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)
						10	50	75	100	120	140	160	200	110	200
1-1	030001	A	1	1	1	100	83.0	56.7	29.5	13.0	8.57	4.44	1.76	13.7	0.547
1-1	030001	A	1	1	2	107	77.7	65.7	30.2	15.6	9.79	5.41	2.49	8.09	0.791
1-1	030001	A	1	1	3	98.6	77.4	57.7	35.8	8.09	7.36	3.22	0.791	6.38	0.061
1-1	030001	A	1	1	4	94.9	81.5	38.2	31.2	7.60	5.17	3.22	0.791	5.17	0.304
1-1	030001	A	1	1	5	111	78.1	64.5	32.9	8.82	5.90	2.74	1.28	10.3	0.547
1-1	030001	A	1	1	6	96.6	76.2	48.0	26.8	7.84	4.20	2.74	1.52	8.09	4.93
1-1	030001	A	1	1	7	105	77.9	58.2	28.3	8.09	4.68	1.76	0.547	8.57	1.76
1-1	030001	A	1	1	8	95.2	74.0	47.2	24.4	8.57	3.71	3.47	0.304	11.2	0.791
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
EC50 (µg/ml). Effective concentration at 50% cytotoxicity. (using a specific sigmoidal equation = model).																123
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 (mg/ml). Effective concentration at 50% cytotoxicity																130

