example.

Neutral Red Cytotoxicity Assay Results (Relative Absorbance Data)
Run-Port Number	Sample ID	Smoke Fraction	Replicate Number	Plate Number	Well Number		Relative Assay Plate Absorbance Readings
							Cigarette Smoke Condensate (µg/mL)								SLS (µg/mL)			EC50 (µg/ml)
						0	10	50	75	100	120	140	160	200	110	200	0
1-1	030001	A	1	1	1	0.046	0.046*	0.039*	0.012*	0.030*	0.003*	0.057*	0.032*	0.003*	0.050*	0.044*	0.040
1-1	030001	A	1	1	2	0.048	107	77.7	65.7	30.2	15.6	9.79	5.41	2.49	8.09	0.791	0.014
1-1	030001	A	1	1	3	0.005	98.6	77.4	57.7	35.8	8.09	7.36	3.22	0.791	6.38	0.061	0.050
1-1	030001	A	1	1	4	0.049	94.9	81.5	38.2	31.2	7.60	5.17	3.22	0.791	5.17	0.304	0.017
1-1	030001	A	1	1	5	0.047	111	78.1	64.5	32.9	8.82	5.90	2.74	1.28	10.3	0.547	0.011
1-1	030001	A	1	1	6	0.032	96.6	76.2	48.0	26.8	7.84	4.20	2.74	1.52	8.09	4.93	0.022
1-1	030001	A	1	1	7	0.044	105	77.9	58.2	28.3	8.09	4.68	1.76	0.547	8.57	1.76	0.056
1-1	030001	A	1	1	8	0.056	0.013*	0.040*	0.008*	0.013*	0.002*	0.029*	0.018*	0.006*	0.016*	0.024*	0.046
Average							101	78.2	54.5	29.9	9.70	6.17	3.37	1.19	8.94	1.22
Std. Dev.							6	2.9	9.4	3.6	2.95	2.19	1.12	0.72	2.72	1.58
Coeff. Var.							5.8	3.6	17.2	11.9	30.4	35.5	33.1	61.0	30.5	129.9
*non-treated control wells
1-1	030001	A	1	2	1		93.4	74.0	56.0	35.1	11.8	8.70	5.10	1.98	5.34	1.02
1-1	030001	A	1	2	2		102	75.2	53.3	35.1	11.8	7.74	5.58	3.66	5.34	1.02
1-1	030001	A	1	2	3		107	79.0	55.0	26.5	10.1	6.06	3.66	1.74	7.02	0.780
1-1	030001	A	1	2	4		104	80.2	55.3	29.8	8.94	3.90	3.66	1.50	6.30	0.780
1-1	030001	A	1	2	5		101	75.7	54.1	26.7	4.86	4.38	1.26	1.02	6.30	1.02
1-1	030001	A	1	2	6		110	85.7	67.7	26.0	5.34	4.62	3.42	1.50	6.30	0.540
1-1	030001	A	1	2	7		104	77.8	64.9	27.7	8.22	3.18	2.46	1.02	6.30	0.780
1-1	030001	A	1	2	8		103	71.6	58.9	25.5	9.42	4.14	3.90	1.26	4.38	0.780
Average							103	77.4	58.1	29.0	8.82	5.34	3.63	1.71	5.91	0.840
Std. Dev.							5	4.4	5.3	4.0	2.63	1.97	1.37	0.86	0.83	0.170
Coeff. Var.							4.6	5.6	9.2	13.6	29.8	36.9	37.6	50.0	14.1	20.2
EC50 (µg/ml). Effective concentration at 50% cytotoxicity. (using a specific sigmoidal equation = model). (could also be EC20 or EC30, EC70, etc.)																		123

to.xpt

do.xpt

di.xpt

Assumption: The intent of this dataset is to provide a summary of trial (study) information. This is not subject-level data.
Assumption: A Trial (study) can have more than one assay type
Assumption: ASSAYID value of ALL indicates that it applies to all assays in the study
Sponsor should be "applicant", how do we represent this? In text explanation in the IG? In definition in CT/Parmcd/Parm? ** Need to have CT discussion, include Erin M.**
TK6, human keratinocyte, is this the "test system"?
Can we draft a starting list of Study Types and Sub Types to submit to CT now?
Is there a dictionary of species and/or strain that we would use? (see example)

