3T3 Neutral Red Uptake cytotoxicity assay

Topic: Acute toxicity, Basal Cytotoxicity

Test Method Number:

TM2007-03 (EU)

Short Name of TM:

3T3 NRU

Year received:

2007

Responsible Organisation:

EURL ECVAM - European Union

Protocol(s)/SOP(s):

139

General Comments:

The validation study assessed the capacity of the 3T3 NRU cytotoxicity assay to identify specifically non-classified chemicals on the basis of the 2000 mg/kg b.w. threshold introduced by the EU CLP Regulation.

Preceding TM:

TM2003-01 - Neutral red uptake for starting doses for acute oral toxicity

Method Description

The 3T3 NRU cytotoxicity assay employs the BALB/c3T3 mouse fibroblast cell line and is based on the ability of viable cells to incorporate and bind the dye Neutral Red (NR), measured spectrophotometrically . The basic concept of basal cytotoxicity assays is that chemicals exert their toxic effects by disrupting structures and functions universal to all cells.
The assay used the test method protocol and the IC50-LD50 regressions of the NRU cytotoxicity method, validated in the NICEATM/ECVAM validation study (see TM2003-01) and was proposed to identify chemicals that would not require classification for acute oral toxicity according to the EU CLP Regulation (i.e. LD50 > 2 000 mg/kg). It was envisaged that use of the 3T3 NRU test method to identify unclassified substances in a testing strategy, to support a read-across, or as part of a weight-of-evidence approach could potentially reduce the amount of animal testing for acute oral toxicity.

Track Approval Status

Submission
Validation
Peer-review
Recommendation
Regulatory acceptance/Standards

Submission

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Date	Stage	Comment	Documents/Links
Jun 2007	Not applicable	See Validation Step

Validation

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Date	Stage	Comment	Documents/Links
Oct 2008	Planning	First validation meeting at EURL ECVAM together with representatives of the test facilities. Validation study conducted by initiative of EURL ECVAM as a follow-up study on the predictive capacity of the 3T3 Neutral Red Uptake cytotoxicity assay to correctly identify substances not classified for acute oral toxicity under the EU CLP system (LD50 > 2 000 mg/kg)
Mar 2011	Finalised	Completion of the EURL ECVAM coordinated validation with Validation Report.

Peer-review

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Date	Stage	Comment	Documents/Links
Jun 2011	Ongoing	Meeting of the ESAC Working Group (WG) 3T3 NRU	ESAC documents
Oct 2011	Ongoing	Endorsement of the WG peer review consensus report at the 35th ESAC plenary meeting.	ESAC Working Group Consensus Report
Mar 2012	Finalised	ESAC Opinion of the ECVAM-coordinated follow-up study to assess the predictive capacity of the validated 3T3 Neutral Red Uptake cytotoxicity assay for acute oral toxicity testing included in the EURL ECVAM Recommendation (see Recommendation step).

Recommendation

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Date	Stage	Comment	Documents/Links
Apr 2013	Published	EURL ECVAM Recommendation on the 3T3 Neutral Red Uptake Cytotoxicity Assay for Acute Oral Toxicity Testing	EURL ECVAM Recommendation

Regulatory acceptance/Standards

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Date	Stage	Comment	Documents/Links
Oct 2015	Adopted/published	Inclusion in the ECHA Guidance on Information Requirements and Chemical Safety Assessment, Chapter R.7a: Endpoint specific guidance.	Endpoint specific guidance (Chapter R.7a)