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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	1-2-B (plate-col-row in the 96-well plate)	CSC-50 (through SLS-200)	1	PLATENUM. (e.g., 1 or 2)	1	1
2	123	NRU	TX	1-2-B	CSC-50	2	WELLNUM (e.g., 1-A through 10-H)	Number representing the location of the well in a multiple-well plate	2-B
3	123	NRU	TX	1-2-B	CSC-50	4	ITVTYPE (CT e.g, Product, positive control, negative control, vehicle control, solvent control)	type of intervention article	PRODUCT
	123	NRU	TX	1-2-B	CSC-50	5	ITVCONC	intervention concentration	50
	123	NRU	TX	1-2-B	CSC-50	6	ITVCONCU	intervention unit	ug/ml
	123	NRU	TX	1-2-B	CSC-50	7	STRAIN	Strain/Substrain	Salmonella enterica enterica
	123	NRU	TX	1-2-B	CSC-50	8	SPDEVID	Sponsor defined device identifier	PUFFMASTER3k
	123	NRU	TX	1-2-B	CSC-50	9	DUREFID	Smoking Regime	Medium Intensity Regimen
	123	NRU	TX	1-2-B	CSC-50	10	RUN	Assay run number	1
	123	NRU	TX	1-2-B	CSC-50	11	SMPLID	Sample ID	030001
	123	NRU	TX	1-2-B	CSC-50	12	SMKEXSYS	Smoke Exposure System	Cambridge filter pad, eluted in DMSO
	123	NRU	TX	1-2-B	CSC-50	13	REPNUM	Replicate Number	1
	123	NRU	TX	1-2-B	CSC-50	14	TRTDMIN	Treatment Duration Min	47.5
	123	NRU	TX	1-2-B	CSC-50	15	TRTDTRG	Treatment Duration Target	48
	123	NRU	TX	1-2-B	CSC-50	16	TRTDMAX	Treatment Duration Max	48.5
	123	NRU	TX	1-2-B	CSC-50	17	TRTDU	Treatment Duration Unit	HOURS
	123	NRU	TX	1-2-B	CSC-50	18	INCBTMP	Incubation Temperature	37
	123	NRU	TX	1-2-B	CSC-50	19	INCBTMPU	Incubation Temperature Unit	C
	~~123~~	~~NRU~~	TX	~~1-2-B~~	~~CSC-50~~	20	~~MEDIA~~	~~Composition of Media~~	~~Sartorius HEK293~~
							WAVELGTH	Spectrophotometer WAVELENGTH IN NANOMETERS	540
	123	NRU	TX	1-2-B	CSC-50	21	HUMID	Atmospheric Relative Humidity Percent	90
	123	NRU	TX	1-2-B	CSC-50	22	ATMCO2	Atmospheric CO2 Percent	5
	123	NRU	TX	1-2-B	CSC-50	23	SPTOBID	Sponsor defined tobacco identifier	CIG01a
	123	NRU	TX	1-2-B	CSC-50	24	EXPTYP	Exposure Type	Air Liquid Interface
	123	NRU	TX	1-2-B	CSC-50	25	SAMTYP	Sample Type	Whole Smoke Conditioned Media
	123	NRU	TX	1-2-C	CSC-50	1	PLATENUM. (e.g., 1 or 2)	1	1
	123	NRU	TX	1-2-C	CSC-50	2	WELLNUM (e.g., 1-A through 10-H)	Number representing the location of the well in the 96-well plate	1-A
	123	NRU	TX	1-2-C	CSC-50	3	INTRVN	name of the intervention article	Cigarette Smoke Condensate
	123	NRU	TX	1-2-C	CSC-50	4	ITVTYPE (CT e.g, Product, positive control, negative control)	type of intervention article	PRODUCT
	123	NRU	TX	1-2-C	CSC-50	5	ITVCONC	intervention concentration	10
	123	NRU	TX	1-2-C	CSC-50	6	ITVCONCU	intervention unit	ug/ml
	123	NRU	TX	1-2-C	CSC-50	7	STRAIN	Strain/Substrain	Salmonella enterica enterica
	123	NRU	TX	1-2-C	CSC-50	8	SPDEVID	Sponsor defined device identifier	PUFFMASTER3k
	123	NRU	TX	1-2-C	CSC-50	9	DUREFID	Smoking Regime	Medium Intensity Regimen
	123	NRU	TX	1-2-C	CSC-50	10	RUN	Assay run number	1
	123	NRU	TX	1-2-C	CSC-50	11	SMPLID	Sample ID	030001
	123	NRU	TX	1-2-C	CSC-50	12	SMKEXSYS	Smoke Exposure System	Cambridge filter pad, eluted in DMSO
	123	NRU	TX	1-2-C	CSC-50	13	REPNUM	Replicate Number	1
	123	NRU	TX	1-2-C	CSC-50	14	TRTDMIN	Treatment Duration Min	47.5
	123	NRU	TX	1-2-C	CSC-50	15	TRTDTRG	Treatment Duration Target	48
	123	NRU	TX	1-2-C	CSC-50	16	TRTDMAX	Treatment Duration Max	48.5
	123	NRU	TX	1-2-C	CSC-50	17	TRTDU	Treatment Duration Unit	HOURS
	123	NRU	TX	1-2-C	CSC-50	18	INCBTMP	Incubation Temperature	37
	123	NRU	TX	1-2-C	CSC-50	19	INCBTMPU	Incubation Temperature Unit	C
	~~123~~	~~NRU~~	TX	~~1-2-C~~		20	~~MEDIA~~	~~Composition of Media~~	~~Sartorius HEK293~~
	123	NRU	TX	1-2-C	CSC-50	21	HUMID	Atmospheric Relative Humidity Percent	90
	123	NRU	TX	1-2-C	CSC-50	22	ATMCO2	Atmospheric CO2 Percent	5
	123	NRU	TX	1-2-C	CSC-50	23	SPTOBID	Sponsor defined tobacco identifier	CIG01a
	123	NRU	TX	1-10-H	CSC-50	24	EXPTYP	Exposure Type	Air Liquid Interface