Row	STUDYID	ASSAYID	DOMAIN	TSSEQ	TSGRPID	TSPARMCD	TSPARM	TSVAL	TSVALNF
1	123	NRU	TS	1		GLPTYP	Good Laboratory Practice Type	FDA
2	123	NRU	TS	2		GLPTYP	Good Laboratory Practice Type	OECD
3	123	NRU	TS	1		STSTDTC	Study Start Date	2022-05-25
4	123	NRU	TS	1		STITLE	Study Title	Determination of the in vitro genotoxicity potential using the in vitro Neutral Red Uptake assay
5	123	NRU	TS	1		SNDIGVER	SEND Implementation Guide Version	TOBACCO IMPLEMENTATION GUIDE VERSION 1.0
6	123	NRU	TS	1		SNDCTVER	SEND Controlled Terminology Version	SEND Terminology 2021-09-30
7	123	NRU	TS	1		SSPONSOR	~~Sponsor~~ Applicant's Organization	Example Applicant Inc.
8	123	NRU	TS	1		SPREFID	~~Sponsor's~~ Applicant's Study Reference ID		NOT APPLICABLE
9	123	NRU	TS	1	1	TSTFNAM	Test Facility Name	Example ~~Tox~~ Testing Lab Name
10	123	NRU	TS	1	1	TSTFLOC	Test Facility Location	10 Somewhere Street, Montgomery, AL 10000
11	123	NRU	TS	1	1	TFCNTRY	Test Facility Country	USA
12	123	NRU	TS	1	1	STDIR	Study Director	Dr. R. Smith
13	123	NRU	TS	1		GLPFL	GLP Flag	Y
14	123	NRU	TS	1		ASTD	Assay Standard	NIH Publication No. 07-4519
15	123	NRU	TS	1		ASTDV	Assay Standard Version	2006-11
16	123	NRU	TS	1		SSTYP	Study Type	GENOTOXICITY IN VITRO
17	123	NRU	TS	1		SSSTYP	Study Sub Type	In Vitro Neutral Red Uptake
18	123	NRU	TS	1		SPECIES	Species	Salmonella enterica
19	123	NRU	TS	1		??	Test System??	TK6 Normal Human Keratinocyte

Assumption: The Trial Sets (TX) domain provides the list of distinct sets of subjects having different experimental factors, treatment factors, inherent characteristics, or distinct ~~sponsor~~ applicant designations as specified in the trial design.
During CT definition/reviews will decide appropriate TXPARM and TXVAL; Treatment duration may be controlled; For now, we just include good example values based on our experience
Where are the details of the smoke regime defined/submitted today? (e.g., puff profile = Bell shaped puffing volumes; Puff Block = 50%, etc.)
Smoke fraction? is this indicator of processing of material from smoke machine..we used Smoke Exposure System - ok?
Incubate cells (37 ºC ±1 ºC, 90% ±10% humidity, and 5% ±1% CO2/air) - these are ranges, do we model for ranges?
- https://ntp.niehs.nih.gov/iccvam/docs/protocols/ivcyto-nhk.pdf

