Disodium 2-(2,4,5,7-tetraiodo-6-oxido-3-oxoxanthen-9-yl)benzoate

Toxicity to microorganisms

Link to relevant study record(s)

Reference

Endpoint:

toxicity to microorganisms

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Data is from peer reviewed journal

Justification for type of information:

Data is from peer reviewed journal

Qualifier:

according to guideline

Guideline:

other: as mentioned below

Principles of method if other than guideline:

Toxicity to micro-organisms study was conducted on Staphylococcus aureus of enterotoxigenic strain ATCC 13565.

GLP compliance:

not specified

Analytical monitoring:

yes

Details on sampling:

- Concentrations: 100 mg/l
- Sampling method: For the preparation of test solution, a test portion (approximately 10 mg) was placed in a 10-ml volumetric flask, which was wrapped in aluminum foil, and dissolved by adding aqueous sodium hydroxide (approx. 0.4% NaOH) (1 ml) and water (9 ml) (high-pressure liquid chromatography grade). The solution obtained by this procedure were found to be stable for at least 6 months in refrigerator at 4°C.

Vehicle:

yes

Test organisms (species):

other: Staphylococcus aureus ATCC 13565

Details on inoculum:

Details on test organisms:
- Laboratory culture: The test organism S. aureus ATCC 13565 was obtained from American Type Culture Collection, Manassas, Va.
- Method of cultivation: For the study, a 300-µl aliquot of dye solution (1 mg/ml) was mixed with 0.7 ml of BHI and then added to a tube of diluted culture. The tubes were aerated vigorously, and the OD was measured every 45 min to check culture growth. The survival rate was measured by plating serial dilutions of a sample taken during the experiment on BHI plates.
- Preparation of inoculum for exposure: For inoculum preparation, brain heart infusion broth (BHI) was used. Cultures were incubated shaking (200 rpm) at 37°C. All the experiments were conducted under conditions of standard room illumination (fluorescent ceiling light, Sylvania Octron 4100K) in tubes containing 3 ml of medium. Growth was analyzed by measuring turbidity (absorbance at a wavelength of 660 to 680 nm) with a spectrophotometer (Spectronic 21D). Experiments were initiated with ̴0.2 ml of inoculum from a fresh culture grown aerobically at an optical density (OD) of ̴1. Colony counts were measured on BHI plates.

Test type:

not specified

Water media type:

not specified

Total exposure duration:

250 min

Dissolved oxygen:

OD was measured every 45 mins to check culture growth

Nominal and measured concentrations:

Nominal concentrations were used in the study

Details on test conditions:

TEST SYSTEM
- Test vessel: Test tubes
- Aeration: The tubes containing the test solutions and cultures were aerated vigorously.
- No. of vessels per concentration (replicates): triplicate
- No. of vessels per vehicle control (replicates): triplicate

- Test concentrations: 100 mg/l

Reference substance (positive control):

not specified

Key result

Duration:

250 min

Dose descriptor:

NOEC

Effect conc.:

100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

growth inhibition

Remarks on result:

other: Other details not known

Validity criteria fulfilled:

not specified

Conclusions:

As the test substance do not have any activity against S. aureus i.e, no difference and no growth rate inhibition was observed between the treated test tubes and the control tubes. The NOEC value was observed to be 100 mg/l.

Executive summary:

Toxicity to micro-organisms study was conducted on Staphylococcus aureus of enterotoxigenic strain ATCC 13565. High-pressure liquid chromatography grade were used for analytical measurements. Inoculum was prepared in brain heart infusion broth (BHI). Cultures were incubated shaking (200 rpm) at 37°C. All the experiments were conducted under conditions of standard room illumination (fluorescent ceiling light, Sylvania Octron 4100K) in tubes containing 3 ml of medium. Growth was analyzed by measuring turbidity (absorbance at a wavelength of 660 to 680 nm) with a spectrophotometer (Spectronic 21D). Experiments were initiated with   ̴ 0.2 ml of inoculum from a fresh culture grown aerobically at an optical density (OD) of   ̴ 1. Colony counts were measured on BHI plates. OD was measured every 45 mins to check culture growth. The survival rate was measured by plating serial dilutions of a sample taken during the experiment on BHI plates. Experiment was performed in triplicate on separate days to ascertain reproducibility. As the test substance do not have any activity against S. aureus i.e, no difference and no growth rate inhibition was observed between the treated test tubes and the control tubes, the NOEC value was observed to be 100 mg/l.