	123	NRU	TX	1-10-H	CSC-50	25	SAMTYP	Sample Type	Whole Smoke Conditioned Media
...				...
	123	NRU	TX	1-10-H	SLS-110	1	PLATENUM	Plate number	1
	123	NRU	TX	1-10-H	SLS-110	2	WELLNUM	Number representing the location of the well in the 96-well plate	10-H
	123	NRU	TX	1-10-H	SLS-110	3	INTRVN	name of the intervention article	sodium laurel sulfate
	123	NRU	TX	1-10-H	SLS-110	4	ITVTYPE	type of intervention article	POSITIVE CONTROL
	123	NRU	TX	1-10-H	SLS-110	5	ITVCONC	intervention concentration	200
	123	NRU	TX	1-10-H	SLS-110	6	ITVCONCU	intervention unit	ug/ml
	123	NRU	TX	1-10-H	SLS-110	7	STRAIN	Strain/Substrain	Salmonella enterica enterica
	123	NRU	TX	1-10-H	SLS-110	8	SPDEVID	Sponsor defined device identifier	PUFFMASTER3k
	123	NRU	TX	1-10-H	SLS-110	9	DUREFID	Smoking Regime	Medium Intensity Regimen
	123	NRU	TX	1-10-H	SLS-110	10	RUN	Assay run number	1
	123	NRU	TX	1-10-H	SLS-110	11	SMPLID	Sample ID	030001
	123	NRU	TX	1-10-H	SLS-110	12	SMKEXSYS	Smoke Exposure System	Cambridge filter pad, eluted in DMSO
	123	NRU	TX	1-10-H	SLS-110	13	REPNUM	Replicate Number	1
	123	NRU	TX	1-10-H	SLS-110	14	TRTDMIN	Treatment Duration Min	47.5
	123	NRU	TX	1-10-H	SLS-110	15	TRTDTRG	Treatment Duration Target	48
	123	NRU	TX	1-10-H	SLS-110	16	TRTDMAX	Treatment Duration Max	48.5
	123	NRU	TX	1-10-H	SLS-110	17	TRTDU	Treatment Duration Unit	HOURS
	123	NRU	TX	1-10-H	SLS-110	18	INCBTMP	Incubation Temperature	37
	123	NRU	TX	1-10-H	SLS-110	19	INCBTMPU	Incubation Temperature Unit	C
	123	NRU	TX	1-10-H	SLS-110	20	~~MEDIA~~	~~Composition of Media~~	~~Sartorius HEK293~~
	123	NRU	TX	1-10-H	SLS-110	21	HUMID	Atmospheric Relative Humidity Percent	90
	123	NRU	TX	1-10-H	SLS-110	22	ATMCO2	Atmospheric CO2 Percent	5
	123	NRU	TX	1-10-H	SLS-110	23	SPTOBID	Sponsor defined tobacco identifier	CIG01a
	123	NRU	TX	1-10-H	SLS-110	24	EXPTYP	Exposure Type	Air Liquid Interface
	123	NRU	TX	1-10-H	SLS-110	25	SAMTYP	Sample Type	Whole Smoke Conditioned Media
...				...
	123	NRU	TX	2-10-H	SLS-110	1	PLATENUM	Plate number	2
	123	NRU	TX	2-10-H	SLS-110	2	WELLNUM	Number representing the location of the well in the 96-well plate	10-H
	123	NRU	TX	2-10-H	SLS-110	3	INTRVN	name of the intervention article	sodium laurel sulfate
	123	NRU	TX	2-10-H	SLS-110	4	ITVTYPE	type of intervention article	POSITIVE CONTROL
	123	NRU	TX	2-10-H	SLS-110	5	ITVCONC	intervention concentration	200
	123	NRU	TX	2-10-H	SLS-110	6	ITVCONCU	intervention unit	ug/ml
	123	NRU	TX	2-10-H	SLS-110	7	STRAIN	Strain/Substrain	Salmonella enterica enterica
	123	NRU	TX	2-10-H	SLS-110	8	SPDEVID	Sponsor defined device identifier	PUFFMASTER3k
	123	NRU	TX	2-10-H	SLS-110	9	DUREFID	Smoking Regime	Medium Intensity Regimen
	123	NRU	TX	2-10-H	SLS-110	10	RUN	Assay run number	1
	123	NRU	TX	2-10-H	SLS-110	11	SMPLID	Sample ID	030001
	123	NRU	TX	2-10-H	SLS-110	12	SMKEXSYS	Smoke Exposure System	Cambridge filter pad, eluted in DMSO
	123	NRU	TX	2-10-H	SLS-110	13	REPNUM	Replicate Number	1
	123	NRU	TX	2-10-H	SLS-110	14	TRTDMIN	Treatment Duration Min	47.5
	123	NRU	TX	2-10-H	SLS-110	15	TRTDTRG	Treatment Duration Target	48
	123	NRU	TX	2-10-H	SLS-110	16	TRTDMAX	Treatment Duration Max	48.5
	123	NRU	TX	2-10-H	SLS-110	17	TRTDU	Treatment Duration Unit	HOURS
	123	NRU	TX	2-10-H	SLS-110	18	INCBTMP	Incubation Temperature	37
	123	NRU	TX	2-10-H	SLS-110	19	INCBTMPU	Incubation Temperature Unit	C
	123	NRU	TX	2-10-H	SLS-110	20	~~MEDIA~~	~~Composition of Media~~	~~Sartorius HEK293~~
	123	NRU	TX	2-10-H	SLS-110	21	HUMID	Atmospheric Relative Humidity Percent	90
	123	NRU	TX	2-10-H	SLS-110	22	ATMCO2	Atmospheric CO2 Percent	5
	123	NRU	TX	2-10-H	SLS-110	23	SPTOBID	Sponsor defined tobacco identifier	CIG01a
	123	NRU	TX	2-10-H	SLS-110	24	EXPTYP	Exposure Type	Air Liquid Interface
	123	NRU	TX	2-10-H	SLS-110	25	SAMTYP	Sample Type	Whole Smoke Conditioned Media
...