Row	STUDYID	ASSAYID	DOMAIN	SETCD	SET (what sponsor calls it)	TXSEQ	TXPARMCD	TXPARM	TXVAL
1	123	NRU	TX	CSC-50	CSC-50	1	TRTDMIN	Treatment Duration Minimum	47.5
2	123	NRU	TX	CSC-50	CSC-50	2	TRTDTRG	Treatment Duration Target	48
3	123	NRU	TX	CSC-50	CSC-50	3	TRTDMAX	Treatment Duration Maximum	48.5
4	123	NRU	TX	CSC-50	CSC-50	4	TRTDU	Treatment Duration Unit	HOURS
5	123	NRU	TX	CSC-50	CSC-50	5	INCBTMP	Incubation Temperature	37
6	123	NRU	TX	CSC-50	CSC-50	6	INCBTMPU	Incubation Temperature Unit	C
7	123	NRU	TX	CSC-50	CSC-50	7	WAVELGTH	Spectrophotometer WAVELENGTH IN NANOMETERS	540
8	123	NRU	TX	CSC-50	CSC-50	8	HUMID	Atmospheric Relative Humidity Percent	90
9	123	NRU	TX	CSC-50	CSC-50	9	ATMCO2	Atmospheric CO2 Percent	5
10	123	NRU	TX	CSC-50	CSC-50	10	SPTOBID	Sponsor defined tobacco identifier	CIG01a
11	123	NRU	TX	CSC-50	CSC-50	11	EXPTYP	Exposure Type	Air Liquid Interface???
12	123	NRU	TX	CSC-50	CSC-50	12	SAMTYP	Sample Type	Whole Smoke Conditioned Media???
13	123	NRU	TX	CSC-50	CSC-50	13	INTRVN	Name of the Intervention Article	Tobacco ProdA
14	123	NRU	TX	CSC-50	CSC-50	14	ITVTYPE (CT e.g, Product, positive control, negative control, vehicle control, solvent control)	type of intervention article	PRODUCT
15	123	NRU	TX	CSC-50	CSC-50	15	ITVCONC	intervention concentration	50
16	123	NRU	TX	CSC-50	CSC-50	16	ITVCONCU	intervention unit	ug/ml
17	123	NRU	TX	CSC-50	CSC-50	17	STRAIN	Strain/Substrain	Salmonella enterica enterica
18	123	NRU	TX	CSC-50	CSC-50	18	SPDEVID	Sponsor defined device identifier	PUFFMASTER3k
19	123	NRU	TX	CSC-50	CSC-50	19	DUREFID	Smoking Regime	Medium Intensity Regimen
20	123	NRU	TX	CSC-50	CSC-50	20	SPTOBID	Sponsor defined tobacco identifier	CIG01a
21	123	NRU	TX	SLS-110	SLS-110	1	TRTDMIN	Treatment Duration Minimum	47.5
22	123	NRU	TX	SLS-110	SLS-110	2	TRTDTRG	Treatment Duration Target	48
23	123	NRU	TX	SLS-110	SLS-110	3	TRTDMAX	Treatment Duration Maximum	48.5
24	123	NRU	TX	SLS-110	SLS-110	4	TRTDU	Treatment Duration Unit	HOURS
25	123	NRU	TX	SLS-110	SLS-110	5	INCBTMP	Incubation Temperature	37
26	123	NRU	TX	SLS-110	SLS-110	6	INCBTMPU	Incubation Temperature Unit	C
27	123	NRU	TX	SLS-110	SLS-110	7	WAVELGTH	Spectrophotometer WAVELENGTH IN NANOMETERS	540
28	123	NRU	TX	SLS-110	SLS-110	8	HUMID	Atmospheric Relative Humidity Percent	90
29	123	NRU	TX	SLS-110	SLS-110	9	ATMCO2	Atmospheric CO2 Percent	5
30	123	NRU	TX	SLS-110	SLS-110	10	SPTOBID	Sponsor defined tobacco identifier	CIG01a
31	123	NRU	TX	SLS-110	SLS-110	11	EXPTYP	Exposure Type	Air Liquid Interface
32	123	NRU	TX	SLS-110	SLS-110	12	SAMTYP	Sample Type	Whole Smoke Conditioned Media
33	123	NRU	TX	SLS-110	SLS-110	13	INTRVN	Name of the Intervention Article	Sodium Laurel Sulfate
34	123	NRU	TX	SLS-110	SLS-110	14	ITVTYPE (CT e.g, Product, positive control, negative control, vehicle control, solvent control)	type of intervention article	POSITIVE CONTROL
35	123	NRU	TX	SLS-110	SLS-110	15	ITVCONC	intervention concentration	200
36	123	NRU	TX	SLS-110	SLS-110	16	ITVCONCU	intervention unit	ug/ml
37	123	NRU	TX	SLS-110	SLS-110	17	STRAIN	Strain/Substrain	Salmonella enterica enterica
38	123	NRU	TX	SLS-110	SLS-110	18	SPDEVID	Sponsor defined device identifier	PUFFMASTER3k
39	123	NRU	TX	SLS-110	SLS-110	19	DUREFID	Smoking Regime	Medium Intensity Regimen
40	123	NRU	TX	SLS-110	SLS-110	20	SPTOBID	Sponsor defined tobacco identifier	CIG01a