Description of key information

Toxicity to micro-organisms study was conducted on Staphylococcus aureus of enterotoxigenic strain ATCC 13565. High-pressure liquid chromatography grade were used for analytical measurements. Inoculum was prepared in brain heart infusion broth (BHI). Cultures were incubated shaking (200 rpm) at 37°C. All the experiments were conducted under conditions of standard room illumination (fluorescent ceiling light, Sylvania Octron 4100K) in tubes containing 3 ml of medium. Growth was analyzed by measuring turbidity (absorbance at a wavelength of 660 to 680 nm) with a spectrophotometer (Spectronic 21D). Experiments were initiated with   ̴ 0.2 ml of inoculum from a fresh culture grown aerobically at an optical density (OD) of   ̴ 1. Colony counts were measured on BHI plates. OD was measured every 45 mins to check culture growth. The survival rate was measured by plating serial dilutions of a sample taken during the experiment on BHI plates. Experiment was performed in triplicate on separate days to ascertain reproducibility. As the test substance do not have any activity against S. aureus i.e, no difference and no growth rate inhibition was observed between the treated test tubes and the control tubes, the NOEC value was observed to be 100 mg/l.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:

100 mg/L

Additional information

Summarized result from the various experimental sources for the determination of effect of test chemical on the growth of microorganisms are as mentioned below:

 

Toxicity to micro-organisms study was conducted on Staphylococcus aureus of enterotoxigenic strain ATCC 13565. High-pressure liquid chromatography grade were used for analytical measurements. Inoculum was prepared in brain heart infusion broth (BHI). Cultures were incubated shaking (200 rpm) at 37°C. All the experiments were conducted under conditions of standard room illumination (fluorescent ceiling light, Sylvania Octron 4100K) in tubes containing 3 ml of medium. Growth was analyzed by measuring turbidity (absorbance at a wavelength of 660 to 680 nm) with a spectrophotometer (Spectronic 21D). Experiments were initiated with   ̴ 0.2 ml of inoculum from a fresh culture grown aerobically at an optical density (OD) of   ̴ 1. Colony counts were measured on BHI plates. OD was measured every 45 mins to check culture growth. The survival rate was measured by plating serial dilutions of a sample taken during the experiment on BHI plates. Experiment was performed in triplicate on separate days to ascertain reproducibility. As the test substance do not have any activity against S. aureus i.e, no difference and no growth rate inhibition was observed between the treated test tubes and the control tubes, the NOEC value was observed to be 100 mg/l.

 

Above study further supported by the supported data from peer reviewed journal. Toxicity study of test chemical on micro-organisms was conducted on 10 streptococcal strains. The assay was performed using paper-disk agar diffusion method under aerobic and anaerobic conditions. The test chemical conc. used for the study was 5000 and 10,000 mg/l (0.5 and 1%) respectively. 10 Streptococcal strains were used for the study. The Streptococcal strains include AHT, BHT, CHT, HHT, HS-10, GF-71, GS-5, E-49, FA-1 and PK-1. Tryptic soy agar enriched with 1% sucrose was used as a medium. The assay was performed using paper-disk agar diffusion method in which the paper disk charged with 0.08 ml of aqueous solution of test chemical was placed on the surface of agar plates inoculated with the respective cultures. The plate was incubated for 16 to 30 hrs at 37°C and the presence of zone of growth inhibition around the paper disks was measured. One set of plates was incubated aerobically; the second set was incubated in anaerobic jars which were filled with a gaseous mixture of 95% nitrogen and 5% carbon dioxide. Based on growth inhibition of test organism Streptococcal strains AHT, BHT, HS-10, GF-71, FA-1 and PK-1 under both aerobic and anaerobic condition, the MIC value was observed to be 5,000 mg/l. Similar effects were observed under aerobic condition for Streptococcal strains GS-5 and E-49, based on which the MIC value was observed to be 5,000 mg/l. Under aerobic condition, traces of inhibition in growth of test organism Streptococcal strain HHT was observed, the LOEC value was determine to be 10,000 mg/l. As growth inhibition of test organism Streptococcal strain GS-5 was observed under anaerobic condition, the MIC value was found to be 10,000 mg/l. Also No effects on growth inhibition of test organism Streptococcal strains HHT and E-49 under anaerobic conditions was observed, the NOEC value was observed to be 10,000 mg/l. Similar effects were observed under both aerobic and anaerobic condition for Streptococcal strains CHT, the NOEC value was determine to be 10,000 mg/l.

