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Ecotoxicological information

Toxicity to microorganisms

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Reference
Endpoint:
toxicity to microorganisms
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
Data is from peer reviewed journal
Reference:
Composition 0
Qualifier:
according to
Guideline:
other: refer below principle
Principles of method if other than guideline:
Toxicity to micro-organisms study was conducted on 11 different human intestinal bacteria.
GLP compliance:
not specified
Test material information:
Composition 1
Specific details on test material used for the study:
- Name of test material (as cited in study report): Sudan II
- Molecular formula (if other than submission substance): C18H16N2O
- Molecular weight (if other than submission substance): 276.337 g/mol
- Smiles notation (if other than submission substance): c12c(\N=N/c3c(cc(C)cc3)C)c(ccc1cccc2)O
- InChl (if other than submission substance): 1S/C18H16N2O/c1-12-7-9-16(13(2)11-12)19-20-18-15-6-4-3-5-14(15)8-10-17(18)21/h3-11,21H,1-2H3
- Structural formula attached as image file (if other than submission substance): No data available
- Substance type: Organic
- Physical state: Solid
Analytical monitoring:
not specified
Details on sampling:
- Concentrations: 100µm (27.357 mg/l)
- Sampling method: Stock solutions of test substance Sudan II was freshly prepared by dissolving each chemical in dimethyl sulfoxide (DMSO).
- Sample storage conditions before analysis: No data available
Vehicle:
yes
Details on test solutions:
Vehical - Dimethyl sulfoxide (DMSO)
Test organisms (species):
other: Bacteroides ovatus, Bifidobacterium infantis, Bifidobatcerium catenulatum, Clostridium indolis, Clostridium perfringens, Clostridium ramosum, Enterococcus faecalis, Escherichia coli, Lactobacillus rhamnosus, Peptostreptococcus magnus
Details on inoculum:
- Laboratory culture: All anaerobic human intestinal bacterial species isolates used in the study was obtained from the American Type Culture Collection (ATCC).

- Method of cultivation: All bacterial seed culture except Lactobacillus rhamnosus was inoculated into Brain Heart Infusion (BHI) medium with an inoculation ratio of 0.01% (v/v) and then 10 ml aliquots of the cultures were transferred to the centrifuge tubes. Later, test compound Sudan II (final conc. 100 µM) was added to the cultures.
Whereas Lactobacillus rhamnosus was inoculated into deMann-Rogosa-Sharpe (MRS) medium instead of BHI medium with 1% (v/v) of the seed culture, and then 10 ml aliquots of the cultures were transferred to the centrifuge tubes. Later Sudan II (final conc. 100 µM) was added to the cultures.
All the above bacterial cultures were incubated at 37ᵒC in the anaerobic chamber without agitation.

- Preparation of inoculum for exposure: All srains, except Lactobacillus rhamnosus, which was cultured on MRS agar plates, were grown on BHI agar supplemented with vitamin K and hemin (Remel) at 37ᵒC overnight in an anaerobic chamber. One colony was picked by a loop and inoculated into a 15ml Falcon centrifuge tube containing 10ml medium(BHI broth supplemented with vitamin K and hemin or MRS). The culture was incubated in static conditions at 37ᵒC overnight in an anaerobic chamber for use as seed culture.

- Pretreatment: No data available

- Initial biomass concentration: No data available
Test type:
not specified
Water media type:
not specified
Total exposure duration:
6 h
Post exposure observation period:
6 and 10 hrs
Hardness:
No data available
Test temperature:
37 deg.C
pH:
No data available
Dissolved oxygen:
No data available
Salinity:
No data available
Nominal and measured concentrations:
No data available
Details on test conditions:
TEST SYSTEM
- Test vessel: Centrifuge tubes
- Type (delete if not applicable): No data available
- Material, size, headspace, fill volume: No data available
- Aeration: No aerating conditions were required during the study.
- Type of flow-through (e.g. peristaltic or proportional diluter): No data available
- Renewal rate of test solution (frequency/flow rate): No data available
- No. of organisms per vessel: No data available
- No. of vessels per concentration (replicates): Triplicates
- No. of vessels per control (replicates): No data available
- No. of vessels per vehicle control (replicates): Triplicates
- Biomass loading rate: bacterial seed culture was inoculated with an inoculation ratio of 1% v/v.

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: No data available
- Particulate matter: No data available
- Metals: No data available
- Pesticides: No data available
- Chlorine: No data available
- Alkalinity: No data available
- Ca/mg ratio: No data available
- Conductivity: No data available
- Culture medium different from test medium: No data available
- Intervals of water quality measurement: No data available

OTHER TEST CONDITIONS
- Adjustment of pH: No data available
- Photoperiod: No data available
- Light intensity: No data available