Entity IDs are unique within the assay (and a study can have many assays)
Replicates vs. Run
- need planned (ei.xpt) separate from the actual (gt.xpt)
- If tests run at the same time, they are replicates and are assigned the same RUNID
- If tests are run at separate times, they are runs and the RUNIDs are distinct
Different runs would be expected to have different ENID and GTDTC values. In ei.xpt, these are distinguished by different RUNID values.
Entities that are replicates of each other would be expected to
- have different ENID values
- be in the same trial set (SETCD values)
- have the same RUNID values

In this example, SETCD and ENID are the same value.

STUDYID	ASSAYID	DOMAIN	ENID (Entity ID)	SETCD (from TX)	RUNID
123	NRU	EI	1-2-B	1-2-B	1
123	NRU	EI	1-2-E?	1-2-E	2
			2-11-G	2-11-G	1
			2-11-F?	2-11-F	2

.

*** Lou Ann:

new pictures with blank media on perimeter

Data values in gt.xpt will be from 2b (1a), 2c (2b), 10g (not 10H) on both plates

Row	STUDYID	ASSAYID	DOMAIN	ENID	GTSEQ	GTTESTCD	GTTEST	GTORRES	GTORRESU	GTSTRESC	GTSTRESN	GTSTRESU
	123	NRU	GT	1-2-B	1	ABSORB	Absorbance Reading	1.7 (0.0 - 2.0)	ug/ml	1.7	1.7	ug/ml
	123	NRU	GT	1-2-B	1	BKCABS	Background Corrected Absorbance Reading	1.6	ug/ml	1.6	1.6	ug/ml
	123	NRU	GT	1-2-B	1	RELABS	Relative Absorbance Reading	77.7	ug/ml	77.7	77.7	ug/ml
	123	NRU	GT	1-2-B	1			77.7	ug/ml	77.7	77.7	ug/ml
	123	NRU	GT	1-2-C	1	RELABS	Relative Absorbance Reading	77.4	ug/ml	77.4	77.4	ug/ml
...
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

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

SET (what sponsor calls it)

(plate-col-row in the 96-well plate)

(through SLS-200)

PLATENUM. (e.g., 1 or 2)

WELLNUM (e.g., 1-A through 10-H)

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

PLATENUM. (e.g., 1 or 2)

WELLNUM (e.g., 1-A through 10-H)

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