ou.xpt. Observational and Experimental Units domain

Experimental unit - The unit to which the treatment is applied, eg. Test tube

Observational unit - The unit on which the response is measured. (This may not be the same as the experimental unit.)

Row	STUDYID	ASSAYID	DOMAIN	SETCD (from TX)	EUID	OBUID
1	123	NRU	OU	CSC-50	1-3-B	1-3-B
2	123	NRU	OU	CSC-50	1-3-C	1-3-C
3	123	NRU	OU	SLS-110	1-11-G	1-11-G
4	123	NRU	OU	SLS-110	2-11-G	2-11-G

cells growing in plate, treat cells in the plate, then add NRU dye, then extract NRU (the OBUID) dye and transfer to another plate (put in spectrophotometer & read).

.

Row	STUDYID	ASSAYID	DOMAIN	SETCD	EUID	OBUID	GTSEQ	GTTESTCD	GTTEST	GTORRES	GTORRESU	GTCOLSRT (coll. summ. result type)	GTSTRESC	GTSTRESN	GTSTRESU
	123	NRU	GT	CSC-50	1-3-B	1-3-B	1	ABSORB	Absorbance Reading	1.7 (0.0 - 2.0)			1.7	1.7
	123	NRU	GT	CSC-50	1-3-B	1-3-B	2	BKCABS	Background Corrected Absorbance Reading	1.6			1.6	1.6
	123	NRU	GT	CSC-50	1-3-B	1-3-B	3	RELABS	Relative Absorbance Reading	77.7	%		77.7	77.7	%
	123	NRU	GT	CSC-50			4	RELABS	Relative Absorbance Reading	78.2	%	MEAN	78.2	78.2	%
	123	NRU	GT	CSC-50			5	RELABS	Relative Absorbance Reading	2.9	%	STANDARD DEVIATION	2.9	2.9	%
	123	NRU	GT	CSC-50			6	RELABS	Relative Absorbance Reading	3.6		COEFFICIENT OF VARIANCE	3.6	3.6
	123	NRU	GT	CSC-50			7	EC50	Effective Concentration of 50 Percent Cytotoxicity	123	ug/ml		123	123	ug/ml
	123	NRU	GT		1-2-C		1	RELABS	Relative Absorbance Reading	77.4			77.4	77.4
...
80	123	NRU	GT		1-11-G		1	RELABS	Relative Absorbance Reading	8.57	ug/ml		8.57	8.57	ug/ml
...					...
160	123	NRU	GT		2-11-G		1	RELABS	Relative Absorbance Reading	6.30	ug/ml		6.30	6.30	ug/ml

Page tree

SEND Example.In vitro Neutral Red Cytotoxicity Assay

Note that Variable Names and Domain abbreviations may change (have not been checked against existing and are not vetted)

SET (what sponsor calls it)

(CT e.g, Product, positive control, negative control, vehicle control, solvent control)

(CT e.g, Product, positive control, negative control, vehicle control, solvent control)