 

Similarly in the third study, toxicity to micro-organisms was conducted on 8 different organisms of yeast. The assay was performed using paper-disk agar diffusion method under aerobic and anaerobic conditions. The test chemical conc. used for the study was 5000 and 10,000 mg/l (0.5 and 1%) respectively. 8 different yeasts were used for the study. These includes Hansenula wingei ATCC 14256 and ATCC 14355, Candida albicans ATCC 752 and ATCC 11651, Saccharomyces cerevisiae ATCC 4098, Saccharomyces fragilis ATCC 8635, Saccharomyces carlsbergensis ATCC 9080 and S. carlsbergensis SRI-206. All yeasts cultures were cultivated on a medium which contained 0.7% yeast extract, 0.5 KH11PO4, 1.5% agar, and 1.0% sucrose. The organism was autoclaved separately and added aseptically to all media. The assay was performed using paper-disk agar diffusion method in which the paper disk charged with 0.08 ml of aqueous solution of test chemical and was placed on the surface of agar plates inoculated with the respective cultures. The plate was incubated for 24 hrs at 25°C and the presence of zone of growth inhibition around the paper disks was measured. One set of plates was incubated aerobically; the second set was incubated in anaerobic jars which were filled with a gaseous mixture of 95% nitrogen and 5% carbon dioxide. Based on growth inhibition of test organism under aerobic condition by the test chemical, the MIC value was observed to be at 5000 mg/l for organism Candida albicans ATCC 752, Candida albicans ATCC 11651, Saccharomyces fragilis ATCC 8635, Saccharomyces carlsbergensis ATCC 9080 and Saccharomyces carlsbergensis SRI-206. As no effects on growth of test organism was observed under anaerobic condition, the NOEC value was consider to be 10, 000 mg/l for test organism Candida albicans ATCC 752, Candida albicans ATCC 11651, Saccharomyces fragilis ATCC 8635, Saccharomyces carlsbergensis ATCC 9080 and Saccharomyces carlsbergensis SRI-206. Similar effects were observed for test organism Hansenula wingei ATCC 14256, Hansenula wingei ATCC 14355 and Saccharomyces cerevisiae ATCC 4098 under both aerobic and anaerobic condition, the NOEC value was determine to be 10,000 mg/l.

 

Similar study was conducted to determine the effect of test chemical on the growth of Paramecium caudate (ciliated) after providing exposure period of 20 min. Test conducted using 1000 mg/l concentration. In this study, test chemical was put in a hollow slide glass and an equal volume of 0.04 M phosphate buffer, pH 7.0, was added. After that 5 to 10 Paramecium caudatum were added, their survival times were measured microscopically. 30 to 40 Paramecium caudate PC for 0.1% concentration were tested by the same method, and the mean survival time and the death rate were calculated. The death rate was defined as the percentage of deaths observed during 20 min. After the exposure of test chemical with Paramecium caudate (ciliated), mortality were observed after 4 minutes i.e. LC100 value was observed to be at concentration of 1000 mg/l.

 

Toxicity to micro-organisms study was conducted on 12 gram – negative bacteria. The assay was performed using paper-disk agar diffusion method under aerobic and anaerobic conditions. The test chemical concentration used for the study was 5000 and 10,000 mg/l (0.5 and 1%) respectively. 12 gram – negative bacteria as a test organism was used for the study. These includes, Escherichia coli ATCC 9637, E. coli ATCC 9723, E. coli ATCC 11303, Proteus vulgaris ATCC 9484, Salmonella typhimurium ATCC 7823, S. typhosa ATCC 9993, Pseudomonas aeruginosa ATCC 12055, P. aeruginosa ATCC 8709, P. aeruginosa ATCC 10145, Shigella boydii ATCC 9905, S. flexneri ATCC 9582 and Aerobacter aerogenes SRI* - 160. Tryptic soy agar enriched with 1% sucrose was used as a medium. The assay was performed using paper-disk agar diffusion method in which the paper disk charged with 0.08 ml of aqueous solution of test chemical was placed on the surface of agar plates inoculated with the respective cultures. The plate was incubated for 16 to 30 hrs at 37°C and the presence of zone of growth inhibition around the paper disks was measured. One set of plates was incubated aerobically; the second set was incubated in anaerobic jars which were filled with a gaseous mixture of 95% nitrogen and 5% carbon dioxide. As no inhibition on the growth of test organisms was observed due to the test chemical exposure for 30 hours, the NOEC value was observed to be 10,000 mg/l.

 

Thus, based on the above all studies from various sources, no effects were observed on the microorganisms, and thus the chemical can be consider to be nontoxic.