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : All parameters were collected as logarithmic signals. Bacterial cell number was analyzed.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: No data available
- Justification for using less concentrations than requested by guideline: No data available
- Range finding study: No data available
- Test concentrations: 27.357 mg/l
- Results used to determine the conditions for the definitive study: No data available
Reference substance (positive control):
not specified
Key result
Duration:
6 h
Dose descriptor:
other: MIC
Effect conc.:
27.357 mg/L
Nominal / measured:
meas. (not specified)
Conc. based on:
test mat.
Basis for effect:
other: Decrease in number of bacterial cells was observed
Reported statistics and error estimates:
The Data (bacterial cell numbers) are presented as mean of triplicate with standard deviations (SD) of <5%. P values < 0.05.
Validity criteria fulfilled:
not specified
Conclusions:
The test substance inhibited the growth of Clostridium perfringens and Lactobacillus rhamnosus with decreased cell numbers of 35% and 47% at 6 hr and 17% and 39% at 10 hr, respectively. Growth of Enterococcus faecalis was inhibited with a decreased cell number value of 11% at 6 hr.At the end the minimum inhibition concentration (MIC) considered to be 27.357 mg/l on the basis of Decrease in number of bacterial cells.
Executive summary:

Toxicity study of micro-organism to the test substance was conducted using 11 different human intestinal bacteria.

 

When test bacteria was exposed to the test substance Sudan II, the growth of C. perfringens and L. rhamnosus was inhibited with decreased cell numbers of 35% and 47% at 6 hr and 17% and 39% at 10 hr, respectively. Growth of E. faecalis was inhibited with a decreased cell number value of 11% at 6 hr.

 

The Data (bacterial cell numbers) are presented as mean of triplicate with standard deviations (SD) of<5%.Pvalues<0.05.

 

The bacterial isolates were preserved at -80ᵒC in 15% glycerol stocks.

 

The bacterial cell number was analyzed on an Accuri C6flow cytometer (FCM) (Accuri Cytometers), with 488 nm excitation from a blue solid state laser at 50mW and the standardfilter setup at 6 and 10 hr incubation times.

 

Only slight inhibition of the growth of bacterial isolates B. ovatus, B. infantis, B. catenulatum, C. indolis,C. ramosum, E. coli,P. magnus and R. obeum respectively were observedat 6 and 10 hr incubation times.

 

Significant inhibition in growth was seen of bacterial isolates such as C. perfringens, L. rhamnosus and E. faecalis.

The minimum inhibition concentration (MIC) considered to be 27.357 mg/l on the basis of Decrease in number of bacterial cells.

Description of key information

The test substance inhibited the growth of Clostridium perfringens and Lactobacillus rhamnosus with decreased cell numbers of 35% and 47% at 6 hr and 17% and 39% at 10 hr, respectively. Growth of Enterococcus faecalis was inhibited with a decreased cell number value of 11% at 6 hr.At the end the minimum inhibition concentration (MIC) considered to be 27.357 mg/l on the basis of Decrease in number of bacterial cells.

Key value for chemical safety assessment

Additional information

By applying weight of evidence approach to two studies which includes experimental results from peer reviewed journal for micro organism toxicity of test chemical 1- (2, 4-dimethylphenylazo)-2-naphthol i.e Sudan II (Cas no. 3118-97-6) were summarized as follows:

First study from peer reviewed journal Anaerobe, Vol. 18, Pg. no. 445-453, 2012 indicate Toxicity study of micro-organism to the test substance was conducted using 11 different human intestinal bacteria.When test bacteria was exposed to the test substance Sudan II, the growth ofC. perfringensandL. rhamnosuswas inhibited with decreased cell numbers of 35% and 47% at 6 hr and 17% and 39% at 10 hr, respectively. Growth ofE. faecaliswas inhibited with a decreased cell number value of 11% at 6 hr.The Data (bacterial cell numbers) are presented as mean of triplicate with standard deviations (SD) of<5%.Pvalues<0.05.The bacterial isolates were preserved at -80ᵒC in 15% glycerol stocks.The bacterial cell number was analyzed on an Accuri C6flow cytometer (FCM) (Accuri Cytometers), with 488 nm excitation from a blue solid state laser at 50mW and the standardfilter setup at 6 and 10 hr incubation times.Only slight inhibition of the growth of bacterial isolatesB. ovatus, B. infantis, B. catenulatum, C. indolis,C. ramosum, E. coli,P. magnus and R. obeum respectively were observedat 6 and 10 hr incubation times.Significant inhibition in growth was seen of bacterial isolates such asC. perfringens, L. rhamnosusandE. faecalis. The minimum inhibition concentration (MIC) considered to be 27.357 mg/l on the basis of Decrease in number of bacterial cells.

And another peer reviewed journal APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Vol. 73, No. 23, Pg. no. 7759–7762, Dec. 2007 for target suggest the toxicity study of micro-organism to the test substance was conducted using human intestinal microflora.When test bacteria was exposed to the test substance Sudan II, a lag phase of 2 to 4 hrs was observed. Final cell density in the medium containing the test substance was found to be about 90%.The MIC value for human intestinal microflora was found to be 10 mg/l. Thus, Sudan II has moderate inhibition effect on the human intestinal microflora indicating that the dye was not toxic to the human intestinal microflora.

Both available studies give the the minimum inhibition concentration (MIC) is in the range 27.357 mg/l to 10 mg/l which indicate the moderate inhibition effects.