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Comet Assay IV & JaCVAM study

The Japanese Center for the Validation of Alternative Methods (JaCVAM) initiative, International Validation Study of the in vivo rat alkaline comet assay commenced in 2006.  This initiative resulted in the publication of the OECD Guideline for Test No. 489: In Vivo Mammalian Alkaline Comet Assay in September 2014.

We have been delighted to see the recent publication of several investigations conducted as part of the JaCVAM exercise, these papers involve Comet Assay IV, the comet assay scoring system from Perceptive Instruments.

Please find the selected papers below. For additional information regarding any of the mentioned publications, we suggest you refer to the original texts.









 

 

p-Chloroaniline, t-butylhydroquinone, and methyl carbamate: Rat in vivo comet test, JaCVAM trial phase 4.2, July 2015

During this investigation, scientists from Huntingdon Life Sciences, UK, tested three compounds, p-Chloroaniline, t-butylhydroquinone, and methyl carbamate as part of the JaCVAM international validation study of in vivo rat alkaline comet assay. The assay slides were scored using the Comet Assay IV image analysis system (Perceptive Instruments). This study concluded that:

  1. p-Chloroaniline produced a statistically significant increase in the % tail intensity.

  2. t-Butylhydroquinone caused a non biologically relevant increase in % tail intensity.

  3. Methyl carbamate did not induce a statistically significant change in % tail intensity.

Results of the International Validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: Individual data for 1,2-dibromoethane, p-anisidine, and o-anthranilic acid in the 2nd step of the 4th phase Validation Study under the JaCVAM initiative,  July 2015

As part of the JaCVAM initiative, researchers from LSI Medience Corporation and Yakult Honsha Co., Ltd, (both Japan) examined three compounds to determine the effectiveness of the comet assay at detecting genotoxic carcinogens. 1,2-dibromoethane (DBE), p-anisidine (ASD), and o-anthranilic acid (ANT) were examined. The image analysis system, Comet Assay IV from Perceptive Instruments was used to analyse comet slides. The key points of this investigation are;

  1. A genotoxic-carcinogen, DBE gave positive results in the assay in both the tissues.

  2. Two non-carcinogens, ASD and ANT showed negative responses in both the tissues.

  3. The combination of the comet and bone marrow micronucleus assays can be done easily.

  4. The combination presents useful information to predict genotoxic rodent carcinogens

Evaluation of p-phenylenediamine, o-phenylphenol sodium salt, and 2,4-diaminotoluene in the rat comet assay as part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet assay, July 2015

Scientists from Janssen Research & Development in Belgium were also involved with the validation of the in vivo rat alkaline comet assay. The compounds p-phenylenediamine dihydrochloride (PPD), o-phenylphenol sodium salt (OPP), and 2,4-diaminotoluene (2,4-DAT), were analysed as part of the JaCVAM initiative. Comet slides were scored using the Comet Assay IV image analysis system (Perceptive Instruments). Highlights of this study include:

  1. Three coded compounds were evaluated in the in vivo rat alkaline comet assay.

  2. PPD and OPP were negative up to 100 and 1000 mg/kg/day, respectively.

  3. 2,4-DAT (genotoxic carcinogen) was weak positive in liver and negative in stomach.

Investigation of sodium arsenite, thioacetamide, and diethanolamine in the alkaline comet assay: Part of the JaCVAM comet validation exercise, April 2015

Researchers from Covance Laboratories Ltd. examined sodium arsenite, thioacetamide, and diethanolamine as part of the JaCVAM-initiative international validation study of the in vivo rat alkaline comet assay.  To analyse the data, a Comet Assay IV system was used. This particular study concluded that:

  1.  Sodium arsenite produced equivocal findings in rat liver but was clearly negative in stomach.

  2. Thioacetamide caused an increase in DNA migration in rat stomach but was negative in liver.

  3.   No evidence of DNA damage was observed in the stomach or liver of rats treated with diethanolamine.

Combination comet/micronucleus assay validation performed by BioReliance under the JaCVAM initiative, March 2015

This paper describes comet validation at BioReliance under the JaCVAM initiative.  Three chemicals were tested, 1,3-dichloropropene, ethionamide & busulfan, and a Comet Assay IV system was used to analyse the comet data.  The scientists concluded that:

1.      1,3-Dichoropropene was positive in comet assay and negative in micronucleus assay.

