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Toxicity to microorganisms

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Endpoint:
activated sludge respiration inhibition testing
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
the study does not need to be conducted because the substance is found to be readily biodegradable and the applied test concentrations are in the range of concentrations that can be expected in the influent of a sewage treatment plant
other:
Reason / purpose:
reference to other study
Endpoint:
toxicity to microorganisms, other
Remarks:
antibacterial assays
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
unknown
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Determination of minimal inhibitory concentration (MIC) with the agar dilution method.
GLP compliance:
not specified
Specific details on test material used for the study:
Sophorolipids were produced by fermentation using C. bombicola ATCC 22214 as the producing organism, and glucose + oleic acid as co-substrates.
Analytical monitoring:
no
Vehicle:
yes
Remarks:
Mueller Hinton Agar containing 3 % NaCl
Details on test solutions:
Stock solution: 10 mg/mL dissolved in 70 % ethanol
Test organisms (species):
other: Bacillus licheniformis, Bacillus pumilus, Pseudomonas luteola, Enterococcus faecium, Staphylococcus xylosus, Enterobacter cloacae, Bacillus mycoides, Aerococcus viridans, Staphylococcus cohnii, Enterobacter sakazakii, Staphylococcus equorum
Details on inoculum:
no details given
Test type:
other: agar dilution method
Water media type:
other: not applicable
Total exposure duration:
18 h
Test temperature:
37 °C
Nominal and measured concentrations:
17 different concentrations were tested (10.000 µg/mL, 5000 µg/mL, 2500 µg/mL, 1250 µg/mL, 625 µg/mL, 312.5 µg/mL, 156 µg/mL, 78 µg/mL, 39 µg/mL, 19.5 µg/mL, 9.76 µg/mL, 4.88 µg/mL, 2.44 µg/mL, 1.22 µg/mL, 0.61 µg/mL, 0.30 µg/mL, 0.15 µg/mL, 0.076 µg/mL) in Mueller Hinton Agar containing 3 % NaCl.
Details on test conditions:
Each bacterial suspension of test microorganisms was diluted in 0.85% saline to obtain 1.00E+07 CFU/mL. In addition, the mixed culture of the test isolates was prepared from these bacterial suspensions. Then, the test agent was separately dissolved in 70 % ethanol to a final concentration of 10.000 µg/mL. Series of two-fold dilutions of these test agents, ranging from 10.000 µg/mL to 0.076 µg/mL, were prepared in MHA containing 3 % NaCl.
One mL volumes of 17 different concentrations were separately added to 19 mL volumes of molten MHA. Next, these agar media were mixed thoroughly and poured into sterile petri dishes. One µL (an inoculum of 1.00E+04 CFU/spot) of diluted bacterial suspension of each test isolate and their mixed culture was separately transfered to agar plates containing 17 different concentrations. Control media without test item were used in all experiments.
Later, the plates were incubated at 37 °C for 18 h, and MICs of antibacterial agents against the test bacteria were determined. Inhibitory effects of 70 % ethanol on the test bacteria and mixed culture were also examined on MHA containing the same volume of ethanol concentrations as other agar media containing antimicrobial agents. All test bacteria and the mixed culture grew in the series of ethanol found in MHA. Dilutions of three antimicrobial agents were prepared according
to the method explained in the EUCAST Definitive Document E.Def3.1 (2000).
Reference substance (positive control):
yes
Key result
Duration:
18 h
Dose descriptor:
other: MIC, minimal inhibitory concentration
Effect conc.:
19.5 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth inhibition
Remarks on result:
other: all strains except Enterobacter cloacae
Key result
Duration:
18 h
Dose descriptor:
other: MIC, minimal inhibitory concentration
Effect conc.:
9.76 mg/L
Nominal / measured:
nominal
Conc. based on:
act. ingr.
Basis for effect:
growth inhibition
Remarks on result:
other: only Enterobacter cloacae
Details on results:
MICs were determined as the lowest concentration of antibacterial inhibiting the visible growth of each microorganism on the agar media containing 3 % NaCl.
The MIC of the test item against all test bacteria and their mixed culture were found as 19.5 mg/L, except E. cloacae (9.76 mg/L).
The present study affirms that the test item inhibited the growth of Gram-positive endospore-forming bacteria, Gram-positive bacteria, Gram-negative bacteria and their mixed culture on MHA.
Results with reference substance (positive control):
not applicable
Reported statistics and error estimates:
not applicable
Validity criteria fulfilled:
not applicable
Remarks:
non-guideline study
Conclusions:
In this study, minimal inhibitory concentrations (MIC) of the test item against 12 test bacteria and their mixed culture were found as 19.5 mg/L, except E. cloacae (9.76 mg/L).
Executive summary:

In the publication by Solaiman et al. (2016), minimal inhibitory concentrations (MIC) were determined as the lowest concentration of antibacterial inhibiting the visible growth of 12 microorganisms on an agar media containing 3 % NaCl. The study did not follow a specific guideline. The study affirmed that the test item inhibited the growth of Gram-positive endospore-forming bacteria, Gram-positive bacteria, Gram-negative bacteria and their mixed culture on MHA.

The MIC of the test item after 18 h against all test bacteria and their mixed culture were found as 19.5 mg/L, except E. cloacae (9.76 mg/L).

Description of key information

The study does not need to be conducted because in a biodegradation study according to OECD 301F, GLP, reliability 1, no inhibitory effects of the test item were observed in the toxicity control.

According to Guidance R.10, the tested concentration at which toxicity to the inoculum can be ruled out with sufficient reliability can be considered as a NOEC for the toxicity to STP microorganisms. Thus, 100 mg/L are considered as the NOEC.

Key value for chemical safety assessment

EC10 or NOEC for microorganisms:
100 mg/L

Additional information

The study does not need to be conducted because in a biodegradation study according to OECD 301F, GLP, reliability 1, no inhibitory effects of the test item were observed in the toxicity control.

According to Guidance R.10, the tested concentration at which toxicity to the inoculum can be ruled out with sufficient reliability can be considered as a NOEC for the toxicity to STP microorganisms. Thus, 100 mg/L are considered as the NOEC.

In the publication by Solaiman et al. (2016), minimal inhibitory concentrations (MIC) were determined as the lowest concentration of antibacterial inhibiting the visible growth of 12 microorganisms on an agar media containing 3 % NaCl. The study did not follow a specific guideline. The study affirmed that the test item inhibited the growth of Gram-positive endospore-forming bacteria, Gram-positive bacteria, Gram-negative bacteria and their mixed culture on MHA. The MIC of the test item after 18 h against all test bacteria and their mixed culture were found as 19.5 mg/L, except E. cloacae (9.76 mg/L).

Though, test organisms and conditions are not in accordance to OECD guidelines, and the results from this study are thus considered not relevant as compared to the NOEC derived from the OECD 301F guideline study.