2.       Ethionamide was negative in both comet and micronucleus assays.

3.       Busulfan was negative in comet assay and positive in micronucleus assay.

 Comet assay evaluation of six chemicals of known genotoxic potential in rats, March 2015

This investigation was a collaboration between scientists based at Integrated Laboratory Systems and the National Institute of Environmental Health Sciences, (both Research Triangle Park, NC, USA) and Biosafety Research Center (Shizuoka, Japan).   They examined six chemicals for potential to induce DNA damage: 2-acetylaminofluorene (2-AAF), N-nitrosodimethylamine (DMN), o-anisidine, 1,2-dimethylhydrazine dihydrochloride (1,2-DMH), sodium chloride, and sodium arsenite. Five genotoxic carcinogens were tested for induction of DNA damage in rats.  Liver and stomach cells of exposed rats were analysed using the comet assay. After staining slides with SYB Gold 100 cells were scored per sample using Comet Assay IV image analysis software.  This particular study concluded that:

  1. Only two (1,2-DMH and DMN) of the five genotoxic carcinogens yielded positive results.

  2. Exposure to 2-AAF or o-anisidine led to equivocal results in the liver of rats.

  3. Negative results for sodium arsenite are consistent with its mechanism of action. 

Use of a standardized JaCVAM in vivo rat comet assay protocol to assess the genotoxicity of three coded test compounds; ampicillin trihydrate, 1,2-dimethylhydrazine dihydrochloride, and N-nitrosodimethylamine, February 2015

Researchers from Consumer and Clinical Radiation Protection Bureau (Health Canada) were also involved with the JaCVAM validation of the in vivo rat alkaline comet assay.  The comet slides were analysed by fluorescence microscopy (Olympus BX60 microscope) at 200× magnification using the Comet Assay IV image analysis system (Perceptive Instruments, UK). The investigators concluded that:

  1. Ampicillin was not genotoxic in the rat liver or stomach.

  2.    1,2-Dimethylhydrazine dihydrochloride was genotoxic in rat liver, but not stomach.

  3.     N-Nitrosodimethylamine was genotoxic in rat liver, but not stomach.

For more information about the studies mentioned here, please follow the below links:

Chloroaniline, t-butylhydroquinone, and methyl carbamate: Rat in vivo comet test, JaCVAM trial phase 4.2, July 2015

Results of the International Validation of the in vivo rodent alkaline comet assay for the detection of genotoxic carcinogens: Individual data for 1,2-dibromoethane, p-anisidine, and o-anthranilic acid in the 2nd step of the 4th phase Validation Study under the JaCVAM initiative, July 2015.

Evaluation of p-phenylenediamine, o-phenylphenol sodium salt, and 2,4-diaminotoluene in the rat comet assay as part of the Japanese Center for the Validation of Alternative Methods (JaCVAM)-initiated international validation study of in vivo rat alkaline comet assay, July 2015

Investigation of sodium arsenite, thioacetamide, and diethanolamine in the alkaline comet assay: Part of the JaCVAM comet validation exercise, April 2015

Combination comet/micronucleus assay validation performed by BioReliance under the JaCVAM initiative, March 2015

Comet assay evaluation of six chemicals of known genotoxic potential in rats, March 2015

 Use of a standardized JaCVAM in vivo rat comet assay protocol to assess the genotoxicity of three coded test compounds; ampicillin trihydrate, 1,2-dimethylhydrazine dihydrochloride, and N-nitrosodimethylamine, February 2015

 

 

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